Publications by year
In Press
No B (In Press). Garbage to be deleted. Nowhere
Studholme DJ (In Press). Population-genomic insights into emergence, crop-adaptation, and dissemination of Pseudomonas syringae pathogens. Microbial Genomics
Studholme DJ (In Press). Signatures of Adaptation to Obligate Biotrophy in the Hyaloperonospora arabidopsidis Genome. Science(330), 1549-1551.
2023
Nakato GV, Wicker E, Coutinho TA, Mahuku G, Studholme DJ (2023). Corrigendum to “A highly specific tool for identification of Xanthomonas vasicola pv. musacearum based on five Xvm-specific coding sequences” [Heliyon 4 (12) (December 2018) Article e01080] (Heliyon (2018) 4(12), (S2405844018364879), (10.1016/j.heliyon.2018.e01080)).
Heliyon,
9(3).
Abstract:
Corrigendum to “A highly specific tool for identification of Xanthomonas vasicola pv. musacearum based on five Xvm-specific coding sequences” [Heliyon 4 (12) (December 2018) Article e01080] (Heliyon (2018) 4(12), (S2405844018364879), (10.1016/j.heliyon.2018.e01080))
In the original published version of this article, an error was present in “Table 1: Polymerase chain reaction primers used to distinguish Xanthomonas campestris pv. musacearum isolates”. Following a recent in silico PCR analysis, the authors realized that the FTR primers and the HNS primers were interchanged in the article version. The authors apologize for the mistake. A corrected Table 1 is displayed below. This correction does not, in any way, compromise the findings of the study, either in terms of the methodology, results, or interpretations drawn from the data therein. Both the HTML and PDF versions of the article have been updated to correct the error.
Abstract.
Williamson HF, Brettschneider J, Caccamo M, Davey RP, Goble C, Kersey PJ, May S, Morris RJ, Ostler R, Pridmore T, et al (2023). Data management challenges for artificial intelligence in plant and agricultural research.
F1000Research,
10, 324-324.
Abstract:
Data management challenges for artificial intelligence in plant and agricultural research
Artificial Intelligence (AI) is increasingly used within plant science, yet it is far from being routinely and effectively implemented in this domain. Particularly relevant to the development of novel food and agricultural technologies is the development of validated, meaningful and usable ways to integrate, compare and visualise large, multi-dimensional datasets from different sources and scientific approaches. After a brief summary of the reasons for the interest in data science and AI within plant science, the paper identifies and discusses eight key challenges in data management that must be addressed to further unlock the potential of AI in crop and agronomic research, and particularly the application of Machine Learning (AI) which holds much promise for this domain.
Abstract.
Erdos Z, Studholme DJ, Raymond B, Sharma MD (2023). De novo genome assembly of Akanthomyces muscarius, a biocontrol agent of insect agricultural pests.
Access Microbiology,
5(6).
Abstract:
De novo genome assembly of Akanthomyces muscarius, a biocontrol agent of insect agricultural pests
The entomopathogenic fungus Akanthomyces muscarius is commonly used in agriculture to manage insect pests. Besides its use as a commercially important biological control agent, it also presents a potential model for studying host–pathogen interactions and the evolution of virulence in a laboratory setting. Here, we describe the first high-quality genome sequence for A. muscarius. We used long- and short-read sequencing to assemble a sequence of 36.1 Mb with an N50 of 4.9 Mb. Genome annotation predicted 12347 genes, with 96.6 % completeness based on the core Hypocrealen gene set. The high-quality assembly and annotation of A. muscarius presented in this study provides an essential tool for future research on this commercially important species.
Abstract.
Erdos Z, Studholme DJ, Raymond B, Sharma M (2023). De-novo genome assembly for Akanthomyces muscarius, a biocontrol agent of insect agricultural pests.
Abstract:
De-novo genome assembly for Akanthomyces muscarius, a biocontrol agent of insect agricultural pests
The entomopathogenic fungus Akanthomyces muscarius is used as a biological control agent for the management of insect pests in agriculture. It also presents a potential model for studying host-pathogen interactions and the evolution of virulence in a laboratory setting. Here, we present the first high-quality genome sequence for A. muscarius. We used long- and short-read sequencing to assemble a sequence of 36.1 Mb with an N50 of 4.9 Mb. Genome annotation predicts 12,347 genes, with 96.6 % completeness based on the core Hypocrealen gene set (Benchmarking Universal Single-Copy Orthologs). The first high-quality assembly and annotation of A. muscarius presented in this study provide a powerful tool for further research on this commercially important species.
Abstract.
Erdos Z, Studholme DJ, Raymond B, Sharma M (2023). De-novo genome assembly for Akanthomyces muscarius, a biocontrol agent of insect agricultural pests.
Abstract:
De-novo genome assembly for Akanthomyces muscarius, a biocontrol agent of insect agricultural pests
The entomopathogenic fungus Akanthomyces muscarius is commonly used in agriculture to manage insect pests. Besides its use as a commercially important biological control agent, it also presents a potential model for studying host-pathogen interactions and virulence evolution in a laboratory setting. Here, we describe the first high-quality genome sequence for A. muscarius. We used long- and short-read sequencing to assemble a sequence of 36.1 Mb with an N50 of 4.9 Mb. Genome annotation predicts 12,347 genes, with 96.6 % completeness based on the core Hypocrealen gene set (Benchmarking Universal Single-Copy Orthologs). The high-quality assembly and annotation of A. muscarius presented in this study provide an essential tool for future research on this commercially important species.
Abstract.
Harrison J, Hussain RMF, Greer SF, Ntoukakis V, Aspin A, Vicente JG, Grant M, Studholme DJ (2023). Draft genome sequences for ten strains of Xanthomonas species that have phylogenomic importance.
Abstract:
Draft genome sequences for ten strains of Xanthomonas species that have phylogenomic importance
Here we report draft-quality genome sequences for pathotype strains of eight plant-pathogenic bacterial pathovars: Xanthomonas campestris pv. asclepiadis, X. campestris pv. cannae, X. campestris pv. esculenti, X. campestris pv. nigromaculans, X. campestris pv. parthenii, X. campestris pv. phormiicola, X. campestris pv. zinniae and X. dyei pv. eucalypti (= X. campestris pv. eucalypti). We also sequenced the type strain of species X. melonis and the unclassified Xanthomonas strain NCPPB 1067. These data will be useful for phylogenomic and taxonomic studies, filling some important gaps in sequence coverage of Xanthomonas phylogenetic diversity. We include representatives of previously under-sequenced pathovars and species-level clades. Furthermore, these genome sequences may be useful in elucidating the molecular basis for important phenotypes such as biosynthesis of coronatine-related toxins and degradation of fungal toxin cercosporin.
Abstract.
Harrison J, Hussain RMF, Greer SF, Ntoukakis V, Aspin A, Vicente JG, Grant M, Studholme DJ (2023). Draft genome sequences for ten strains of Xanthomonas species that have phylogenomic importance.
Access Microbiology,
5(7).
Abstract:
Draft genome sequences for ten strains of Xanthomonas species that have phylogenomic importance
Here we report draft-quality genome sequences for pathotype strains of eight plant-pathogenic bacterial pathovars:
.
. Xanthomonas campestris
.
. pv. asclepiadis,
.
. X. campestris
.
. pv. cannae,
.
. X. campestris
.
. pv. esculenti,
.
. X. campestris
.
. pv. nigromaculans,
.
. X. campestris
.
. pv. parthenii,
.
. X. campestris
.
. pv. phormiicola,
.
. X. campestris
.
. pv. zinniae and
.
. X. dyei
.
. pv. eucalypti (=
.
. X. campestris
.
. pv. eucalypti). We also sequenced the type strain of species
.
. X. melonis
.
. and the unclassified
.
. Xanthomonas
.
. strain NCPPB 1067. These data will be useful for phylogenomic and taxonomic studies, filling some important gaps in sequence coverage of
.
. Xanthomonas
.
. phylogenetic diversity. We include representatives of previously under-sequenced pathovars and species-level clades. Furthermore, these genome sequences may be useful in elucidating the molecular basis for important phenotypes, such as biosynthesis of coronatine-related toxins and degradation of fungal toxin cercosporin.
Abstract.
Vicente JG, McHugh J, Bryning A, Carroll S, Harrison J, Studholme D (2023). First identification of Xanthomonas nasturtii as the cause of black rot of watercress in Hawaii.
Plant DisAbstract:
First identification of Xanthomonas nasturtii as the cause of black rot of watercress in Hawaii.
Watercress (Nasturtium officinale) has been in continuous production in Hawaii for over a century and is part of the local diet. Black rot of watercress was first identified as caused by Xanthomonas nasturtii in Florida (Vicente et al. 2017), but symptoms of this disease have also been regularly observed in Hawaii production in all islands, mostly during the rainy season from December to April in areas with poor air circulation (McHugh & Constantinides, 2004). Initially, this disease was attributed to X. campestris due to similar symptoms to black rot of brassicas. Samples of watercress with symptoms that could be attributed to a bacterial disease including yellow spots and lesions on leaves and stunting and deformation of plants in more advanced stages, were collected from a farm in Aiea in the island of Oahu, Hawaii, in October 2017. Isolations were performed at the University of Warwick. Fluid from macerated leaves was streaked into plates of King's B (KB) medium and Yeast Dextrose Calcium Carbonate Agar (YDC). After 48-72 hrs incubation at 28°C, the plates showed a range of mixed colonies. Single cream-yellow mucoid colonies were sub-cultured several times and pure isolates including WHRI 8984 were stored at -76°C as previously described (Vicente et al. 2017). Colony morphology was observed in KB plates and, in contrast to the type strain from Florida (WHRI 8853 = NCPPB 4600), isolate WHRI 8984 did not cause browning of the medium. Pathogenicity was tested on four-week old watercress and Savoy cabbage cv. Wirosa F1 plants by inoculations on leaves as previously described (Vicente et al. 2017). WHRI 8984 did not produce symptoms when inoculated on cabbage but produced typical symptoms on watercress. A re-isolation from a leaf showing a V-shaped lesion, produced isolates with the same morphology, including isolate WHRI 10007A, that was also shown to be pathogenic to watercress therefore completing the Koch's postulates. Fatty acid profiling was performed on WHRI 8984 and 10007A and controls grown on trypticase soy broth agar (TSBA) plates at 28°C for 48 hrs as described by Weller et al. (2000). Profiles were compared with the RTSBA6 v6.21 library; as the database does not include X. nasturtii, the results were only interpreted at the genus level, and both isolates were shown to be Xanthomonas sp. For molecular analysis, DNA was extracted and the gyrB partial gene was amplified and sequenced as described by Parkinson et al. (2007). Comparisons with sequences available in the National Centre for Biotechnology Information (NCBI) databases using the Basic Local Alignment Search Tool (BLAST) showed that partial gyrB of WHRI 8984 and 10007A were identical to the type strain from Florida therefore confirming that they belong to X. nasturtii. For whole genome sequencing, genomic libraries for WHRI 8984 were prepared using Illumina's Nextera XT v2 kit and sequenced on a HiSeq Rapid Run flowcell. The sequences were processed as previously described (Vicente et al. 2017) and the whole genome assembly has been deposited in GenBank (accession QUZM00000000.1); the phylogenetic tree shows that WHRI 8984 is close, but not identical to the type strain. This is the first identification of X. nasturtii in watercress crops in Hawaii. Control of this disease generally involves the use of copper bactericides and minimizing moisture on leaves by reducing overhead irrigation and increasing air circulation (McHugh & Constantinides, 2004); seed testing might help to select batches that are disease free and, in longer term, breeding for disease resistance might produce cultivars that can be part of management strategies.
Abstract.
Author URL.
Reyes-Herrera PH, Torres-Bedoya E, Lopez-Alvarez D, Burbano-David D, Carmona SL, Bebber DP, Studholme DJ, Betancourt M, Soto-Suarez M (2023). Genome Sequence Data Reveal at Least Two Distinct Incursions of the Tropical Race 4 Variant of Fusarium Wilt into South America.
Phytopathology,
113(1), 90-97.
Abstract:
Genome Sequence Data Reveal at Least Two Distinct Incursions of the Tropical Race 4 Variant of Fusarium Wilt into South America.
The global banana industry is threatened by one of the most devastating diseases: Fusarium wilt of banana. Fusarium wilt of banana is caused by the soilborne fungus Fusarium oxysporum f. sp. cubense (Foc), which almost annihilated the banana production in the late 1950s. A new strain of Foc, known as tropical race 4 (TR4), attacks a wide range of banana varieties, including Cavendish clones, which are the source of 99% of banana exports. In 2019, Foc TR4 was reported in Colombia, and more recently (2021) in Peru. In this study, we sequenced three fungal isolates identified as Foc TR4 from La Guajira (Colombia) and compared them against 19 whole-genome sequences of Foc TR4 publicly available, including four genome sequences recently released from Peru. To understand the genetic relatedness of the Colombian Foc TR4 isolates and those from Peru, we conducted a phylogenetic analysis based on a genome-wide set of single nucleotide polymorphisms (SNPs). Additionally, we compared the genomes of the 22 available Foc TR4 isolates, looking for the presence-absence of gene polymorphisms and genomic regions. Our results reveal that (i) the Colombian and Peruvian isolates are genetically distant, which could be better explained by independent incursions of the pathogen to the continent, and (ii) there is a high correspondence between the genetic relatedness and geographic origin of Foc TR4. The profile of present/absent genes and the distribution of missing genomic regions showed a high correspondence to the clades recovered in the phylogenetic analysis, supporting the results obtained by SNP-based phylogeny.
Abstract.
Author URL.
Cotton S, McHugh MP, Dewar R, Haas JG, Templeton K, Consortium TCGU, Robson SC, Connor TR, Loman NJ, Golubchik T, et al (2023). Investigation of hospital discharge cases and SARS-CoV-2 introduction into Lothian care homes. Journal of Hospital Infection, 135, 28-36.
Erdos Z, Studholme DJ, Sharma MD, Chandler D, Bass C, Raymond B (2023). Manipulating multi-level selection in a fungal entomopathogen reveals social conflicts and a method for improving biocontrol traits.
Abstract:
Manipulating multi-level selection in a fungal entomopathogen reveals social conflicts and a method for improving biocontrol traits
AbstractChanges in parasite virulence are commonly expected to lead to trade-offs in other life history traits that can affect fitness. Understanding these trade-offs is particularly important if we want to manipulate the virulence of microbial biological control agents. Theoretically, selection across different spatial scales, i.e. between- and within-hosts, shapes these trade- offs. However, trade-offs are also dependent on parasite biology. Despite their applied importance the evolution of virulence in fungal parasites is poorly understood: virulence can be unstable in culture and commonly fails to increase in simple passage experiments. We hypothesized that manipulating selection intensity at different scales would reveal virulence trade-offs in a fungal pathogen of aphids,Akanthomyces muscarius. Starting with a genetically diverse stock we selected for infectivity, parasite yield or speed of kill by manipulating competition within and between hosts and between groups of hosts over 7 rounds of infection. We characterized ancestral and evolved lineages by whole genome sequencing and by measuring virulence, growth rate, sporulation and fitness. While several lineages showed increases in virulence, we saw none of the trade-offs commonly found in obligately-killing parasites. Phenotypically similar lineages within treatments often shared multiple single-nucleotide variants, indicating strong convergent evolution. The most dramatic phenotypic changes were in timing of sporulation and spore productionin vitro.We found that early sporulation led to reduced competitive fitness but could increase yield of spores on media, a trade-off characteristic of social conflict. Notably, the selection regime with strongest between-group competition and lowest genetic diversity produced the most consistent shift to early sporulation, as predicted by social evolution theory. Mutli-level selection therefore revealed social interactions novel to fungi and showed that these biocontrol agents have the genomic flexibility to improve multiple traits -virulence and spore production - that are often in conflict in other parasites.Author summaryUnderstanding the ecological forces that shape virulence is a key challenge in evolutionary biology. Here we investigated how competition at different levels of selection (within-hosts, between-hosts, between populations) could alter investment in virulence in a fungal entomopathogen. We predicted that cooperative investment in virulence would increase at higher scales of competition and aimed to further our understanding of potential trade-offs shaping life-history of a fungal insect pathogens. We found moderate increases in virulence in different selection regimes but importantly, none of the commonly expected trade-offs, such as that between spore production and virulence or a relationship between virulence and growth rate, which is consistent with cooperation. However, we found convergent genetic changes and significant differences in timing and production of spores, dependent how we manipulated scales of selection. Our data suggests that this is driven by social conflict regarding the timing of sporulation. This carries fundamental importance for understanding how varying selection pressure at different scales shape pathogen life history. In addition, these results also have applied importance for understanding how to improve and select for beneficial traits in biocontrol agents.
Abstract.
Devran Z, Özalp T, Studholme DJ, Tör M (2023). Mapping of the gene in tomato conferring resistance to root-knot nematodes at high soil temperature.
Frontiers in Plant Science,
14Abstract:
Mapping of the gene in tomato conferring resistance to root-knot nematodes at high soil temperature
Root-knot nematodes (RKNs, Meloidogyne spp.) can cause severe yield losses in tomatoes. The Mi-1.2 gene in tomato confers resistance to the Meloidogyne species M. incognita, M. arenaria and M. javanica, which are prevalent in tomato growing areas. However, this resistance breaks down at high soil temperatures (>28°C). Therefore, it is imperative that new resistance sources are identified and incorporated into commercial breeding programmes. We identified a tomato line, MT12, that does not have Mi-1.2 but provides resistance to M. incognita at 32°C soil temperature. An F2 mapping population was generated by crossing the resistant line with a susceptible line, MT17; the segregation ratio showed that the resistance is conferred by a single dominant gene, designated RRKN1 (Resistance to Root-Knot Nematode 1). The RRKN1 gene was mapped using 111 Kompetitive Allele Specific PCR (KASP) markers and characterized. Linkage analysis showed that RRKN1 is located on chromosome 6 and flanking markers placed the locus within a 270 kb interval. These newly developed markers can help pyramiding R-genes and generating new tomato varieties resistant to RKNs at high soil temperatures.
Abstract.
Harrison J, Hussain RMF, Aspin A, Grant MR, Vicente JG, Studholme DJ (2023). Phylogenomic Analysis Supports the Transfer of 20 Pathovars from Xanthomonas campestris into Xanthomonas euvesicatoria.
Taxonomy,
3(1), 29-45.
Abstract:
Phylogenomic Analysis Supports the Transfer of 20 Pathovars from Xanthomonas campestris into Xanthomonas euvesicatoria
The Gram-negative bacterial genus Xanthomonas includes numerous infra-specific taxa known as pathovars, which are defined primarily on host range and disease symptoms. With the advent of molecular sequence data, many pathovars have been transferred from X. campestris into other Xanthomonas species to better harmonise taxonomy and phylogeny. We performed whole-genome shotgun sequencing on pathotype strains of the following X. campestris pathovars: blepharidis, carissae, clerodendri, convolvuli, coriandri, daturae, euphorbiae, fici, heliotropii, ionidii, lawsoniae, mirabilis, obscurae, paulliniae, pennamericanum, spermacoces, uppalii, vernoniae, viegasii and zingibericola. These genomes showed more than 98% average nucleotide identity with the type-strain of X. euvesicatoria and less than 88% with the type-strain of X. campestris. We propose the transfer of these pathovars into X. euvesicatoria and present an emended species description for X. euvesicatoria.
Abstract.
Bagratee T, Studholme D (2023). Targeted genome sequencing for tuberculosis drug susceptibility testing in South Africa: a proposed diagnostic pipeline.
Abstract:
Targeted genome sequencing for tuberculosis drug susceptibility testing in South Africa: a proposed diagnostic pipeline
In July 2023, the World Health Organization (WHO) began recommending targeted next-generation sequencing (tNGS), due to its ability to detect resistance to many drugs with a single test. In March 2023, South Africa further adopted the GeneXpert® XDR cartridge, which detects mutations associated with resistance to second-line injectable drugs. Here, we consider the feasibility for implementing tNGS in South Africa, what such a facility might look like and the specific context of this upper-middle-income country. Whilst the WHO now recommends tNGS for TB diagnostics and DST, there are many economic and infrastructural challenges opposing its deployment. In lieu of this, we instead recommend a stratified diagnostic pipeline that utilises South Africa's existing GeneXpert technologies, in an attempt to reduce the costs associated with implementation of tNGS.
Abstract.
Wacker T, Helmstetter N, Wilson D, Fisher MC, Studholme DJ, Farrer RA (2023). Two-speed genome evolution drives pathogenicity in fungal pathogens of animals.
Proceedings of the National Academy of Sciences,
120(2).
Abstract:
Two-speed genome evolution drives pathogenicity in fungal pathogens of animals
. The origins and evolution of virulence in amphibian-infecting chytrids
. Batrachochytrium dendrobatidis
. (
. Bd
. ) and
. Batrachochytrium salamandrivorans
. (
. Bsal)
. are largely unknown. Here, we use deep nanopore sequencing of
. Bsal
. and comparative genomics against 21 high-quality genome assemblies that span the fungal Chytridiomycota. We discover that
. Bsal
. has the most repeat-rich genome of the Chytridiomycota, comprising 40.9% repetitive elements; this genome has expanded to more than 3× the length of its conspecific
. Bd
. with autonomous and fully functional LTR/Gypsy elements contributing significantly to the expansion. The M36 metalloprotease virulence factors are highly expanded (
. n
. = 177) in
. Bsal
. most of which (53%) are flanked by transposable elements, suggesting they have a repeat-associated expansion. We find enrichment upstream of M36 metalloprotease genes of three novel repeat families belonging to the repeat superfamily of LINEs that are implicated with gene copy number variations. Additionally,
. Bsal
. has a highly compartmentalized genome architecture, with virulence factors enriched in gene-sparse/repeat-rich compartments, while core conserved genes are enriched in gene-rich/repeat-poor compartments. Genes upregulated during infection are primarily found in the gene-sparse/repeat-rich compartment in both
. Bd
. and
. Bsal
. Furthermore, genes with signatures of positive selection in
. Bd
. are enriched in repeat-rich regions, suggesting these regions are a cradle for the evolution of chytrid pathogenicity. These are the hallmarks of two-speed genome evolution, and this study provides evidence of two-speed genomes in an animal pathogen, shedding light on the evolution of fungal pathogens of vertebrates driving global declines and extinctions.
.
Abstract.
2022
Baker S, Dougan G, Hess C, Kingston N, Lehner PJ, Lyons PA, Matheson NJ, Owehand WH, Saunders C, Summers C, et al (2022). Author Correction: SARS-CoV-2 evolution during treatment of chronic infection. Nature, 608(7922), e23-e23.
Baker S, Dougan G, Hess C, Kingston N, Lehner PJ, Lyons PA, Matheson NJ, Owehand WH, Saunders C, Summers C, et al (2022). Author Correction: Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies. Nature, 608(7922), e24-e24.
Kläser K, Molteni E, Graham M, Canas LS, Österdahl MF, Antonelli M, Chen L, Deng J, Murray B, Kerfoot E, et al (2022). COVID-19 due to the B.1.617.2 (Delta) variant compared to B.1.1.7 (Alpha) variant of SARS-CoV-2: a prospective observational cohort study.
Scientific Reports,
12(1).
Abstract:
COVID-19 due to the B.1.617.2 (Delta) variant compared to B.1.1.7 (Alpha) variant of SARS-CoV-2: a prospective observational cohort study
The Delta (B.1.617.2) variant was the predominant UK circulating SARS-CoV-2 strain between May and December 2021. How Delta infection compares with previous variants is unknown. This prospective observational cohort study assessed symptomatic adults participating in the app-based COVID Symptom Study who tested positive for SARS-CoV-2 from May 26 to July 1, 2021 (Delta overwhelmingly the predominant circulating UK variant), compared (1:1, age- and sex-matched) with individuals presenting from December 28, 2020 to May 6, 2021 (Alpha (B.1.1.7) the predominant variant). We assessed illness (symptoms, duration, presentation to hospital) during Alpha- and Delta-predominant timeframes; and transmission, reinfection, and vaccine effectiveness during the Delta-predominant period. 3581 individuals (aged 18 to 100 years) from each timeframe were assessed. The seven most frequent symptoms were common to both variants. Within the first 28 days of illness, some symptoms were more common with Delta versus Alpha infection (including fever, sore throat, and headache) and some vice versa (dyspnoea). Symptom burden in the first week was higher with Delta versus Alpha infection; however, the odds of any given symptom lasting ≥ 7 days was either lower or unchanged. Illness duration ≥ 28 days was lower with Delta versus Alpha infection, though unchanged in unvaccinated individuals. Hospitalisation for COVID-19 was unchanged. The Delta variant appeared more (1.49) transmissible than Alpha. Re-infections were low in all UK regions. Vaccination markedly reduced the risk of Delta infection (by 69-84%). We conclude that COVID-19 from Delta or Alpha infections is similar. The Delta variant is more transmissible than Alpha; however, current vaccines showed good efficacy against disease. This research framework can be useful for future comparisons with new emerging variants.
Abstract.
Sambles C, Suarez-Fernandez M, Lopez-Moya F, Vicente Lopez-Llorca L, Studholme DJ (2022). Chitosan induces differential transcript usage of chitosanase 3 encoding gene (<i>csn3</i>) in the biocontrol fungus <i>Pochonia chlamydosporia</i> 123.
BMC GENOMICS,
23(1).
Author URL.
Grant M (2022). Culturing Xanthomonas v1.
Abstract:
Culturing Xanthomonas v1
This is the protocol for culturing strains of Xanthomonas species in preparation for extracting genomic DNA for sequencing.
Abstract.
Grant M (2022). DNA extraction using the Qiagen 67563 MagAttract HMW DNA Kit (48) v1.
Abstract:
DNA extraction using the Qiagen 67563 MagAttract HMW DNA Kit (48) v1
This protocol is for extraction of genomic DNA from Xanthomonas culture for DNA sequencing.
Abstract.
J Studholme D (2022). De-novo assembly of Xanthomonas genomes from Illumina NovaSeq reads v1.
Abstract:
De-novo assembly of Xanthomonas genomes from Illumina NovaSeq reads v1
This protocol describes the de-novo assembly of Xanthomonas genome sequences from short-read genomic shotgun sequencing data. It includes quality control of the raw sequence reads, assembly and finally polishing of the assembly based on alignment of reads against the preliminary assembly.
Abstract.
Harrison J, Hussain RMF, Greer S, Ntoukakis V, Aspin A, Vicente J, Grant M, Studholme DJ (2022). Draft genome sequences for ten strains of Xanthomonas species that have phylogenomic importance.
Abstract:
Draft genome sequences for ten strains of Xanthomonas species that have phylogenomic importance
Here we report draft-quality genome sequences for pathotype strains of eight plant-pathogenic bacterial pathovars: Xanthomonas campestris pv. asclepiadis, X. campestris pv. cannae, X. campestris pv. esculenti, X. campestris pv. nigromaculans, X. campestris pv. parthenii, X. campestris pv. phormicola, and and X. campestris pv. zinniae. We also sequenced type strains of species X. melonis and X. dyei pv. eucalypti and unclassified Xanthomonas strain NCPPB 1067. These data will be useful for phylogenomic and taxonomic studies, filling some important gaps in sequence coverage of the Xanthomonas phylogenetic diversity. We include representatives of previously un-sequenced pathovars and species-level clades. Furthermore, these genome sequences may be useful in elucidating the molecular basis for important phenotypes such as biosynthesis of coronatine-related toxins and degradation of fungal toxin cercosporin.
Abstract.
Lebas B, Adams I, Al Rwahnih M, Baeyen S, Bilodeau G, Blouin AG, Boonham N, Candresse T, Chandelier A, De Jonghe K, et al (2022). Facilitating the adoption of high-throughput sequencing technologies as a plant pest diagnostic test in laboratories: A step-by-step description.
EPPO Bulletin,
52(2), 394-418.
Abstract:
Facilitating the adoption of high-throughput sequencing technologies as a plant pest diagnostic test in laboratories: A step-by-step description
High-throughput sequencing (HTS) is a powerful tool that enables the simultaneous detection and potential identification of any organisms present in a sample. The growing interest in the application of HTS technologies for routine diagnostics in plant health laboratories is triggering the development of guidelines on how to prepare laboratories for performing HTS testing. This paper describes general and technical recommendations to guide laboratories through the complex process of preparing a laboratory for HTS tests within existing quality assurance systems. From nucleic acid extractions to data analysis and interpretation, all of the steps are covered to ensure reliable and reproducible results. These guidelines are relevant for the detection and identification of any plant pest (e.g. arthropods, bacteria, fungi, nematodes, invasive plants or weeds, protozoa, viroids, viruses), and from any type of matrix (e.g. pure microbial culture, plant tissue, soil, water), regardless of the HTS technology (e.g. amplicon sequencing, shotgun sequencing) and of the application (e.g. surveillance programme, phytosanitary certification, quarantine, import control). These guidelines are written in general terms to facilitate the adoption of HTS technologies in plant pest routine diagnostics and enable broader application in all plant health fields, including research. A glossary of relevant terms is provided among the Supplementary Material.
Abstract.
Lascelles DM, Roberts MR, Cruz L, Cruz J, Studholme DJ, Harrison J, Greer SF, Grant M, Holden JM, Carter B, et al (2022). First report of black rot caused by <i>Xanthomonas nasturtii</i> on watercress in Spain and Portugal. New Disease Reports, 46(2).
Venkatesan L, Muzemil S, Fiche F, Grant M, Studholme DJ (2022). Genome Resources for Ensete ventricosum (Enset) and Related Species. In (Ed) Underutilised Crop Genomes, Springer Nature, 355-371.
Reyes-Herrera PH, Torres-Bedoya E, Lopez-Alvarez D, Burbano-David D, Carmona SL, Bebber DP, Studholme DJ, Betancourt M, Soto-Suarez M (2022). Genome sequence data reveal at least two distinct incursions of the tropical race 4 (TR4) variant of<i>Fusarium</i>wilt into South America.
Abstract:
Genome sequence data reveal at least two distinct incursions of the tropical race 4 (TR4) variant ofFusariumwilt into South America
AbstractThe global banana industry is threatened by one of the most devastating diseases: Fusarium wilt (FWB). FWB is caused by the soil-borne fungusFusarium oxysporumf. sp.cubense(Foc), which almost annihilated the banana production in the late 1950s. A new strain ofFoc, known as tropical race 4 (TR4), attacks a wide range of banana varieties including Cavendish clones which are the source of 99% of banana exports. In 2019,FocTR4 was reported in Colombia, and more recently (2021) in Peru. In this study, we sequenced three fungal isolates identified asFocTR4 from La Guajira (Colombia) and compared them against 19 whole-genome sequences ofFocTR4 publicly available, including four genome sequences recently released from Peru. To understand the genetic relatedness of the ColombianFocTR4 isolates and those from Peru, we conducted a phylogenetic analysis based on a genome-wide set of single nucleotide polymorphisms (SNPs). Additionally, we compared the genomes of the 22 availableFocTR4 isolates looking for the presence-absence of gene polymorphisms and genomic regions. Our results reveal that (i) the Colombian and Peruvian isolates are genetically distant, which could be better explained by independent incursions of the pathogen to the continent, and (ii) there is a high correspondence between the genetic relatedness and geographic origin ofFocTR4. The profile of present/absent genes and the distribution of missing genomic regions showed a high correspondence to the clades recovered in the phylogenetic analysis, supporting the results obtained by SNP-based phylogeny.
Abstract.
Aggarwal D, Page AJ, Schaefer U, Savva GM, Myers R, Volz E, Ellaby N, Platt S, Groves N, Gallagher E, et al (2022). Genomic assessment of quarantine measures to prevent SARS-CoV-2 importation and transmission. Nature Communications, 13(1).
Aggarwal D, Warne B, Jahun AS, Hamilton WL, Fieldman T, du Plessis L, Hill V, Blane B, Watkins E, Wright E, et al (2022). Genomic epidemiology of SARS-CoV-2 in a UK university identifies dynamics of transmission.
Nature Communications,
13(1).
Abstract:
Genomic epidemiology of SARS-CoV-2 in a UK university identifies dynamics of transmission
AbstractUnderstanding SARS-CoV-2 transmission in higher education settings is important to limit spread between students, and into at-risk populations. In this study, we sequenced 482 SARS-CoV-2 isolates from the University of Cambridge from 5 October to 6 December 2020. We perform a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. We observe limited viral introductions into the university; the majority of student cases were linked to a single genetic cluster, likely following social gatherings at a venue outside the university. We identify considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and following a national lockdown. Transmission clusters were largely segregated within the university or the community. Our study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics.
Abstract.
Nickbakhsh S, Hughes J, Christofidis N, Griffiths E, Shaaban S, Enright J, Smollett K, Nomikou K, Palmalux N, Tong L, et al (2022). Genomic epidemiology of SARS-CoV-2 in a university outbreak setting and implications for public health planning.
Scientific Reports,
12(1).
Abstract:
Genomic epidemiology of SARS-CoV-2 in a university outbreak setting and implications for public health planning
Whole genome sequencing of SARS-CoV-2 has occurred at an unprecedented scale, and can be exploited for characterising outbreak risks at the fine-scale needed to inform control strategies. One setting at continued risk of COVID-19 outbreaks are higher education institutions, associated with student movements at the start of term, close living conditions within residential halls, and high social contact rates. Here we analysed SARS-CoV-2 whole genome sequences in combination with epidemiological data to investigate a large cluster of student cases associated with University of Glasgow accommodation in autumn 2020, Scotland. We identified 519 student cases of SARS-CoV-2 infection associated with this large cluster through contact tracing data, with 30% sequencing coverage for further analysis. We estimated at least 11 independent introductions of SARS-CoV-2 into the student population, with four comprising the majority of detected cases and consistent with separate outbreaks. These four outbreaks were curtailed within a week following implementation of control measures. The impact of student infections on the local community was short-term despite an underlying increase in community infections. Our study highlights the need for context-specific information in the formation of public health policy for higher educational settings.
Abstract.
Kontellas G (2022). Glycolytic and unique energy producing enzymes of trematode parasites are potential drug targets: bioinformatics and structural studies.
Abstract:
Glycolytic and unique energy producing enzymes of trematode parasites are potential drug targets: bioinformatics and structural studies
The goal of this project was the investigation of glycolytic enzymes that are vital in the survival and proliferation of adult Fasciola hepatica. These enzymes are triose-phosphate isomerase, phosphoglycerate kinase and cofactor dependent phosphoglycerate mutase. Detailed knowledge about the biochemistry and structure of the above-mentioned fluke enzymes can enable the subsequent development of new and more efficient drugs. The structural comparison of these F. hepatica enzymes with their host mammalian counterparts will elucidate fine structural features that could lead to drugs specifically targeting the trematode enzymes and leaving the host ones unaffected.
In this project, triose-phosphate isomerase. from F. hepatica was over-expressed and purified at 95 % of purity, suitable for crystallographic studies. Enzyme kinetics were studied in a range of temperatures (25 – 42 ⁰C) for the substrate glyceraldehyde-3-phosphate. The highest affinity, and correspondingly the lowest Km value, was observed at 25 ⁰C. Optimal pH value for triose-phosphate isomerase was 7.6. The inhibition of triose-phosphate isomerase by the drug triclabendazole is relatively weak according to the performed inhibition assays where the Ki was established. The inhibition mode is potentially non-competitive. Triose-phosphate isomerase was crystallised and the structure was solved at the highest resolution to date of 1.59 Å. Two residues participating in the formation of the dimer interface were found not to be conserved in mammalian hosts. Consequently, the specific dimer interactions formed in F. hepatica triose-phosphate isomerase but are absent in host enzyme, may be playing a role in the binding of triclabendazole to the fluke enzyme.
As the phosphoglycerate kinase published gene model from F. hepatica was incorrect or incomplete, the correct sequence of the gene was predicted. A method for over-expressing the enzyme in milligram quantities was successfully developed. The enzyme was purified at 92 % of purity and kinetic parameters were established over a range of temperatures (25 – 42 ⁰C). Highest activity of phosphoglycerate kinase was observed at 25 ⁰C and the highest affinity according to the established values of Km at different temperatures, was observed at 39 ⁰C. Optimal pH value for phosphoglycerate kinase. was 7.6. Clorsulon was shown to be a weak uncompetitive inhibitor of phosphoglycerate kinase and the equilibrium dissociation constant of enzyme-substrate-inhibitor complex (αKi) was established.
Phosphoglycerate mutase from F. hepatica was over-expressed and purified at 93 % of purity, suitable for crystallographic studies. Enzyme kinetics were studied in a range of temperatures (25 – 42 ⁰C) with the highest activity observed at 39 ⁰C and the highest affinity at 42 ⁰C. Optimal pH value for phosphoglycerate mutase was 7.6. The inhibition studies show a relatively weak inhibition of. phosphoglycerate mutase. by Clorsulon according to the calculated Ki. The inhibition mode of Clorsulon is likely non-competitive. The first crystal structure of phosphoglycerate mutase from. F. hepatica was solved at the low resolution of 3.16 Å. The asymmetric unit of the crystal and the size exclusion chromatography experiments revealed that there are four identical chains forming dimers that are part of a tetrameric assembly.
An analysis of the available. F. hepatica transcriptome was performed to identify over-expressed non-mammalian proteins during the adult stage that could be important for survival of the parasite. This analysis identified two fluke proteins. that share the CAP (cysteine-rich secretory proteins, antigen 5, and pathogenesis related-1 proteins) domain. CAP superfamily proteins in parasitic helminths are thought to be involved in manipulation of host defence responses. Thus, these two proteins could be considered as vaccine targets.
Recent studies have improved the understanding of rhodoquinone synthesis that is absent in mammals. One of the enzymes that is shared among the rhodoquinone and ubiquinone pathways and suggested to be a potential drug target is 4-hydroxybenzoate polyprenyltransferase or COQ2. In this project COQ2 from F. hepatica has been predicted using AlphaFold, a protein structure prediction algorithm with atomic accuracy. Structural comparison of FhCOQ2 with the predicted structure of human COQ2 has revealed two non-conserved residues in the putative active site of the FhCOQ2 enzyme. The two F. hepatica residues might indicate the potentially different substrate specificity of FhCOQ2 utilising tryptophan metabolites for RQ synthesis.
Abstract.
Twohig KA, Nyberg T, Zaidi A, Thelwall S, Sinnathamby MA, Aliabadi S, Seaman SR, Harris RJ, Hope R, Lopez-Bernal J, et al (2022). Hospital admission and emergency care attendance risk for SARS-CoV-2 delta (B.1.617.2) compared with alpha (B.1.1.7) variants of concern: a cohort study.
The Lancet Infectious Diseases,
22(1), 35-42.
Abstract:
Hospital admission and emergency care attendance risk for SARS-CoV-2 delta (B.1.617.2) compared with alpha (B.1.1.7) variants of concern: a cohort study
Background: the SARS-CoV-2 delta (B.1.617.2) variant was first detected in England in March, 2021. It has since rapidly become the predominant lineage, owing to high transmissibility. It is suspected that the delta variant is associated with more severe disease than the previously dominant alpha (B.1.1.7) variant. We aimed to characterise the severity of the delta variant compared with the alpha variant by determining the relative risk of hospital attendance outcomes. Methods: This cohort study was done among all patients with COVID-19 in England between March 29 and May 23, 2021, who were identified as being infected with either the alpha or delta SARS-CoV-2 variant through whole-genome sequencing. Individual-level data on these patients were linked to routine health-care datasets on vaccination, emergency care attendance, hospital admission, and mortality (data from Public Health England's Second Generation Surveillance System and COVID-19-associated deaths dataset; the National Immunisation Management System; and NHS Digital Secondary Uses Services and Emergency Care Data Set). The risk for hospital admission and emergency care attendance were compared between patients with sequencing-confirmed delta and alpha variants for the whole cohort and by vaccination status subgroups. Stratified Cox regression was used to adjust for age, sex, ethnicity, deprivation, recent international travel, area of residence, calendar week, and vaccination status. Findings: Individual-level data on 43 338 COVID-19-positive patients (8682 with the delta variant, 34 656 with the alpha variant; median age 31 years [IQR 17–43]) were included in our analysis. 196 (2·3%) patients with the delta variant versus 764 (2·2%) patients with the alpha variant were admitted to hospital within 14 days after the specimen was taken (adjusted hazard ratio [HR] 2·26 [95% CI 1·32–3·89]). 498 (5·7%) patients with the delta variant versus 1448 (4·2%) patients with the alpha variant were admitted to hospital or attended emergency care within 14 days (adjusted HR 1·45 [1·08–1·95]). Most patients were unvaccinated (32 078 [74·0%] across both groups). The HRs for vaccinated patients with the delta variant versus the alpha variant (adjusted HR for hospital admission 1·94 [95% CI 0·47–8·05] and for hospital admission or emergency care attendance 1·58 [0·69–3·61]) were similar to the HRs for unvaccinated patients (2·32 [1·29–4·16] and 1·43 [1·04–1·97]; p=0·82 for both) but the precision for the vaccinated subgroup was low. Interpretation: This large national study found a higher hospital admission or emergency care attendance risk for patients with COVID-19 infected with the delta variant compared with the alpha variant. Results suggest that outbreaks of the delta variant in unvaccinated populations might lead to a greater burden on health-care services than the alpha variant. Funding: Medical Research Council; UK Research and Innovation; Department of Health and Social Care; and National Institute for Health Research.
Abstract.
Mirghasempour SA, Studholme DJ, Chen W, Cui D, Mao B (2022). Identification and Characterization of Fusarium nirenbergiae Associated with Saffron Corm Rot Disease.
Plant Dis,
106(2), 486-495.
Abstract:
Identification and Characterization of Fusarium nirenbergiae Associated with Saffron Corm Rot Disease.
Saffron (Crocus sativus L.) is the most expensive spice plant and is distributed widely around the world. However, its production is limited by corm rot, a disastrous disease, attributed to Fusarium oxysporum in many regions of the world. In 2020, extensive surveys were carried out in Zhejiang, Shanghai, Anhui, and Guizhou provinces as saffron growing areas of China. Fourteen single-spore isolates were obtained from rotted corms and identified as F. nirenbergiae according to morphological appearance and multilocus phylogenetic analysis with translation elongation factor 1-α (tef1), DNA-directed RNA polymerase II largest subunit (rpb2), and β-tubulin (tub2). Results of the pathogenicity assay supported the conclusion that F. nirenbergiae is the pathogen responsible for corm rot. In this study, we obtained the whole genome sequence of two highly virulent F. nirenbergiae strains via the Illumina HiSeq platform. Genome sequence assemblies of approximately 52.7 and 52.2 Mb were generated for isolates WY5 and SH1, respectively. To the best of our knowledge, this is the first report of F. nirenbergiae causing C. sativus corm rot in China and indeed worldwide. Results from this research contribute to our understanding of genetic diversity, genomic information, and host determination, which will enable researchers to design appropriate management measures for this hazardous disease.
Abstract.
Author URL.
Snowden J (2022). Molecular and Behavioural Characterisation of Dothistroma septosporum in Great Britain and New Zealand.
Abstract:
Molecular and Behavioural Characterisation of Dothistroma septosporum in Great Britain and New Zealand
Dothistroma septosporum is a foliar pathogen that causes Dothistroma needle blight (DNB), affecting over 80 species of tree. The pathogen is of serious concern; it has spread globally and is of particular significance in pines used in the timber industry leading to quarantine status in several countries. This study examined isolates of D. septosporum originating in Great Britain and New Zealand with molecular and behavioural characterisation and identification based on phenotype and genotype. Subcultures grown from 19 archived isolates from five distinct genetic population clusters of D. septosporum from Great Britain and New Zealand were used to create inocula for in vitro and in planta experiments. An in vitro biomass experiment investigated the dependency of fungal growth on temperature, the isolates and their country of origin, population cluster and latitude of the original sample. A second experiment was carried out in planta, inoculating freshly collected Scots pine (Pinus sylvestris Linnaeus), lodgepole pine (Pinus contorta var. latifolia Engelm. ex S. Watson) and Corsican pine (Pinus nigra subsp. laricio (Poir.) Palib. ex Maire) needles with D. septosporum conidial suspensions from the chosen isolates. Both experiments were conducted over the temperature range 15°C to 40°C. The fungal DNA concentration recovered from lesions on the needles was estimated using qPCR, and the pathogen species and genotypes for each sample were determined from β-tubulin 2 marker gene sequences and BLAST comparisons. The results for both experiments were statistically analysed using linear mixed-effects modelling to determine any relationship between temperature, the isolates and their country of origin, population cluster and latitude of the original sample, and species of inoculated pine needle. In the case of the in planta results, the mean concentration of DNA recovered from lesions on the three species of inoculated pine needles was also compared as a proxy measure for the fungal burden of the infection in the needles.
This study has shown the temperature-dependent growth and fungal burden of D. septosporum for Great Britain and New Zealand population clusters peaks between 20°C and 25°C, and the growth falls away significantly at temperatures on either side of this small range. There appears to be some variation in temperature for peak growth and tolerance of temperature above and below this peak, depending on the population cluster. Differences in the profiles of fungal mass produced in the biomass experiment and the DNA concentrations estimated from lesions in the needle bioassay provided additional information on the variation between population clusters and isolates, both as a function of temperature, and the pine host. A significant difference in growth and fungal burden between isolates from Great Britain and New Zealand was also found at 30°C, and additionally in the pattern of results between in vitro and in planta experiments at this temperature. It is suggested that these differences might be due to gene expression and the role of effector proteins and dothistromin in determining the growth and fungal burden of the pathogen, based on evidence from other studies. A description of the Dothistroma septosporum developmental stages was proposed for in vitro and in planta, which accounts for growth and pathogenicity phenotype behaviour with temperature. It was established that isolates of D. septosporum do not achieve higher levels of infection on species of pine needles that correspond with the original host from which the isolate was sampled, compared to the other two species of pine.
The results of this study could help in the management of DNB around the world as the climate changes. Based on the behaviour of the pathogen in response to temperature, consideration needs to be given to the susceptibility of host species and the pathogenicity of different population clusters of D. septosporum. This study has shown that the lesions on needles on some species of pine had much higher fungal burdens than others at lower temperatures and this could assist forest management in planting less susceptible species appropriate to expected summer temperatures.
Abstract.
Mirghasempour SA, Studholme DJ, Chen W, Zhu W, Mao B (2022). Molecular and Pathogenic Characterization of Fusarium Species Associated with Corm Rot Disease in Saffron from China. Journal of Fungi, 8(5).
J Studholme D (2022). Phylogenomic analysis of Xanthomonas v1.
Abstract:
Phylogenomic analysis of Xanthomonas v1
This is a protocol for using PhaME to generate a phylogenomic tree from a set of Xanthomonas spp. genome sequences.
Abstract.
Robson SC, Connor TR, Loman NJ, Golubchik T, Martinez Nunez RT, Bonsall D, Rambaut A, Snell LB, Ludden C, Corden S, et al (2022). Publisher Correction: Genomic reconstruction of the SARS CoV-2 epidemic in England. Nature, 606(7915), e18-e18.
Willett BJ, Grove J, MacLean OA, Wilkie C, De Lorenzo G, Furnon W, Cantoni D, Scott S, Logan N, Ashraf S, et al (2022). Publisher Correction: SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway (Nature Microbiology, (2022), 7, 8, (1161-1179), 10.1038/s41564-022-01143-7).
Nature Microbiology,
7(10).
Abstract:
Publisher Correction: SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway (Nature Microbiology, (2022), 7, 8, (1161-1179), 10.1038/s41564-022-01143-7)
In the version of this article initially published, the author affiliation information was incomplete, neglecting to note that Brian J. Willett, Joe Grove, Oscar A. MacLean, Craig Wilkie, Giuditta De Lorenzo, Wilhelm Furnon, Diego Cantoni, Sam Scott, Nicola Logan and Shirin Ashraf contributed equally and that John Haughney, David L. Robertson, Massimo Palmarini, Surajit Ray and Emma C. Thomson jointly supervised the work, as now indicated in the HTML and PDF versions of the article.
Abstract.
Willett BJ, Grove J, MacLean OA, Wilkie C, De Lorenzo G, Furnon W, Cantoni D, Scott S, Logan N, Ashraf S, et al (2022). SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway.
Nature Microbiology,
7(8), 1161-1179.
Abstract:
SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway
Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant.
Abstract.
Ashford F, Best A, Dunn SJ, Ahmed Z, Siddiqui H, Melville J, Wilkinson S, Mirza J, Cumley N, Stockton J, et al (2022). SARS-CoV-2 Testing in the Community: Testing Positive Samples with the TaqMan SARS-CoV-2 Mutation Panel to Find Variants in Real Time. Journal of Clinical Microbiology, 60(4), e02408-e02421.
Eales O, Page AJ, de Oliveira Martins L, Wang H, Bodinier B, Haw D, Jonnerby J, Atchison C, Robson SC, Connor TR, et al (2022). SARS-CoV-2 lineage dynamics in England from September to November 2021: high diversity of Delta sub-lineages and increased transmissibility of AY.4.2.
BMC Infectious Diseases,
22(1).
Abstract:
SARS-CoV-2 lineage dynamics in England from September to November 2021: high diversity of Delta sub-lineages and increased transmissibility of AY.4.2
Background: Since the emergence of SARS-CoV-2, evolutionary pressure has driven large increases in the transmissibility of the virus. However, with increasing levels of immunity through vaccination and natural infection the evolutionary pressure will switch towards immune escape. Genomic surveillance in regions of high immunity is crucial in detecting emerging variants that can more successfully navigate the immune landscape. Methods: We present phylogenetic relationships and lineage dynamics within England (a country with high levels of immunity), as inferred from a random community sample of individuals who provided a self-administered throat and nose swab for rt-PCR testing as part of the REal-time Assessment of Community Transmission-1 (REACT-1) study. During round 14 (9 September–27 September 2021) and 15 (19 October–5 November 2021) lineages were determined for 1322 positive individuals, with 27.1% of those which reported their symptom status reporting no symptoms in the previous month. Results: We identified 44 unique lineages, all of which were Delta or Delta sub-lineages, and found a reduction in their mutation rate over the study period. The proportion of the Delta sub-lineage AY.4.2 was increasing, with a reproduction number 15% (95% CI 8–23%) greater than the most prevalent lineage, AY.4. Further, AY.4.2 was less associated with the most predictive COVID-19 symptoms (p = 0.029) and had a reduced mutation rate (p = 0.050). Both AY.4.2 and AY.4 were found to be geographically clustered in September but this was no longer the case by late October/early November, with only the lineage AY.6 exhibiting clustering towards the South of England. Conclusions: As SARS-CoV-2 moves towards endemicity and new variants emerge, genomic data obtained from random community samples can augment routine surveillance data without the potential biases introduced due to higher sampling rates of symptomatic individuals.
Abstract.
2021
O'Sullivan DM, Doyle RM, Temisak S, Redshaw N, Whale AS, Logan G, Huang J, Fischer N, Amos GCA, Preston MD, et al (2021). An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities.
Sci Rep,
11(1).
Abstract:
An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities.
Despite the advent of whole genome metagenomics, targeted approaches (such as 16S rRNA gene amplicon sequencing) continue to be valuable for determining the microbial composition of samples. Amplicon microbiome sequencing can be performed on clinical samples from a normally sterile site to determine the aetiology of an infection (usually single pathogen identification) or samples from more complex niches such as human mucosa or environmental samples where multiple microorganisms need to be identified. The methodologies are frequently applied to determine both presence of micro-organisms and their quantity or relative abundance. There are a number of technical steps required to perform microbial community profiling, many of which may have appreciable precision and bias that impacts final results. In order for these methods to be applied with the greatest accuracy, comparative studies across different laboratories are warranted. In this study we explored the impact of the bioinformatic approaches taken in different laboratories on microbiome assessment using 16S rRNA gene amplicon sequencing results. Data were generated from two mock microbial community samples which were amplified using primer sets spanning five different variable regions of 16S rRNA genes. The PCR-sequencing analysis included three technical repeats of the process to determine the repeatability of their methods. Thirteen laboratories participated in the study, and each analysed the same FASTQ files using their choice of pipeline. This study captured the methods used and the resulting sequence annotation and relative abundance output from bioinformatic analyses. Results were compared to digital PCR assessment of the absolute abundance of each target representing each organism in the mock microbial community samples and also to analyses of shotgun metagenome sequence data. This ring trial demonstrates that the choice of bioinformatic analysis pipeline alone can result in different estimations of the composition of the microbiome when using 16S rRNA gene amplicon sequencing data. The study observed differences in terms of both presence and abundance of organisms and provides a resource for ensuring reproducible pipeline development and application. The observed differences were especially prevalent when using custom databases and applying high stringency operational taxonomic unit (OTU) cut-off limits. In order to apply sequencing approaches with greater accuracy, the impact of different analytical steps needs to be clearly delineated and solutions devised to harmonise microbiome analysis results.
Abstract.
Author URL.
Volz E, Mishra S, Chand M, Barrett JC, Johnson R, Geidelberg L, Hinsley WR, Laydon DJ, Dabrera G, Toole AO, et al (2021). Assessing transmissibility of SARS-CoV-2 lineage B.1.1.7 in England.
NATURE,
593(7858), 266-+.
Author URL.
Graham MS, Sudre CH, May A, Antonelli M, Murray B, Varsavsky T, Kläser K, Canas LS, Molteni E, Modat M, et al (2021). Changes in symptomatology, reinfection, and transmissibility associated with the SARS-CoV-2 variant B.1.1.7: an ecological study. The Lancet Public Health, 6(5), e335-e345.
Suarez‐Fernandez M, Sambles C, Lopez‐Moya F, Nueda MJ, Studholme DJ, Lopez‐Llorca LV (2021). Chitosan modulates <i>Pochonia chlamydosporia</i> gene expression during nematode egg parasitism.
Environmental Microbiology,
23(9), 4980-4997.
Abstract:
Chitosan modulates Pochonia chlamydosporia gene expression during nematode egg parasitism
SummaryClimate change makes plant‐parasitic nematodes (PPN) an increasing threat to commercial crops. PPN can be managed sustainably by the biocontrol fungus Pochonia chlamydosporia (Pc). Chitosan generated from chitin deacetylation enhances PPN parasitism by Pc. In this work, we investigate the molecular mechanisms of Pc for chitosan resistance and root‐knot nematode (RKN) parasitism, using transcriptomics. Chitosan and RKN modify the expression of Pc genes, mainly those involved in oxidation–reduction processes. Both agents significantly modify the expression of genes associated to 113 GO terms and 180 Pc genes. Genes encoding putative glycoproteins (Pc adhesives) to nematode eggshell, as well as genes involved in redox, carbohydrate and lipid metabolism trigger the response to chitosan. We identify genes expressed in both the parasitic and endophytic phases of the Pc lifecycle; these include proteases, chitosanases and transcription factors. Using the Pathogen—Host Interaction database (PHI‐base), our previous RNA‐seq data and RT‐PCR of Pc colonizing banana we have investigated genes expressed both in the parasitic and endophytic phases of Pc lifecycle.
Abstract.
Chen NWG, Ruh M, Darrasse A, Foucher J, Briand M, Costa J, Studholme DJ, Jacques M-A (2021). Common bacterial blight of bean: a model of seed transmission and pathological convergence.
Mol Plant Pathol,
22(12), 1464-1480.
Abstract:
Common bacterial blight of bean: a model of seed transmission and pathological convergence.
BACKGROUND: Xanthomonas citri pv. fuscans (Xcf) and Xanthomonas phaseoli pv. phaseoli (Xpp) are the causal agents of common bacterial blight of bean (CBB), an important disease worldwide that remains difficult to control. These pathogens belong to distinct species within the Xanthomonas genus and have undergone a dynamic evolutionary history including the horizontal transfer of genes encoding factors probably involved in adaptation to and pathogenicity on common bean. Seed transmission is a key point of the CBB disease cycle, favouring both vertical transmission of the pathogen and worldwide distribution of the disease through global seed trade. TAXONOMY: Kingdom: Bacteria; phylum: Proteobacteria; class: Gammaproteobacteria; order: Lysobacterales (also known as Xanthomonadales); family: Lysobacteraceae (also known as Xanthomonadaceae); genus: Xanthomonas; species: X. citri pv. fuscans and X. phaseoli pv. phaseoli (Xcf-Xpp). HOST RANGE: the main host of Xcf-Xpp is the common bean (Phaseolus vulgaris). Lima bean (Phaseolus lunatus) and members of the Vigna genus (Vigna aconitifolia, Vigna angularis, Vigna mungo, Vigna radiata, and Vigna umbellata) are also natural hosts of Xcf-Xpp. Natural occurrence of Xcf-Xpp has been reported for a handful of other legumes such as Calopogonium sp. Pueraria sp. pea (Pisum sativum), Lablab purpureus, Macroptilium lathyroides, and Strophostyles helvola. There are conflicting reports concerning the natural occurrence of CBB agents on tepary bean (Phaseolus acutifolius) and cowpea (Vigna unguiculata subsp. unguiculata). SYMPTOMS: CBB symptoms occur on all aerial parts of beans, that is, seedlings, leaves, stems, pods, and seeds. Symptoms initially appear as water-soaked spots evolving into necrosis on leaves, pustules on pods, and cankers on twigs. In severe infections, defoliation and wilting may occur. DISTRIBUTION: CBB is distributed worldwide, meaning that it is frequently encountered in most places where bean is cultivated in the Americas, Asia, Africa, and Oceania, except for arid tropical areas. Xcf-Xpp are regulated nonquarantine pathogens in Europe and are listed in the A2 list by the European and Mediterranean Plant Protection Organization (EPPO). GENOME: the genome consists of a single circular chromosome plus one to four extrachromosomal plasmids of various sizes, for a total mean size of 5.27 Mb with 64.7% GC content and an average predicted number of 4,181 coding sequences. DISEASE CONTROL: Management of CBB is based on integrated approaches that comprise measures aimed at avoiding Xcf-Xpp introduction through infected seeds, cultural practices to limit Xcf-Xpp survival between host crops, whenever possible the use of tolerant or resistant bean genotypes, and chemical treatments, mainly restricted to copper compounds. The use of pathogen-free seeds is essential in an effective management strategy and requires appropriate sampling, detection, and identification methods. USEFUL WEBSITES: https://gd.eppo.int/taxon/XANTPH, https://gd.eppo.int/taxon/XANTFF, and http://www.cost.eu/COST_Actions/ca/CA16107.
Abstract.
Author URL.
Williamson HF, Brettschneider J, Caccamo M, Davey RP, Goble C, Kersey PJ, May S, Morris RJ, Ostler R, Pridmore T, et al (2021). Data management challenges for artificial intelligence in plant and agricultural research.
F1000Research,
10, 324-324.
Abstract:
Data management challenges for artificial intelligence in plant and agricultural research
Artificial Intelligence (AI) is increasingly used within plant science, yet it is far from being routinely and effectively implemented in this domain. Particularly relevant to the development of novel food and agricultural technologies is the development of validated, meaningful and usable ways to integrate, compare and visualise large, multi-dimensional datasets from different sources and scientific approaches. After a brief summary of the reasons for the interest in data science and AI within plant science, the paper identifies and discusses eight key challenges in data management that must be addressed to further unlock the potential of AI in crop and agronomic research, and particularly the application of Machine Learning (AI) which holds much promise for this domain.
Abstract.
Williamson HF, Brettschneider J, Caccamo M, Davey RP, Goble C, Kersey PJ, May S, Morris RJ, Ostler R, Pridmore T, et al (2021). Data management challenges for artificial intelligence in plant and agricultural research.
F1000Research,
10Abstract:
Data management challenges for artificial intelligence in plant and agricultural research.
Artificial Intelligence (AI) is increasingly used within plant science, yet it is far from being routinely and effectively implemented in this domain. Particularly relevant to the development of novel food and agricultural technologies is the development of validated, meaningful and usable ways to integrate, compare and visualise large, multi-dimensional datasets from different sources and scientific approaches. After a brief summary of the reasons for the interest in data science and AI within plant science, the paper identifies and discusses eight key challenges in data management that must be addressed to further unlock the potential of AI in crop and agronomic research, and particularly the application of Machine Learning (AI) which holds much promise for this domain.
Abstract.
Christakis CA, Daskalogiannis G, Chatzaki A, Markakis EA, Mermigka G, Sagia A, Rizzo GF, Catara V, Lagkouvardos I, Studholme DJ, et al (2021). Endophytic Bacterial Isolates from Halophytes Demonstrate Phytopathogen Biocontrol and Plant Growth Promotion Under High Salinity.
Front Microbiol,
12Abstract:
Endophytic Bacterial Isolates from Halophytes Demonstrate Phytopathogen Biocontrol and Plant Growth Promotion Under High Salinity.
Halophytic endophytes potentially contribute to the host's adaptation to adverse environments, improving its tolerance against various biotic and abiotic stresses. Here, we identified the culturable endophytic bacteria of three crop wild relative (CWR) halophytes: Cakile maritima, Matthiola tricuspidata, and Crithmum maritimum. In the present study, the potential of these isolates to improve crop adaptations to various stresses was investigated, using both in vitro and in-planta approaches. Endophytic isolates were identified by their 16S rRNA gene sequence and evaluated for their ability to: grow in vitro in high levels of NaCl; inhibit the growth of the economically important phytopathogens Verticillium dahliae, Ralstonia solanacearum, and Clavibacter michiganensis and the human pathogen Aspergillus fumigatus; provide salt tolerance in-planta; and provide growth promoting effect in-planta. Genomes of selected isolates were sequenced. In total, 115 endophytic isolates were identified. At least 16 isolates demonstrated growth under increased salinity, plant growth promotion and phytopathogen antagonistic activity. Three showed in-planta suppression of Verticillium growth. Furthermore, representatives of three novel species were identified: two Pseudomonas species and one Arthrobacter. This study provides proof-of-concept that the endophytes from CWR halophytes can be used as "bio-inoculants," for the enhancement of growth and stress tolerance in crops, including the high-salinity stress.
Abstract.
Author URL.
Volz E, Hill V, McCrone JT, Price A, Jorgensen D, O’Toole Á, Southgate J, Johnson R, Jackson B, Nascimento FF, et al (2021). Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity. Cell, 184(1), 64-75.e11.
Muzemil S, Chala A, Tesfaye B, Studholme DJ, Grant M, Yemataw Z, Mekonin S, Olango TM (2021). Evaluation of 20 enset (<i>Ensete ventricosum)</i> landraces for response to <i>Xanthomonas vasicola</i> pv. <i>Musacearum</i> infection.
EUROPEAN JOURNAL OF PLANT PATHOLOGY,
161(4), 821-836.
Author URL.
Elliott P, Haw D, Wang H, Eales O, Walters CE, Ainslie KEC, Atchison C, Fronterre C, Diggle PJ, Page AJ, et al (2021). Exponential growth, high prevalence of SARS-CoV-2, and vaccine effectiveness associated with the Delta variant.
Science,
374(6574).
Abstract:
Exponential growth, high prevalence of SARS-CoV-2, and vaccine effectiveness associated with the Delta variant.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections were rising during early summer 2021 in many countries as a result of the Delta variant. We assessed reverse transcription polymerase chain reaction swab positivity in the Real-time Assessment of Community Transmission–1 (REACT-1) study in England. During June and July 2021, we observed sustained exponential growth with an average doubling time of 25 days, driven by complete replacement of the Alpha variant by Delta and by high prevalence at younger, less-vaccinated ages. Prevalence among unvaccinated people [1.21% (95% credible interval 1.03%, 1.41%)] was three times that among double-vaccinated people [0.40% (95% credible interval 0.34%, 0.48%)]. However, after adjusting for age and other variables, vaccine effectiveness for double-vaccinated people was estimated at between ~50% and ~60% during this period in England. Increased social mixing in the presence of Delta had the potential to generate sustained growth in infections, even at high levels of vaccination.
Abstract.
Author URL.
Vöhringer HS, Sanderson T, Sinnott M, De Maio N, Nguyen T, Goater R, Schwach F, Harrison I, Hellewell J, Ariani CV, et al (2021). Genomic reconstruction of the SARS-CoV-2 epidemic in England.
Nature,
600(7889), 506-511.
Abstract:
Genomic reconstruction of the SARS-CoV-2 epidemic in England
AbstractThe evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus leads to new variants that warrant timely epidemiological characterization. Here we use the dense genomic surveillance data generated by the COVID-19 Genomics UK Consortium to reconstruct the dynamics of 71 different lineages in each of 315 English local authorities between September 2020 and June 2021. This analysis reveals a series of subepidemics that peaked in early autumn 2020, followed by a jump in transmissibility of the B.1.1.7/Alpha lineage. The Alpha variant grew when other lineages declined during the second national lockdown and regionally tiered restrictions between November and December 2020. A third more stringent national lockdown suppressed the Alpha variant and eliminated nearly all other lineages in early 2021. Yet a series of variants (most of which contained the spike E484K mutation) defied these trends and persisted at moderately increasing proportions. However, by accounting for sustained introductions, we found that the transmissibility of these variants is unlikely to have exceeded the transmissibility of the Alpha variant. Finally, B.1.617.2/Delta was repeatedly introduced in England and grew rapidly in early summer 2021, constituting approximately 98% of sampled SARS-CoV-2 genomes on 26 June 2021.
Abstract.
Kahveci E, Devran Z, oezkaynak E, Hong Y, Studholme DJ, Toer M (2021). Genomic-Assisted Marker Development Suitable for <i>CsCvy</i>-<i>1</i> Selection in Cucumber Breeding.
FRONTIERS IN PLANT SCIENCE,
12 Author URL.
Wang S, Vetukuri RR, Kushwaha SK, Hedley PE, Morris J, Studholme DJ, Welsh LRJ, Boevink PC, Birch PRJ, Whisson SC, et al (2021). Haustorium formation and a distinct biotrophic transcriptome characterize infection of <i>Nicotiana benthamiana</i> by the tree pathogen <i>Phytophthora kernoviae</i>.
MOLECULAR PLANT PATHOLOGY,
22(8), 954-968.
Author URL.
Meng B, Kemp SA, Papa G, Datir R, Ferreira IATM, Marelli S, Harvey WT, Lytras S, Mohamed A, Gallo G, et al (2021). Recurrent emergence of SARS-CoV-2 spike deletion H69/V70 and its role in the Alpha variant B.1.1.7.
Cell Reports,
35(13).
Abstract:
Recurrent emergence of SARS-CoV-2 spike deletion H69/V70 and its role in the Alpha variant B.1.1.7
We report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike ΔH69/V70 in multiple independent lineages, often occurring after acquisition of receptor binding motif replacements such as N439K and Y453F, known to increase binding affinity to the ACE2 receptor and confer antibody escape. In vitro, we show that, although ΔH69/V70 itself is not an antibody evasion mechanism, it increases infectivity associated with enhanced incorporation of cleaved spike into virions. ΔH69/V70 is able to partially rescue infectivity of spike proteins that have acquired N439K and Y453F escape mutations by increased spike incorporation. In addition, replacement of the H69 and V70 residues in the Alpha variant B.1.1.7 spike (where ΔH69/V70 occurs naturally) impairs spike incorporation and entry efficiency of the B.1.1.7 spike pseudotyped virus. Alpha variant B.1.1.7 spike mediates faster kinetics of cell-cell fusion than wild-type Wuhan-1 D614G, dependent on ΔH69/V70. Therefore, as ΔH69/V70 compensates for immune escape mutations that impair infectivity, continued surveillance for deletions with functional effects is warranted.
Abstract.
Kemp SA, Collier DA, Datir RP, Ferreira IATM, Gayed S, Jahun A, Hosmillo M, Rees-Spear C, Mlcochova P, Lumb IU, et al (2021). SARS-CoV-2 evolution during treatment of chronic infection.
Nature,
592(7853), 277-282.
Abstract:
SARS-CoV-2 evolution during treatment of chronic infection
The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.
Abstract.
Collier DA, De Marco A, Ferreira IATM, Meng B, Datir RP, Walls AC, Kemp SA, Bassi J, Pinto D, Silacci-Fregni C, et al (2021). Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies.
NATURE,
593(7857), 136-+.
Author URL.
Torres Bedoya E, Bebber DP, Studholme DJ (2021). Taxonomic Revision of the Banana Fusarium Wilt TR4 Pathogen is Premature.
Phytopathology,
111(12), 2141-2145.
Abstract:
Taxonomic Revision of the Banana Fusarium Wilt TR4 Pathogen is Premature.
Taxonomic revisions for pathogens of crops should be based on robust underpinning evidence. Recently, a substantial revision was proposed for the taxonomy of the causative agent of Fusarium wilt on banana. We reanalyzed the data on which this revision was based and discovered that the data do not robustly support the proposals. Several apparent discrepancies and errors in the published phylogenies cast further doubt on the conclusions drawn from them. Although we do not assert that the authors' conclusions are incorrect, we posit that the taxonomic changes are premature, given the data currently in the public domain.[Formula: see text] Copyright © 2021 the Author(s). This is an open access article distributed under the CC BY 4.0 International license.
Abstract.
Author URL.
Trew J (2021). The Enterocytozoonidae: the emergence of a microsporidian clade into the aquatic and terrestrial food-chain.
Abstract:
The Enterocytozoonidae: the emergence of a microsporidian clade into the aquatic and terrestrial food-chain
Pathogens of livestock have proven to be a major concern, in terms of human health, economic sustainability and food availability. They have been shown to be a limiting factor in regard to these three factors and, as such, will become a growing problem as the global population continues to expand. Controlling and predicting pathogen outbreaks is vital to the sustained growth of populations and has become an important topic of study. However, it is a complex process with a multitude of varying factors, that cannot be solved through one method.
A specific family of Microsporidia, the Enterocytozoonidae, has received a lot of attention in regard to their effect on human health and aquaculture. Enterocytozoonids are largely found in marine environments and mainly infect aquatic hosts. Many of the organisms infected by enterocytozoonids are economically important to aquaculture, with a number infecting wild-caught fish and farmed crustacea. Two species, in particular, have received a lot of academic and medical attention, Enterocytozoon bieneusi and Enterocytozoon hepatopenaei. However, the family as a whole is understudied and the diversity described is hypothesised not to be a true representation of the family’s diversity or distribution.
The aim of this thesis is to assess the potential risk the Enterocytozoonidae have on aquaculture. This has been done in three different ways: i) it described and catalogued the current diversity and distribution in South West UK estuaries and a greater spatial scale using metagenomic databases, ii) using population genomics, it investigates the biogeography of, E. hepatopenaei, a pathogen of two shrimp (Penaeus vannamei and Penaeus monodon) widely farmed in South East Asia, with the aim to infer transmission routes into shrimp ponds, iii) and lastly, it investigated the loss and gain of orthogroups within the Enterocytozoonidae associated with the lineage wide host-shift to aquatic host from terrestrial hosts.
Overall, this thesis found a greater distribution and abundance for Enterospora canceri and E. hepatopenaei in the locations sampled, and greater diversity within the Enterocytozoondiae than was previously described. Suggesting an alternative host (s) for En. canceri, likely planktonic crustacea, as prevalence in the current described host was very low (3%). Use of metagenomic databases in this study also expanded upon the known distribution and diversity of the Enterocytozoonidae, showing a largely marine-based distribution. Suggesting an Atlantic-based radiation. It found that E. hepatopenaei is likely to be endemic to the countries examined, due to strong geographic signals. However, analysis suggests some transmission between Thailand India and China. Lastly, it identified candidate genes, unique to lineages within the Enterocytozoonidae, that could contribute to the family’s success in the invasion of their respective host cells.
A greater diversity and abundance for novel sequences more related to the Enterocytozoon/Enterospora branch of the Enterocytozoondiae was also observed, suggesting, at least in estuaries in the Southwest of the United Kingdom, that this clade may be more diverse. Possibly due to their described main hosts being planktonic crustaceans. Which may lend to the success of the two most prevalent species in the family, E. hepatopenaei and E. bieneusi.
This study found that the Enterocytozoonidae likely pose a continued threat to aquatic livestock, largely due to their widespread nature, overlapping with human influenced environments, and evident opportunistic propensity to host-shift. In addition, also being present inland in freshwater makes preventative measures difficult, as they could be found in a range of water sources.
Abstract.
de Silva TI, Liu G, Lindsey BB, Dong D, Moore SC, Hsu NS, Shah D, Wellington D, Mentzer AJ, Angyal A, et al (2021). The impact of viral mutations on recognition by SARS-CoV-2 specific T cells.
iScience,
24(11).
Abstract:
The impact of viral mutations on recognition by SARS-CoV-2 specific T cells
We identify amino acid variants within dominant SARS-CoV-2 T cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T cells assessed by IFN-γ and cytotoxic killing assays. Complete loss of T cell responsiveness was seen due to Q213K in the A∗01:01-restricted CD8+ ORF3a epitope FTSDYYQLY207-215; due to P13L, P13S, and P13T in the B∗27:05-restricted CD8+ nucleocapsid epitope QRNAPRITF9-17; and due to T362I and P365S in the A∗03:01/A∗11:01-restricted CD8+ nucleocapsid epitope KTFPPTEPK361-369. CD8+ T cell lines unable to recognize variant epitopes have diverse T cell receptor repertoires. These data demonstrate the potential for T cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T cell as well as humoral immunity.
Abstract.
Malesevic M, Stanisavljevic N, Miljkovic M, Jovcic B, Filipic B, Studholme DJ, Kojic M (2021). The large plasmidome of Lactococcus lactis subsp. lactis bv. diacetylactis S50 confers its biotechnological properties.
Int J Food Microbiol,
337Abstract:
The large plasmidome of Lactococcus lactis subsp. lactis bv. diacetylactis S50 confers its biotechnological properties.
Plasmids are autonomous episomally replicating genetic elements, which carry backbone genes important for the replication and maintenance within their host, and accessory genes that might confer an advantage to their host under specific selective pressure in its ecological niche. The genome of dairy isolate L. lactis subsp. lactis bv. diacetylactis S50 was sequenced using the PacBio SMRT Cell Seq-RSII platform and revealed to possess one of the largest plasmidomes among L. lactis strains studied so far, harboring six plasmids: pS6 (5553 bp), pS7a (7308 bp), pS7b (7266 bp), pS19 (19,027 bp), pS74 (74,256 bp) and pS127 (127,002 bp) in total representing 8.9% of genome size (240,412 bp). Based on predicted plasmid replication proteins and origins it appears that all six plasmids replicate via the theta-type mechanism. The two the largest plasmids (pS74 and pS127), carry a number of genes known to be important for growth and survival in the dairy environment. These genes encode technological functions such as bacteriocin production, protein degradation, magnesium and cobalt/nickel transporters, selenium binding, exopolysaccharides (EPS) production, bacteriophage and stress resistance. Beside genes for replication, the small plasmids (pS6, pS7a, pS7a, and pS19) also carry genes important for mobilization and host survival such as type I restriction-modification (R-M) system, metal transporters, enzymes and transcriptional regulators. All plasmids in S50 strain are mobilizable, containing an oriT sequences, while pS127 is self-conjugative and allows for mobilization of the other plasmids. Small plasmids are prone to structural and segregational instability, while pS127 appeared to be segregationally stable thanks to the possession of two partition systems. The main characteristic of plasmid pS74 is EPS production, while plasmid pS127 is characterized by proteinase and multiple bacteriocins, tra locus, phage abortive systems and metal transporters. In addition to LcnA and LcnB, plasmid pS127 encodes several bacteriocin-pheromone molecules and a new bacteriocin named LcnS50, with narrow spectrum of action limited to lactococci, that has been successfully cloned and heterologously expressed.
Abstract.
Author URL.
Catara V, Cubero J, Pothier JF, Bosis E, Bragard C, Đermić E, Holeva MC, Jacques MA, Petter F, Pruvost O, et al (2021). Trends in molecular diagnosis and diversity studies for phytosanitary regulated xanthomonas.
Microorganisms,
9(4).
Abstract:
Trends in molecular diagnosis and diversity studies for phytosanitary regulated xanthomonas
Bacteria in the genus Xanthomonas infect a wide range of crops and wild plants, with most species responsible for plant diseases that have a global economic and environmental impact on the seed, plant, and food trade. Infections by Xanthomonas spp. cause a wide variety of non‐specific symptoms, making their identification difficult. The coexistence of phylogenetically close strains, but drastically different in their phenotype, poses an added challenge to diagnosis. Data on future climate change scenarios predict an increase in the severity of epidemics and a geographical expansion of pathogens, increasing pressure on plant health services. In this context, the effectiveness of integrated disease management strategies strongly depends on the availability of rapid, sensitive, and specific diagnostic methods. The accumulation of genomic information in recent years has facilitated the identification of new DNA markers, a cornerstone for the development of more sensitive and specific methods. Nevertheless, the challenges that the taxonomic complexity of this genus represents in terms of diagnosis together with the fact that within the same bacterial species, groups of strains may interact with distinct host species demonstrate that there is still a long way to go. In this review, we describe and discuss the current molecular‐based methods for the diagnosis and detection of regulated Xanthomonas, taxonomic and diversity studies in Xanthomonas and genomic approaches for molecular diagnosis.
Abstract.
Wacker T, Helmstetter N, Wilson D, Fisher MC, Studholme DJ, Farrer RA (2021). Two-speed genome expansion drives the evolution of pathogenicity in animal fungal pathogens.
Abstract:
Two-speed genome expansion drives the evolution of pathogenicity in animal fungal pathogens
AbstractThe origins of virulence in amphibian-infecting chytrids Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are largely unknown. Here, we use deep nanopore sequencing of Bsal and comparative genomics against 21 high-quality genome assemblies that span the fungal Chytridiomycota. Bsal has the most repeat-rich genome, comprising 40.9% repetitive elements, which has expanded to more than 3X the length of its conspecific Bd. M36 metalloprotease virulence factors are highly expanded in Bsal and 53% of the 177 unique genes are flanked by transposable elements, suggesting repeat-driven expansion. The largest M36 sub-family are mostly (84%) flanked upstream by a novel LINE element, a repeat superfamily implicated with gene copy number variations. We find that Bsal has a highly compartmentalized genome architecture, with virulence factors enriched in gene-sparse/repeat-rich compartments, while core conserved genes occur in gene-rich/repeat-poor compartments. This is a hallmark of two-speed genome evolution. Furthermore, genes with signatures of positive selection in Bd are enriched in repeat-rich regions, suggesting they are a cradle for chytrid pathogenicity evolution, and Bd also has a two-speed genome. This is the first evidence of two-speed genomes in any animal pathogen, and sheds new light on the evolution of fungal pathogens of vertebrates driving global declines and extinctions.
Abstract.
Masoli JAH, Jeffries A, Temperton B, Auckland C, Michelsen M, Warwick-Dugdale J, Manley R, Farbos A, Ellard S, Knight B, et al (2021). Viral genetic sequencing identifies staff transmission of COVID-19 is important in a community hospital outbreak.
Abstract:
Viral genetic sequencing identifies staff transmission of COVID-19 is important in a community hospital outbreak
AbstractBackgroundWe have successfully used whole-genome sequencing to provide additional information for transmission pathways in infectious spread. We report and interpret genomic sequencing results in clinical context from a large outbreak of COVID-19 with 46 cases across staff and patients in a community hospital in the UK.MethodsFollowing multiple symptomatic cases within a two-week period, all staff and patients were screened by RT-PCR and staff subsequently had serology tests.ResultsThirty staff (25%) and 16 patients (62%) tested positive for COVID-19. Genomic sequencing data showed significant overlap of viral haplotypes in staff who had overlapping shift patterns. Patient haplotypes were more distinct from each other but had overlap with staff haplotypes.ConclusionsThis study includes clinical and genomic epidemiological detail that demonstrates the value of a combined approach. Viral genetic sequencing has identified that staff transmission of COVID-19 was important in this community hospital outbreak.Key pointsDetailed analysis of a large community hospital outbreak in older adults and staff with concurrent clinical and genomic data, including working patterns.Staff transmission was important in this community hospital outbreak.We found plausible associations between staff and patient cases.
Abstract.
2020
Mirghasempour SA, Huang S, Studholme DJ, Brady CL (2020). A Grain Rot of Rice in Iran Caused by a Xanthomonas Strain Closely Related to X. sacchari.
Plant Dis,
104(6), 1581-1583.
Abstract:
A Grain Rot of Rice in Iran Caused by a Xanthomonas Strain Closely Related to X. sacchari.
Rice grain rot disease was detected for the first time in Mazandaran Province, Iran. The bacteria isolated from infected rice plants showed grains rotted and darkening. A Xanthomonas strain closely connected to X. sacchari was identified using molecular and whole genome sequencing approaches confirmed as the causal agent by fulfilling Koch's postulates.
Abstract.
Author URL.
Lange A, Paris JR, Gharbi K, Cézard T, Miyagawa S, Iguchi T, Studholme DJ, Tyler CR (2020). A newly developed genetic sex marker and its application to understanding chemically induced feminisation in roach (<i>Rutilus rutilus</i>).
Molecular Ecology Resources,
20(4), 1007-1022.
Abstract:
A newly developed genetic sex marker and its application to understanding chemically induced feminisation in roach (Rutilus rutilus)
AbstractOestrogenic wastewater treatment works (WwTW) effluents discharged into UK rivers have been shown to affect sexual development, including inducing intersex, in wild roach (Rutilus rutilus). This can result in a reduced breeding capability with potential population level impacts. In the absence of a sex probe for roach it has not been possible to confirm whether intersex fish in the wild arise from genetic males or females, or whether sex reversal occurs in the wild, as this condition can be induced experimentally in controlled exposures to WwTW effluents and a steroidal oestrogen. Using restriction site‐associated DNA sequencing (RAD‐seq), we identified a candidate for a genetic sex marker and validated this marker as a sex probe through PCR analyses of samples from wild roach populations from nonpolluted rivers. We also applied the sex marker to samples from roach exposed experimentally to oestrogen and oestrogenic effluents to confirm suspected phenotypic sex reversal from males to females in some treatments, and also that sex‐reversed males are able to breed as females. We then show, unequivocally, that intersex in wild roach populations results from feminisation of males, but find no strong evidence for complete sex reversal in wild roach at river sites contaminated with oestrogens. The discovered marker has utility for studies in roach on chemical effects, wild stock assessments, and reducing the number of fish used where only one sex is required for experimentation. Furthermore, we show that the marker can be applied nondestructively using a fin clip or skin swab, with animal welfare benefits.
Abstract.
Aanensen DM, Abudahab K, Adams A, Afifi S, Alam MT, Alderton A, Alikhan N-F, Allan J, Almsaud M, Alrezaihi A, et al (2020). An integrated national scale SARS-CoV-2 genomic surveillance network.
LANCET MICROBE,
1(3), E99-E100.
Author URL.
Bez C, Javvadi SG, Bertani I, Devescovi G, Guarnaccia C, Studholme DJ, Geller AM, Levy A, Venturi V (2020). AzeR, a transcriptional regulator that responds to azelaic acid in Pseudomonas nitroreducens.
Microbiology (Reading),
166(1), 73-84.
Abstract:
AzeR, a transcriptional regulator that responds to azelaic acid in Pseudomonas nitroreducens.
Azelaic acid is a dicarboxylic acid that has recently been shown to play a role in plant-bacteria signalling and also occurs naturally in several cereals. Several bacteria have been reported to be able to utilize azelaic acid as a unique source of carbon and energy, including Pseudomonas nitroreducens. In this study, we utilize P. nitroreducens as a model organism to study bacterial degradation of and response to azelaic acid. We report genetic evidence of azelaic acid degradation and the identification of a transcriptional regulator that responds to azelaic acid in P. nitroreducens DSM 9128. Three mutants possessing transposons in genes of an acyl-CoA ligase, an acyl-CoA dehydrogenase and an isocitrate lyase display a deficient ability in growing in azelaic acid. Studies on transcriptional regulation of these genes resulted in the identification of an IclR family repressor that we designated as AzeR, which specifically responds to azelaic acid. A bioinformatics survey reveals that AzeR is confined to a few proteobacterial genera that are likely to be able to degrade and utilize azelaic acid as the sole source of carbon and energy.
Abstract.
Author URL.
Morcrette H, Kovacs-Simon A, Tennant RK, Love J, Wagley S, Yang ZR, Studholme DJ, Soyer OS, Champion OL, Butler CS, et al (2020). Campylobacter jejuni 11168H Exposed to Penicillin Forms Persister Cells and Cells with Altered Redox Protein Activity.
Frontiers in Cellular and Infection Microbiology,
10Abstract:
Campylobacter jejuni 11168H Exposed to Penicillin Forms Persister Cells and Cells with Altered Redox Protein Activity
The formation of persister cells is one mechanism by which bacteria can survive exposure to environmental stresses. We show that Campylobacter jejuni 11168H forms persister cells at a frequency of 10−3 after exposure to 100 × MIC of penicillin G for 24 h. Staining the cell population with a redox sensitive fluorescent dye revealed that penicillin G treatment resulted in the appearance of a population of cells with increased fluorescence. We present evidence, to show this could be a consequence of increased redox protein activity in, or associated with, the electron transport chain. These data suggest that a population of penicillin G treated C. jejuni cells could undergo a remodeling of the electron transport chain in order to moderate membrane hyperpolarization and intracellular alkalization; thus reducing the antibiotic efficacy and potentially assisting in persister cell formation.
Abstract.
Sidda JD, Song L, Parker JL, Studholme DJ, Sambles C, Grant M (2020). Diversity of secoiridoid glycosides in leaves of UK and Danish ash provide new insight for ash dieback management.
Scientific Reports,
10(1).
Abstract:
Diversity of secoiridoid glycosides in leaves of UK and Danish ash provide new insight for ash dieback management
Secoiridoid glycosides are anti-feeding deterrents of the Oleaceae family recently highlighted as potential biomarkers in Danish ash trees to differentiate between those tolerant and susceptible to the fungal disease ash dieback. With the knowledge that emerald ash borer has recently entered Europe from Russia, and that extensive selection trials are ongoing in Europe for ash dieback tolerant European ash (Fraxinus excelsior), we undertook comprehensive screening of secoiridoid glycosides in leaf extracts of trees tolerant and susceptible to ash dieback sampled from sites in the UK and Denmark. Here we report an unexpected diversity of secoiridoid glycosides in UK trees and higher levels of secoiridoid glycosides in the UK sample group. While it is unlikely that secoiridoid glycosides generally can serve as reliable markers for ash dieback susceptibility, there are differences between tolerant and susceptible groups for specific secoiridoids. We predict that the high levels—and structural diversity—of secoiridoids present in the UK group may provide a robust reservoir of anti-feeding deterrents to mitigate future herbivore threats such as the Emerald ash borer.
Abstract.
Guo Y, Dupont P-Y, Mesarich CH, Yang B, McDougal RL, Panda P, Dijkwel P, Studholme DJ, Sambles C, Win J, et al (2020). Functional analysis of RXLR effectors from the New Zealand kauri dieback pathogen Phytophthora agathidicida.
Mol Plant Pathol,
21(9), 1131-1148.
Abstract:
Functional analysis of RXLR effectors from the New Zealand kauri dieback pathogen Phytophthora agathidicida.
New Zealand kauri is an ancient, iconic, gymnosperm tree species that is under threat from a lethal dieback disease caused by the oomycete Phytophthora agathidicida. To gain insight into this pathogen, we determined whether proteinaceous effectors of P. agathidicida interact with the immune system of a model angiosperm, Nicotiana, as previously shown for Phytophthora pathogens of angiosperms. From the P. agathidicida genome, we defined and analysed a set of RXLR effectors, a class of proteins that typically have important roles in suppressing or activating the plant immune system. RXLRs were screened for their ability to activate or suppress the Nicotiana plant immune system using Agrobacterium tumefaciens transient transformation assays. Nine P. agathidicida RXLRs triggered cell death or suppressed plant immunity in Nicotiana, of which three were expressed in kauri. For the most highly expressed, P. agathidicida (Pa) RXLR24, candidate cognate immune receptors associated with cell death were identified in Nicotiana benthamiana using RNA silencing-based approaches. Our results show that RXLRs of a pathogen of gymnosperms can interact with the immune system of an angiosperm species. This study provides an important foundation for studying the molecular basis of plant-pathogen interactions in gymnosperm forest trees, including kauri.
Abstract.
Author URL.
Sambles C, Venkatesan L, Shittu OM, Harrison J, Moore K, Tripathi L, Grant M, Warmington R, Studholme DJ (2020). Genome sequencing data for wild and cultivated bananas, plantains and abacá.
Data in Brief,
33Abstract:
Genome sequencing data for wild and cultivated bananas, plantains and abacá
We performed shotgun genome sequencing on a total of 19 different Musa genotypes including representatives of wild banana species Musa acuminata and M. balibisiana, allopolyploid bananas and plantains, Fe'i banana, pink banana (also known as hairy banana) and abacá (also known as hemp banana). We aligned sequence reads against a previously sequenced reference genome and assessed ploidy and, in the case of allopolyploids, the contributions of the a and B genomes; this provides important quality-assurance data about the taxonomic identities of the sequenced plant material. These data will be useful for phylogenetics, crop improvement, studies of the complex story of intergenomic recombination in AAB and ABB allotriploid bananas and plantains and can be integrated into resources such as the Banana Genome Hub.
Abstract.
Stulberg MJ, Santillana G, Studholme DJ, Kasiborski B, Ortiz-Castro M, Broders K, Arias S, Block C, Munkvold G, Rascoe J, et al (2020). Genomics-Informed Molecular Detection of Xanthomonas vasicola pv. vasculorum Strains Causing Severe Bacterial Leaf Streak of Corn.
Phytopathology,
110(6), 1174-1179.
Abstract:
Genomics-Informed Molecular Detection of Xanthomonas vasicola pv. vasculorum Strains Causing Severe Bacterial Leaf Streak of Corn.
Xanthomonas vasicola pv. vasculorum (syn. X. campestris pv. vasculorum) was initially identified as the causal agent of bacterial leaf streak of corn in South Africa. The pathovar vasculorum causes disease on sugarcane and corn, but a subset of these strains was noted for its increased disease severity in corn. This subset was reclassified as X. campestris pv. zeae in the early 1990s and was found to have slightly different biochemical and genetic properties than isolates from sugarcane. There has been an emergence of X. campestris pv. zeae-like strains of X. vasicola pv. vasculorum in both the United States and Argentina since 2010. We performed whole genome sequencing on U.S. isolates to confirm their identity. Informed by comparative genomics, we then developed specific TaqMan qPCR and loop-mediated isothermal amplification (LAMP) assays for the detection of this specific subset of X. vasicola pv. vasculorum strains. The qPCR 4909 assay was tested against 27 xanthomonads (diverse representation), 32 DNA extractions from corn leaves confirmed as positive or negative for the bacterium, 41 X. vasicola pv. vasculorum isolates from corn in the United States and Argentina, and 31 additional bacteria associated with corn, sugarcane, or sorghum. In all cases the assay was shown to be specific for the X. vasicola pv. vasculorum isolates that cause more severe disease on corn. We then tested the LAMP 166 assay against the 27 xanthomonads and 32 corn leaf DNA samples, and we found this assay was also specific for this subset of X. vasicola pv. vasculorum isolates. We also developed a live/dead cells distinction protocol using propidium monoazide prior to DNA extraction for analyzing seed washes using these assays. These two detection assays can be useful for both diagnosticians and researchers to specifically identify the X. vasicola pv. vasculorum isolates that cause more severe symptoms on corn.
Abstract.
Author URL.
Christakis CA, Daskalogiannis G, Chatzakis A, Markakis EA, Sagia A, Rizzo GF, Catara V, Lagkouvardos I, Studholme DJ, Sarris PF, et al (2020). Halophytic bacterial endophytome: a potential source of beneficial microbes for a sustainable agriculture.
Abstract:
Halophytic bacterial endophytome: a potential source of beneficial microbes for a sustainable agriculture
AbstractHalophytes have evolved several strategies to survive in saline environments; however, additional support from their associated microbiota could help combat adverse conditions. Endophytic communities of halophytes may be different than those in other plants because salinity acts as an environmental filter. At the same time, they may contribute to the host’s adaptation to adverse environmental conditions and can improve host tolerance against various biotic and abiotic stresses, which may be of importance in modern and sustainable agriculture.In this study the culturable endophytic bacteria of three halophytic species Cakile maritima, Matthiola tricuspidata and Crithmum maritimum were isolated and identified. Endophytic bacteria were isolated from roots and leaves of the sampled plants. Significant differences were observed in bacterial species abundance among different plant species and tissue from which the isolates were obtained. In total, 115 strains were identified by analysis of complete 16S rDNA sequences, while the majority of these isolates were derived from the root samples.The strains were evaluated for their ability to: 1) grow in-vitro in high levels of NaCl; 2) inhibit the growth of the economically important plant pathogenic fungus Verticillium dahliae in vitro and in planta, the human pathogenic fungus Aspergillus fumigatus in vitro, as well as, the economically important plant bacterial pathogens Ralstonia solanacearum and Clavibacter michiganensis in vitro; 3) provide salt tolerance in planta; 4) provide growth promoting effect in planta.Additionally, the genomes of twelve selected isolates, exhibiting interesting features, were sequenced and analysed. Three novel bacterial species were identified that belong to the genus Pseudomonas (two strains) and Arthrobacter (one strain).The outcome of our study is the proof-of-concept that the crop wild relatives (CWR) halophytic microbiome could potentially serve as a source of beneficial microorganisms that could be used (as unique species or as artificial communities) as Bio-Inoculants, for the enhancement of plant growth and stress tolerance in crops, including the high-salinity stress.This is very important in the era of ecosystem degradation and climate change, where the maximizing microbial functions in agroecosystems could be a prerequisite for the future of global sustainable agriculture. Globally, there is a strong need for the identification and bio-banking of novel beneficial endophytic microbes with as many desirable characters, for the development of a new environmentally friendly global strategy in food production that will be based in the sustainable agriculture with low chemical inputs and a low environmental impact.
Abstract.
Kioukis A, Michalopoulou VA, Briers L, Pirintsos S, Studholme DJ, Pavlidis P, Sarris PF (2020). Intraspecific diversification of the crop wild relative Brassica cretica Lam. using demographic model selection.
BMC Genomics,
21(1).
Abstract:
Intraspecific diversification of the crop wild relative Brassica cretica Lam. using demographic model selection.
BACKGROUND: Crop wild relatives (CWRs) contain genetic diversity, representing an invaluable resource for crop improvement. Many of their traits have the potential to help crops to adapt to changing conditions that they experience due to climate change. An impressive global effort for the conservation of various CWR will facilitate their use in crop breeding for food security. The genus Brassica is listed in Annex I of the International Treaty on Plant Genetic Resources for Food and Agriculture. Brassica oleracea (or wild cabbage), a species native to southern and western Europe, has become established as an important human food crop plant because of its large reserves stored over the winter in its leaves. Brassica cretica Lam. (Bc) is a CWR in the brassica group and B. cretica subsp. nivea (Bcn) has been suggested as a separate subspecies. The species Bc has been proposed as a potential gene donor to brassica crops, including broccoli, cabbage, cauliflower, oilseed rape, etc. RESULTS: We sequenced genomes of four Bc individuals, including two Bcn and two Bc. Demographic analysis based on our whole-genome sequence data suggests that populations of Bc are not isolated. Classification of the Bc into distinct subspecies is not supported by the data. Using only the non-coding part of the data (thus, the parts of the genome that has evolved nearly neutrally), we find the gene flow between different Bc population is recent and its genomic diversity is high. CONCLUSIONS: Despite predictions on the disruptive effect of gene flow in adaptation, when selection is not strong enough to prevent the loss of locally adapted alleles, studies show that gene flow can promote adaptation, that local adaptations can be maintained despite high gene flow, and that genetic architecture plays a fundamental role in the origin and maintenance of local adaptation with gene flow. Thus, in the genomic era it is important to link the selected demographic models with the underlying processes of genomic variation because, if this variation is largely selectively neutral, we cannot assume that a diverse population of crop wild relatives will necessarily exhibit the wide-ranging adaptive diversity required for further crop improvement.
Abstract.
Author URL.
Hamilton PB, Lockyer AE, Uren Webster TM, Studholme DJ, Paris JR, Baynes A, Nicol E, Dawson DA, Moore K, Farbos A, et al (2020). Investigation into Adaptation in Genes Associated with Response to Estrogenic Pollution in Populations of Roach (Rutilus rutilus) Living in English Rivers.
Environ Sci Technol,
54(24), 15935-15945.
Abstract:
Investigation into Adaptation in Genes Associated with Response to Estrogenic Pollution in Populations of Roach (Rutilus rutilus) Living in English Rivers.
Exposure of male fish to estrogenic substances from wastewater treatment works (WwTWs) results in feminization and reduced reproductive fitness. Nevertheless, self-sustaining populations of roach (Rutilus rutilus) inhabit river stretches polluted with estrogenic WwTW effluents. In this study, we examine whether such roach populations have evolved adaptations to tolerate estrogenic pollution by comparing frequency differences in single-nucleotide polymorphisms (SNPs) between populations sampled from rivers receiving either high- or low-level WwTW discharges. SNPs within 36 "candidate" genes, selected for their involvement in estrogenic responses, and 120 SNPs in reference genes were genotyped in 465 roaches. There was no evidence for selection in highly estrogen-dependent candidate genes, including those for the estrogen receptors, aromatases, and vitellogenins. The androgen receptor (ar) and cytochrome P450 1A genes were associated with large shifts in allele frequencies between catchments and in individual populations, but there is no clear link to estrogen pollution. Selection at ar in the effluent-dominated River Lee may have resulted from historical contamination with endocrine-disrupting pesticides. Critically, although our results suggest population-specific selection including at genes related to endocrine disruption, there was no strong evidence that the selection resulted from exposure to estrogen pollution.
Abstract.
Author URL.
Kojic M, Jovcic B, Miljkovic M, Novovic K, Begovic J, Studholme DJ (2020). Large-scale chromosome flip-flop reversible inversion mediates phenotypic switching of expression of antibiotic resistance in lactococci.
Microbiol Res,
241Abstract:
Large-scale chromosome flip-flop reversible inversion mediates phenotypic switching of expression of antibiotic resistance in lactococci.
Bacteria can gain resistance to antimicrobials by acquiring and expressing genetic elements that encode resistance determinants such as efflux pumps and drug-modifying enzymes, thus hampering treatment of infection. Previously we showed that acquisition of spectinomycin resistance in a lactococcal strain was correlated with a reversible genomic inversion, but the precise location and the genes affected were unknown. Here we use long-read whole-genome sequencing to precisely define the genomic inversion and we use quantitative PCR to identify associated changes in gene expression levels. The boundaries of the inversion fall within two identical copies of a prophage-like sequence, located on the left and right replichores; this suggests possible mechanisms for inversion through homologous recombination or prophage activity. The inversion is asymmetrical in respect of the axis between the origin and terminus of the replication and modulates the expression of a SAM-dependent methyltransferase, whose heterologous expression confers resistance to spectinomycin in lactococci and that is up-regulated on exposure to spectinomycin. This study provides one of the first examples of phase variation via large-scale chromosomal inversions that confers a switch in antimicrobial resistance in bacteria and the first outside of Staphylococcus aureus.
Abstract.
Author URL.
Nakato GV, Studholme DJ, Blomme G, Grant M, Coutinho TA, Were EM, Wicker E, Mahuku G (2020). SNP‐based genotyping and whole‐genome sequencing reveal previously unknown genetic diversity in <i>Xanthomonas vasicola</i> pv. <i>musacearum</i>, causal agent of banana xanthomonas wilt, in its presumed Ethiopian origin.
Plant Pathology,
70(3), 534-543.
Abstract:
SNP‐based genotyping and whole‐genome sequencing reveal previously unknown genetic diversity in Xanthomonas vasicola pv. musacearum, causal agent of banana xanthomonas wilt, in its presumed Ethiopian origin
AbstractFor decades, Xanthomonas vasicola pv. musacearum (Xvm) has been an economically important bacterial pathogen on enset in Ethiopia. Since 2001, Xvm has also been responsible for significant losses to banana crops in several East and Central African countries, with devastating consequences for smallholder farmers. Understanding the genetic diversity within Xvm populations is essential for the smart design of transnationally reasoned, durable, and effective management practices. Previous studies have revealed limited genetic diversity in Xvm, with East African isolates from banana each falling into one of two closely related clades previously designated as sublineages SL 1 and SL 2, the former of which had also been detected on banana and enset in Ethiopia. Given the presumed origin of Xvm in Ethiopia, we hypothesized that both clades might be found in that country, along with additional genotypes not seen in Central and East African bananas. Genotyping of 97 isolates and whole‐genome sequencing of 15 isolates revealed not only the presence of SL 2 in Ethiopia, but additional diversity beyond SL 1 and SL 2 in four new clades. Moreover, SL 2 was detected in the Democratic Republic of Congo, where previously SL 1 was the only clade reported. These results demonstrate a greater range of genetic diversity among Xvm isolates than previously reported, especially in Ethiopia, and further support the hypothesis that the East/Central Africa xanthomonas wilt epidemic has been caused by a restricted set of genotypes drawn from a highly diverse pathogen pool in Ethiopia.
Abstract.
Filipic B, Novovic K, Studholme DJ, Malesevic M, Mirkovic N, Kojic M, Jovcic B (2020). Shotgun metagenomics reveals differences in antibiotic resistance genes among bacterial communities in Western Balkans glacial lakes sediments.
J Water Health,
18(3), 383-397.
Abstract:
Shotgun metagenomics reveals differences in antibiotic resistance genes among bacterial communities in Western Balkans glacial lakes sediments.
Long-term overuse of antibiotics has driven the propagation and spreading of antibiotic resistance genes (ARGs) such as efflux pumps in the environment, which can be transferred to clinically relevant pathogens. This study explored the abundance and diversity of ARGs and mobile genetic elements within bacterial communities from sediments of three Western Balkans glacial lakes: Plav Lake (high impact of human population), Black Lake (medium impact of human population) and Donje Bare Lake (remote lake, minimal impact of human population) via shotgun metagenomics. Assembled metagenomic sequences revealed that Resistance-Nodulation-Division (RND) efflux pumps genes were most abundant in metagenome from the Plav Lake. The Integron Finder bioinformatics tool detected 38 clusters of attC sites lacking integron-integrases (CALIN) elements: 20 from Plav Lake, four from Black Lake and 14 from Donje Bare Lake. A complete integron sequence was recovered only from the assembled metagenome from Plav Lake. Plasmid contents within the metagenomes were similar, with proportions of contigs being plasmid-related: 1.73% for Plav Lake, 1.59% for Black Lake and 1.64% for Donje Bare Lake. The investigation showed that RNDs and mobile genetic elements content correlated with human population impact.
Abstract.
Author URL.
Thines M, Sharma R, Rodenburg SYA, Gogleva A, Judelson HS, Xia X, van den Hoogen J, Kitner M, Klein J, Neilen M, et al (2020). The Genome of Peronospora belbahrii Reveals High Heterozygosity, a Low Number of Canonical Effectors, and TC-Rich Promoters.
Mol Plant Microbe Interact,
33(5), 742-753.
Abstract:
The Genome of Peronospora belbahrii Reveals High Heterozygosity, a Low Number of Canonical Effectors, and TC-Rich Promoters.
Along with Plasmopara destructor, Peronosopora belbahrii has arguably been the economically most important newly emerging downy mildew pathogen of the past two decades. Originating from Africa, it has started devastating basil production throughout the world, most likely due to the distribution of infested seed material. Here, we present the genome of this pathogen and results from comparisons of its genomic features to other oomycetes. The assembly of the nuclear genome was around 35.4 Mbp in length, with an N50 scaffold length of around 248 kbp and an L50 scaffold count of 46. The circular mitochondrial genome consisted of around 40.1 kbp. From the repeat-masked genome, 9,049 protein-coding genes were predicted, out of which 335 were predicted to have extracellular functions, representing the smallest secretome so far found in peronosporalean oomycetes. About 16% of the genome consists of repetitive sequences, and, based on simple sequence repeat regions, we provide a set of microsatellites that could be used for population genetic studies of P. belbahrii. P. belbahrii has undergone a high degree of convergent evolution with other obligate parasitic pathogen groups, reflecting its obligate biotrophic lifestyle. Features of its secretome, signaling networks, and promoters are presented, and some patterns are hypothesized to reflect the high degree of host specificity in Peronospora species. In addition, we suggest the presence of additional virulence factors apart from classical effector classes that are promising candidates for future functional studies.
Abstract.
Author URL.
Studholme DJ, Wicker E, Abrare SM, Aspin A, Bogdanove A, Broders K, Dubrow Z, Grant M, Jones JB, Karamura G, et al (2020). Transfer of Xanthomonas campestris pv. arecae and X. campestris pv. musacearum to X. vasicola (Vauterin) as X. vasicola pv. arecae comb. nov. and X. vasicola pv. musacearum comb. nov. and Description of X. vasicola pv. vasculorum pv. nov.
Phytopathology,
110(6), 1153-1160.
Abstract:
Transfer of Xanthomonas campestris pv. arecae and X. campestris pv. musacearum to X. vasicola (Vauterin) as X. vasicola pv. arecae comb. nov. and X. vasicola pv. musacearum comb. nov. and Description of X. vasicola pv. vasculorum pv. nov.
We present an amended description of the bacterial species Xanthomonas vasicola to include the causative agent of banana Xanthomonas wilt, as well as strains that cause disease on Areca palm, Tripsacum grass, sugarcane, and maize. Genome-sequence data reveal that these strains all share more than 98% average nucleotide with each other and with the type strain. Our analyses and proposals should help to resolve the taxonomic confusion that surrounds some of these pathogens and help to prevent future use of invalid names.[Formula: see text] Copyright © 2020 the Author(s). This is an open access article distributed under the CC BY 4.0 International license.
Abstract.
Author URL.
Harrison J (2020). Use of Next Generation Sequencing to Investigate the Genomics of Bacterial Pathogens.
Abstract:
Use of Next Generation Sequencing to Investigate the Genomics of Bacterial Pathogens.
This thesis explores the use of next generation sequencing as a tool to investigate the genomics of bacterial pathogens of plants and humans.
Firstly, second-generation sequencing was applied to the evolution of distantly related bacterial species that have converged on common host plants (Xanthomonas bacteria on sugarcane and common-bean plants). This revealed evidence of recent horizontal gene transfer between X. phaseoli pv. phaseoli and X. citri pv. fuscans and between X. axonopodis pv. vasculorum and X. vasicola. distantly related sugarcane pathogens. Furthermore, we discovered that strains isolated from lablab bean (a close relative of common bean) form a previously unknown third distinct clade (and perhaps pathovar) and whole-genome comparisons suggested horizontal gene transfer played an important role in the evolution of host specificity in xanthomonad pathogens.
Next, second-generation sequencing was used to rapidly gain insight into novel emerging bacterial pathogens, namely unusually virulent Asian strains of the human pathogen Campylobacter jejuni and a xanthomonad causing unusual symptoms on common bean in African country of Rwanda. A type six secretion system was shown to be associated with a more serious form of campylobacteriosis and a molecular marker for an intact type six secretion system was identified. This was shown to be more prevalent in strains isolated from Asia than strains isolated in the UK, a finding which has serious implications for chicken import. Further to this the genome sequence of a newly emerging Xanthomonas bean pathogen isolated from a recent outbreak in Rwanda is presented. Analysis of the Rwandan Xanthomonas genome shows it represents the first sequenced isolate in a novel species level clade, which was subsequently named as Xanthomonas cannabis and is genetically distinct from previously known bean pathogens.
Lastly, the performance of the third-generation sequencing platform Oxford Nanopore MinION was assessed which will prove to be an exciting resource to perform bacterial genomic studies in the future. In summary, this work exemplifies the value of sequencing-based approaches for rapidly and cheaply gaining insights into evolution of bacterial pathogens
Abstract.
2019
Glover R (2019). Biomonitoring and surveillance with short- and long-read metabarcoding.
Abstract:
Biomonitoring and surveillance with short- and long-read metabarcoding
The aim of this project was to develop applied metabarcoding methods to aid both the monitoring of environmental water quality and surveillance of airborne fungal phytopathogens. An Illumina short-read metabarcoding method was developed which is now in active use by the UK Environment Agency to determine the abundance of diatom species, feeding into the classification of water bodies for the EU Water Framework Directive. Further work was undertaken to future proof this method by comparing the diatom assemblages of three English rivers as determined by light microscopy, the developed Illumina short-read metabarcoding method and long-read nanopore metabarcoding. The river and method comparison study showed that the light microscopy was the outlier and potentially largest source of error as the two separate metabarcoding methods performed very similarly. A study was undertaken to compare the airborne fungal communities in six locations in eastern England over a one month period in 2015 to assess metabarcoding as a potential surveillance tool for the introduction of phytopathogens and the utility of the current UK pollen network for sample acquisition. This study highlighted issues with contamination at stages within the metabarcoding laboratory preparation protocols which made the bioinformatics analysis problematic; however, recommendations are made for procedures to reduce contamination in metabarcoding studies. The final study characterised a week's data from the eastern England fungal spore samples with a novel long PCR amplifying the entire ribosomal tandem repeat - including the intergenic spacer - to investigate the regions full utility in the light of nanopore sequencing.
Abstract.
Miljkovic M, Jovanovic S, O'Connor PM, Mirkovic N, Jovcic B, Filipic B, Dinic M, Studholme DJ, Fira D, Cotter PD, et al (2019). Brevibacillus laterosporus strains BGSP7, BGSP9 and BGSP11 isolated from silage produce broad spectrum multi-antimicrobials.
PLoS One,
14(5).
Abstract:
Brevibacillus laterosporus strains BGSP7, BGSP9 and BGSP11 isolated from silage produce broad spectrum multi-antimicrobials.
Bacteria active against multi-drug resistant pathogens, isolated by direct selection of colonies from clover silage samples, produce zones of inhibition against two Gram-negative (Klebsiella pneumoniae Ni9 and Pseudomonas aeruginosa MMA83) and two Gram-positive (Staphylococcus aureus ATCC25923 and Listeria monocytogenes ATCC19111) pathogens. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 produced the largest zones of inhibition against all four pathogens when grown in LB broth with aeration at 37°C. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 were identified as Brevibacillus laterosporus and pulsed field gel electrophoresis and extracellular protein profiles showed that three different strains (BGSP7, BGSP9 and BGSP11) were isolated. A semi-native SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) gel overlay assay showed that BGSP7 and BGSP9 produce small antimicrobial molecules of about 1.5 kDa, while BGSP11 produces antimicrobial molecules of 1.5 and 6 kDa active against S. aureus ATCC25923. Amino acid analysis of two antimicrobial molecules (1583.73 Da; from BGSP7 and 1556.31 Da; from BGSP11) revealed that they have a similar composition and differ only by virtue of the presence of a methionine which is present only in BGSP11 molecule. Genome sequencing of the three isolates revealed the presence of gene clusters associated with the production of non-ribosomally synthesized peptides (brevibacillin, bogorol, gramicidin S, plipastatin and tyrocin) and bacteriocins (laterosporulin, a lactococcin 972-like bacteriocin, as well as putative linocin M18, sactipeptide, UviB and lantipeptide-like molecules). Ultimately, the purification of a number of antimicrobial molecules from each isolate suggests that they can be considered as potent biocontrol strains that produce an arsenal of antimicrobial molecules active against Gram-positive and Gram-negative multi-resistant pathogens, fungi and insects.
Abstract.
Author URL.
Kioukis A, Michalopoulou VA, Briers L, Pirintsos S, Studholme DJ, Pavlidis P, Sarris PF (2019). Draft Genome Sequence and intraspecific diversification of the wild crop relative<i>Brassica cretica</i>Lam. using demographic model selection.
Abstract:
Draft Genome Sequence and intraspecific diversification of the wild crop relativeBrassica creticaLam. using demographic model selection
AbstractCrop wild relatives contain great levels of genetic diversity, representing an invaluable resource for crop improvement. Many of their traits have the potential to help crops become more resistant and resilient, and adapt to the new conditions that they will experience due to climate change. An impressive global effort occurs for the conservation of various wild crop relatives and facilitates their use in crop breeding for food security.The genusBrassicais listed in Annex I of the International Treaty on Plant Genetic Resources for Food and Agriculture.Brassica oleracea(or wild cabbage) is a species native to coastal southern and western Europe that has become established as an important human food crop plant because of its large reserves stored over the winter in its leaves.Brassica creticaLam. is a wild relative crop in the brassica group andB. creticasubsp.niveahas been suggested as a separate subspecies. The speciesB. creticahas been proposed as a potential gene donor to a number of crops in the brassica group, including broccoli, Brussels sprout, cabbage, cauliflower, kale, swede, turnip and oilseed rape.Here, we present the draftde novogenome assemblies of fourB. creticaindividuals, including twoB. creticasubsp.niveaand twoB. cretica.De novoassembly of Illumina MiSeq genomic shotgun sequencing data yielded 243,461 contigs totalling 412.5 Mb in length, corresponding to 122 % of the estimated genome size ofB. cretica(339 Mb). According to synteny mapping and phylogenetic analysis of conserved genes,B. creticagenome based on our sequence data reveals approximately 30.360 proteins.Furthermore, our demographic analysis based on whole genome data, suggests that distinct populations ofB. creticaare not isolated. Our findings suggest that the classification of theB. creticain distinct subspecies is not supported from the genome sequence data we analyzed.
Abstract.
Ward G (2019). Drop in the Ocean: Molecular Approaches for Exploring the Diversity and Distribution of the Ascetosporea.
Abstract:
Drop in the Ocean: Molecular Approaches for Exploring the Diversity and Distribution of the Ascetosporea
The protistan class Ascetosporea (Rhizaria, Endomyxa) comprises five orders of parasites of aquatic invertebrates: Haplosporida, Paramyxida, Mikrocytida, Paradinida and Claustrosporida. The group includes a number of species known for their devastating effects as pathogens of economically significant bivalve species, the most notorious of which are the oyster pathogens Marteilia refringens (Paramyxida), Bonamia ostreae (Haplosporida) and Mikrocytos mackini (Mikrocytida). Due to its significance to aquaculture, interest in the group is growing, and a number of recent environmental DNA (eDNA)-based studies indicate vast, uncharacterised diversity within the class.
Traditionally species discovery and description has relied on microscopy studies of infected invertebrate tissue, and as such our understanding of the group is biased towards pathogens of commercially exploited or easily studied invertebrates, with comparatively little known about the diversity of Ascetosporea outside such hosts. Many species, particularly those infecting less “important” hosts, have not been molecularly characterised, hindering phylogenetic studies and limiting our understanding of the relationship between morphology and phylogeny.
This study uses primer-based screens of a wide range of environmental and invertebrate-associated samples to explore the diversity, distribution and host range of the four best-known ascetosporean orders, and in doing so links sequence data to described species, and allows the molecular and morphological characterisation of novel haplosporidian species in bivalve hosts. PCR screens also uncovered a large radiation of novel sequence types within Paradinida, in littoral and coastal environmental samples, and demonstrated the abundance and diversity of haplosporidians in freshwater and terrestrial sample types.
This study also focuses on the development and application of sensitive complementary molecular and microscopy-based methods for the detection of parasite life-stages in host tissues, and consideration given to the role of molecular methods in facilitating rapid, accurate diagnosis of pathogens in important hosts.
Abstract.
Muzemil S, Chala A, Tesfaye B, Studholme DJ, Grant M, Yemataw Z, Olango TM (2019). Evaluation of 20 enset (<i>Ensete ventricosum</i>) landraces for response to <i>Xanthomonas vasicola</i> pv. <i>musacearum</i> infection.
Abstract:
Evaluation of 20 enset (Ensete ventricosum) landraces for response to Xanthomonas vasicola pv. musacearum infection
AbstractBacterial wilt, caused by Xanthomonas vasicola pv. musacearum (Xvm), formerly X. campestris pv. musacearum, is the most threatening and economically important disease of enset (Ensete ventricosum), the multipurpose food security crop orphan to south and southwestern Ethiopia. Xvm has also had a major impact on banana and plantain production in East Africa following its detection in Uganda in 2001 and subsequent spread. Effective control of this disease currently relies on integrated disease management (IDM) strategies including minimization of field pathogen inoculum and deployment of wilt resistant enset landraces. Identifying landraces with stable and durable Xvm resistance will greatly accelerate breeding of varieties that can be included as a component of IDM. In this study, 20 enset landraces previously reported to exhibit lower susceptibility to Xvm were grown in pots under open field conditions and inoculated with an aggressive Xvm inoculum isolated from a disease hotspot area. Longitudinal and survival analyses were applied to each landrace, based on disease units representing a combination of area-under-disease progress stairs, disease index and apparent infection rate. Considerable variation was observed among the 20 landraces; however, none exhibited full immunity to Xvm infection. Three landraces, viz. Hae’la, Mazia and Lemat (HML), showed lowest susceptibility to Xvm as evidenced by lower disease units and higher survival rates. Landraces Kuro, Gezewet, Bededet, and Alagena showed similar levels of Xvm infection as did HML, but with lower survival rates. By contrast, landrace Arkia showed the highest infection level and lowest survival rate, suggesting a high degree of susceptibility to Xvm. This study identifies new material that can be used in future breeding programmes to develop Xvm-resistant enset varieties.
Abstract.
Wagley S, Scott AE, Ireland PM, Prior JL, Atkins TP, Bancroft GJ, Studholme DJ, Titball RW (2019). Genome Resequencing of Laboratory Stocks of Burkholderia pseudomallei K96243.
Microbiol Resour Announc,
8(9).
Abstract:
Genome Resequencing of Laboratory Stocks of Burkholderia pseudomallei K96243.
We have resequenced the genomes of four Burkholderia pseudomallei K96243 laboratory cultures and compared them to the reported genome sequence that was published in 2004. Compared with the reference genome, these laboratory cultures harbored up to 42 single-nucleotide variants and up to 11 indels, including a 31.7-kb deletion in one culture.
Abstract.
Author URL.
Studholme DJ, Panda P, Sanfuentes Von Stowasser E, González M, Hill R, Sambles C, Grant M, Williams NM, McDougal RL (2019). Genome sequencing of oomycete isolates from Chile supports the New Zealand origin of Phytophthora kernoviae and makes available the first Nothophytophthora sp. genome.
Mol Plant Pathol,
20(3), 423-431.
Abstract:
Genome sequencing of oomycete isolates from Chile supports the New Zealand origin of Phytophthora kernoviae and makes available the first Nothophytophthora sp. genome.
Genome sequences were generated for six oomycete isolates collected from forests in Valdivia, Chile. Three of the isolates were identified morphologically as Phytophthora kernoviae, whereas two were similar to other clade 10 Phytophthora species. One isolate was tentatively identified as Nothophytophthora valdiviana based on nucleotide sequence similarity in the cytochrome oxidase 1 gene. This is the first genome sequence for this recently described genus. The genome assembly was more fragmented and contained many duplicated genes when compared with the other Phytophthora sequences. Comparative analyses were performed with genomic sequences of the P. kernoviae isolates from the UK and New Zealand. Although the potential New Zealand origin of P. kernoviae has been suggested, new isolations from Chile had cast doubt on this hypothesis. We present evidence supporting P. kernoviae as having originated in New Zealand. However, investigation of the diversity of oomycete species in Chile has been limited and warrants further exploration. We demonstrate the expediency of genomic analyses in determining phylogenetic relationships between isolates within new and often scantly represented taxonomic groups, such as Phytophthora clade 10 and Nothophytophthora. Data are available on GenBank via BioProject accession number PRJNA352331.
Abstract.
Author URL.
Warmington RJ, Kay W, Jeffries A, O'Neill P, Farbos A, Moore K, Bebber DP, Studholme DJ (2019). High-Quality Draft Genome Sequence of the Causal Agent of the Current Panama Disease Epidemic.
Microbiol Resour Announc,
8(36).
Abstract:
High-Quality Draft Genome Sequence of the Causal Agent of the Current Panama Disease Epidemic.
We present a high-quality draft genome assembly for Fusarium oxysporum f. sp. cubense tropical race 4 (Fusarium odoratissimum), assembled from PacBio reads and consisting of 15 contigs with a total assembly size of 48.59 Mb. This strain appears to belong to vegetative compatibility group complex 01213/16.
Abstract.
Author URL.
Minardi D, Studholme DJ, Oidtmann B, Pretto T, van der Giezen M (2019). Improved method for genotyping the causative agent of crayfish plague (Aphanomyces astaci) based on mitochondrial DNA.
Parasitology,
146(8), 1022-1029.
Abstract:
Improved method for genotyping the causative agent of crayfish plague (Aphanomyces astaci) based on mitochondrial DNA.
Aphanomyces astaci causes crayfish plague, which is a devastating disease of European freshwater crayfish. The likely first introduction of A. astaci into Europe was in the mid-19th century in Italy, presumably with the introduction of North American crayfish. These crayfish can carry A. astaci in their cuticle as a benign infection. Aphanomyces astaci rapidly spread across Europe causing the decline of the highly susceptible indigenous crayfish species. Random amplified polymorphic DNA-PCR analysis of A. astaci pure cultures characterized five genotype groups (A, B, C, D and E). Current A. astaci genotyping techniques (microsatellites and genotype-specific regions, both targeting nuclear DNA) can be applied directly to DNA extracted from infected cuticles but require high infection levels. Therefore, they are not suitable for genotyping benign infections in North American crayfish (carriers). In the present study, we combine bioinformatics and molecular biology techniques to develop A. astaci genotyping molecular markers that target the mitochondrial DNA, increasing the sensitivity of the genotyping tools. The assays were validated on DNA extracts of A. astaci pure cultures, crayfish tissue extractions from crayfish plague outbreaks and tissue extractions from North American carriers. We demonstrate the presence of A. astaci genotype groups a and B in UK waters.
Abstract.
Author URL.
Chaudhari Y, Cairns TC, Sidhu Y, Attah V, Thomas G, Csukai M, Talbot NJ, Studholme DJ, Haynes K (2019). The <i>Zymoseptoria tritici</i> ORFeome: a functional genomics community resource.
Abstract:
The Zymoseptoria tritici ORFeome: a functional genomics community resource
AbstractLibraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for Zymoseptoria tritici, which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, cloned into the Gateway® Entry Vector pDONR207, and sequenced, providing a total of 3022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors (n = 410) and secondary metabolite biosynthetic proteins (n = 171), in addition to genes residing at dispensable chromosomes (n= 122), or those that are preferentially expressed during plant infection (n = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway® Destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The Z. tritici ORFeome constitutes a powerful resource for functional genomics, and offers unparalleled opportunities to understand the biology of Z. tritici.
Abstract.
Chaudhari Y, Cairns TC, Sidhu Y, Attah V, Thomas G, Csukai M, Talbot NJ, Studholme DJ, Haynes K (2019). The Zymoseptoria tritici ORFeome: a Functional Genomics Community Resource.
Mol Plant Microbe Interact,
32(12), 1564-1570.
Abstract:
The Zymoseptoria tritici ORFeome: a Functional Genomics Community Resource.
Libraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for Zymoseptoria tritici, which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4,075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, were cloned into the Gateway entry vector pDONR207, and were sequenced, providing a total of 3,022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors (n = 410) and secondary metabolite biosynthetic proteins (n = 171) in addition to genes residing at dispensable chromosomes (n = 122) or those that are preferentially expressed during plant infection (n = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The Z. tritici ORFeome constitutes a powerful resource for functional genomics and offers unparalleled opportunities to understand the biology of Z. tritici.[Formula: see text] Copyright © 2019 the Author(s). This is an open access article distributed under the CC BY 4.0 International license.
Abstract.
Author URL.
Messer A (2019). The evaluation of metagenomic analysis software, using in-silico and in-vitro mock community datasets, for the accurate study of bio-aerosol samples.
Abstract:
The evaluation of metagenomic analysis software, using in-silico and in-vitro mock community datasets, for the accurate study of bio-aerosol samples.
The bio-aerosol is an important medium for the potential dispersal of biological warfare agents within the battlefield space. In order to better protect the military personnel who work within this environment it is imperative that we increase our understanding of this matrix, especially the naturally occurring variation and its causes. Understanding the naturally occurring variation within the bio-aerosol will enable future and current biological detection platforms to be put through better test and evaluation processes, thus reducing the potential for false alarms and false negatives. Analysing bio-aerosol samples collected across a temporal gradient through a metagenomics approach will enable the natural variation to be better understood. However, metagenomic analysis tools have been shown to have contradictory reviews within the literature, it is therefore essential to identify the most suitable analysis approach.
Here I developed a metagenomic analysis pipeline which delivers high confidence taxonomic identification to species level, as well as accurate measures of diversity and homogeneity. The analysis pipeline that was developed takes the output from multiple tools thus reducing the number of false positives, delivering high confidence taxonomic identification. The analysis pipeline also gives a more accurate measure of diversity and homogeneity compared to any of the tools being used individually. This improved accuracy will deliver superior results when measuring the change in abundance of species identified within the bio-aerosol in sampling regimes carried out at Dstl. These improvements will lead to more accurate test bio-aerosols being developed for biological detection platform evaluation. Fundamentally this will improve the UK military’s capability to detect biological warfare releases within the battlespace.
Abstract.
Thines M, Sharma R, Rodenburg SYA, Gogleva A, Judelson HS, Xia X, van den Hoogen J, Kitner M, Klein J, Neilen M, et al (2019). The genome of Peronospora belbahrii reveals high heterozygosity, a low number of canonical effectors and CT-rich promoters.
2018
Nakato GV, Wicker E, Coutinho TA, Mahuku G, Studholme DJ (2018). A highly specific tool for identification of Xanthomonas vasicola pv. musacearum based on five Xvm-specific coding sequences.
Heliyon,
4(12).
Abstract:
A highly specific tool for identification of Xanthomonas vasicola pv. musacearum based on five Xvm-specific coding sequences
Xanthomonas vasicola pv. musacearum (Xvm) is a bacterial pathogen responsible for the economically important Xanthomonas wilt disease on banana and enset crops in Sub-Saharan Africa. Given that the symptoms are similar to those of other diseases, molecular diagnosis is essential to unambiguously identify this pathogen and distinguish it from closely related strains not pathogenic on these hosts. Currently, Xvm identification is based on polymerase chain reaction (PCR) with GspDm primers, targeting the gene encoding general secretory protein D. Experimental results and examination of genomic sequences revealed poor specificity of the GspDm PCR. Here, we present and validate five new Xvm-specific primers amplifying only Xvm strains.
Abstract.
Woods-Tör A, Studholme DJ, Cevik V, Telli O, Holub EB, Tör M (2018). A suppressor/avirulence gene combination in hyaloperonospora arabidopsidis determines race specificity in Arabidopsis thaliana.
Frontiers in Plant Science,
9Abstract:
A suppressor/avirulence gene combination in hyaloperonospora arabidopsidis determines race specificity in Arabidopsis thaliana
The pathosystem of Arabidopsis thaliana and diploid biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa) has been a model for investigating the molecular basis of Flor’s gene-for-gene hypothesis. The isolates Hpa-Noks1 and Hpa-Cala2 are virulent on Arabidopsis accession RMX-A02 whilst an F1 generated from a cross between these two isolates was avirulent. The F2 progeny segregated 3,1 (avirulent, virulent), indicating a single major effect AVR locus in this pathogen. SNP-based linkage mapping confirmed a single AVR locus within a 14kb map interval containing two genes encoding putative effectors. The Hpa-Cala2 allele of one gene, designated H. arabidopsidis cryptic1 (HAC1), encodes a protein with a signal peptide and an RxLR/dEER motif, and triggers a defense response in RMX-A02. The second gene is heterozygous in Hpa-Cala2. One allele, designated Suppressor of HAC1Cala2 (S-HAC1Cala2) encodes a protein with a signal peptide and a dKEE motif with no RxLR motif; the other allele (s-hac1Cala2) encodes a protein with a signal peptide, a dEEE motif and is divergent in sequence from the S-HAC1Cala2 allele. In selfed progeny from Hpa-Cala2, dominant S-HAC1Cala2 allele carrying progeny correlates with virulence in RMX-A02, whereas homozygous recessive s-hac1Cala2 carrying progeny were avirulent. Genetic investigations suggested other heterozygous suppressor loci might exist in the Hpa-Cala2 genome.
Abstract.
Dorati F, Barrett GA, Sanchez-Contreras M, Arseneault T, José MS, Studholme DJ, Murillo J, Caballero P, Waterfield NR, Arnold DL, et al (2018). Coping with Environmental Eukaryotes; Identification of Pseudomonas syringae Genes during the Interaction with Alternative Hosts or Predators.
Microorganisms,
6(2).
Abstract:
Coping with Environmental Eukaryotes; Identification of Pseudomonas syringae Genes during the Interaction with Alternative Hosts or Predators.
Understanding the molecular mechanisms underpinning the ecological success of plant pathogens is critical to develop strategies for controlling diseases and protecting crops. Recent observations have shown that plant pathogenic bacteria, particularly Pseudomonas, exist in a range of natural environments away from their natural plant host e.g. water courses, soil, non-host plants. This exposes them to a variety of eukaryotic predators such as nematodes, insects and amoebae present in the environment. Nematodes and amoeba in particular are bacterial predators while insect herbivores may act as indirect predators, ingesting bacteria on plant tissue. We therefore postulated that bacteria are probably under selective pressure to avoid or survive predation and have therefore developed appropriate coping mechanisms. We tested the hypothesis that plant pathogenic Pseudomonas syringae are able to cope with predation pressure and found that three pathovars show weak, but significant resistance or toxicity. To identify the gene systems that contribute to resistance or toxicity we applied a heterologous screening technique, called Rapid Virulence Annotation (RVA), for anti-predation and toxicity mechanisms. Three cosmid libraries for P. syringae pv. aesculi, pv. tomato and pv. phaseolicola, of approximately 2000 cosmids each, were screened in the susceptible/non-toxic bacterium Escherichia coli against nematode, amoebae and an insect. A number of potential conserved and unique genes were identified which included genes encoding haemolysins, biofilm formation, motility and adhesion. These data provide the first multi-pathovar comparative insight to how plant pathogens cope with different predation pressures and infection of an insect gut and provide a foundation for further study into the function of selected genes and their role in ecological success.
Abstract.
Author URL.
Michalopoulou VA, Vicente JG, Studholme DJ, Sarris PF (2018). Draft genome sequences of pathotype strains for three pathovars belonging to three xanthomonas species.
Microbiology Resource Announcements,
7(12).
Abstract:
Draft genome sequences of pathotype strains for three pathovars belonging to three xanthomonas species
We present here the draft genome sequences of type/pathotype strains for three Xanthomonas species and pathovars with different host specificities, the Hedera helix L. pathogen Xanthomonas hortorum pv. hederae WHRI 7744 (NCPPB 939T), the rice pathogen X. oryzae pv. oryzicola WHRI 5234 (NCPPB 1585), and the cotton pathogen X. citri subsp. malvacearum WHRI 5232 (NCPPB 633).
Abstract.
Schaum E, Buckling A, Studholme D, Smirnoff N, Yvon-Durocher G (2018). Environmental fluctuations accelerate molecular evolution of thermal tolerance in a marine diatom. Nature Communications
Yemataw Z, Muzemil S, Ambachew D, Tripathi L, Tesfaye K, Chala A, Farbos A, O'Neill P, Moore K, Grant M, et al (2018). Genome sequence data from 17 accessions of Ensete ventricosum, a staple food crop for millions in Ethiopia.
Data in Brief,
18, 285-293.
Abstract:
Genome sequence data from 17 accessions of Ensete ventricosum, a staple food crop for millions in Ethiopia
We present raw sequence reads and genome assemblies derived from 17 accessions of the Ethiopian orphan crop plant enset (Ensete ventricosum (Welw.) Cheesman) using the Illumina HiSeq and MiSeq platforms. Also presented is a catalogue of single-nucleotide polymorphisms inferred from the sequence data at an average density of approximately one per kilobase of genomic DNA.
Abstract.
Morcrette H, Morgan MS, Farbos A, O'Neill P, Moore K, Titball RW, Studholme DJ (2018). Genome sequence of Staphylococcus aureus Ex1, isolated from a patient with spinal osteomyelitis.
Genome Announcements,
6(26).
Abstract:
Genome sequence of Staphylococcus aureus Ex1, isolated from a patient with spinal osteomyelitis
Here, we present the genome sequence of Staphylococcus aureus Ex1, isolated in 2015 from a patient with spinal osteomyelitis at the Royal Devon and Exeter Hospital in the United Kingdom. The availability of the Ex1 genome sequence provides a resource for studying the basis for spinal infection and horizontal gene transfer in S. aureus.
Abstract.
Devran Z, Kahveci E, Hong Y, Studholme DJ, Tör M (2018). Identifying molecular markers suitable for Frl selection in tomato breeding.
Theor Appl Genet,
131(10), 2099-2105.
Abstract:
Identifying molecular markers suitable for Frl selection in tomato breeding.
Modern plant breeding heavily relies on the use of molecular markers. In recent years, next generation sequencing (NGS) emerged as a powerful technology to discover DNA sequence polymorphisms and generate molecular markers very rapidly and cost effectively, accelerating the plant breeding programmes. A single dominant locus, Frl, in tomato provides resistance to the fungal pathogen Fusarium oxysporum f. sp. radicis-lycopersici (FORL), causative agent of Fusarium crown and root rot. In this study, we describe the generation of molecular markers associated with the Frl locus. An F2 mapping population between an FORL resistant and a susceptible cultivar was generated. NGS technology was then used to sequence the genomes of a susceptible and a resistant parent as well the genomes of bulked resistant and susceptible F2 lines. We zoomed into the Frl locus and mapped the locus to a 900 kb interval on chromosome 9. Polymorphic single-nucleotide polymorphisms (SNPs) within the interval were identified and markers co-segregating with the resistant phenotype were generated. Some of these markers were tested successfully with commercial tomato varieties indicating that they can be used for marker-assisted selection in large-scale breeding programmes.
Abstract.
Author URL.
Yemataw Z, Tesfaye K, Grant M, Studholme DJ, Chala A (2018). Multivariate analysis of morphological variation in enset (Ensete ventricosum (Welw.) Cheesman) reveals regional and clinal variation in germplasm from south and south western Ethiopia.
Australian Journal of Crop Science,
12(12), 1849-1858.
Abstract:
Multivariate analysis of morphological variation in enset (Ensete ventricosum (Welw.) Cheesman) reveals regional and clinal variation in germplasm from south and south western Ethiopia
Enset (Ensete ventricosum (Welw.) Cheesman) is cultivated by millions of people across Ethiopia in diverse agro-ecological and cultural settings, selecting for various agronomic traits. However, as for other underutilized crops, our understanding of the diversity and utilization of enset remains limited. This work sought to redress this limitation by estimating morphological diversity among enset accessions collected from major enset growing regions, including across altitudinal gradients. In total, landraces comprising 387 accessions originating from nine regions of Ethiopia were characterized using multivariate analysis of 15 quantitative traits. Cluster analysis grouped accessions in to five distinct classes with maximum number of accessions 338 in cluster (I) and minimum 1 in cluster (V). The clustering of accessions did not show grouping on the basis of region of origin. The first four principal components accounted for ~74% of the total variance. Linear discriminant analysis indicated that around 40.8% (160 accessions) and 45.2% (175 accession) of the studied accessions were correctly classified to their respective regions of origin altitude groups, respectively. The breadth of phenotypic differences in these 15 traits suggests significant degrees of genetic variation. These traits will be exploited to identify potential donors for future enset improvement efforts.
Abstract.
Minardi D, Studholme DJ, van der Giezen M, Pretto T, Oidtmann B (2018). New genotyping method for the causative agent of crayfish plague (Aphanomyces astaci) based on whole genome data.
Journal of Invertebrate Pathology,
156, 6-13.
Abstract:
New genotyping method for the causative agent of crayfish plague (Aphanomyces astaci) based on whole genome data
The oomycete Aphanomyces astaci causes crayfish plague, the most important disease of European freshwater crayfish species. Presumably introduced into Europe 150 years ago with the import of North American crayfish, A. astaci is highly pathogenic to European crayfish species. Five genotypes (A, B, C, D, and E) have been defined based on random amplified polymorphic DNA analysis (RAPD-PCR) from A. astaci pure cultures. The distinction of genotypes is an essential tool to conduct molecular epidemiological studies on crayfish plague and it has been used to clarify and better understand the history and spread of this disease in Europe. Whereas RAPD-PCR requires DNA from pure culture isolates, the development of genotyping tools that can be applied to DNA extracted from clinical samples allows a much wider application of genotyping studies, including revisiting historic samples. In this study, we present a new approach that adds to currently available methods for genotyping A. astaci strains directly from clinical crayfish samples. Whole-genome sequencing of A. astaci strains representing all currently known genotypes was employed, genomic regions unique to the respective genotype identified, and a PCR-based genotyping assay designed, which focuses on the presence/absence of PCR product after amplification with the genotype-specific primers. Our diagnostic methodology was tested using DNA extracts from pure A. astaci cultures, other Aphanomyces species and additional oomycetes, samples from a recent Italian crayfish plague outbreak and additional historical samples available in the Centre for Environment, Fisheries and Aquaculture Science laboratory. The new markers were reliable for pure culture and clinical samples from a recent outbreak and successfully discriminated genotype A, B, D, and E. The marker for genotype C required an additional sequencing step of the generated PCR product to confirm genotype.
Abstract.
2017
Sambles CM, Salmon DL, Florance H, Howard TP, Smirnoff N, Nielsen LR, McKinney LV, Kjær ED, Buggs RJA, Studholme DJ, et al (2017). Ash leaf metabolomes reveal differences between trees tolerant and susceptible to ash dieback disease.
Sci Data,
4Abstract:
Ash leaf metabolomes reveal differences between trees tolerant and susceptible to ash dieback disease.
European common ash, Fraxinus excelsior, is currently threatened by Ash dieback (ADB) caused by the fungus, Hymenoscyphus fraxineus. To detect and identify metabolites that may be products of pathways important in contributing to resistance against H. fraxineus, we performed untargeted metabolomic profiling on leaves from five high-susceptibility and five low-susceptibility F. excelsior individuals identified during Danish field trials. We describe in this study, two datasets. The first is untargeted LC-MS metabolomics raw data from ash leaves with high-susceptibility and low-susceptibility to ADB in positive and negative mode. These data allow the application of peak picking, alignment, gap-filling and retention-time correlation analyses to be performed in alternative ways. The second, a processed dataset containing abundances of aligned features across all samples enables further mining of the data. Here we illustrate the utility of this dataset which has previously been used to identify putative iridoid glycosides, well known anti-herbivory terpenoid derivatives, and show differential abundance in tolerant and susceptible ash samples.
Abstract.
Author URL.
Schaum C-E, Buckling A, Smirnoff N, Studholme DJ, Yvon-Durocher G (2017). Environmental fluctuations accelerate molecular evolution of thermal tolerance in a marine diatom.
Abstract:
Environmental fluctuations accelerate molecular evolution of thermal tolerance in a marine diatom
Diatoms contribute roughly 20% of global primary production, but the factors determining their ability to adapt to global warming are unknown. Here we quantify the capacity for adaptation to warming in the marine diatom Thalassiosira pseudonana. We found that evolutionary rescue under severe warming was slow but adaptation to more realistic scenarios, where temperature increases were moderate or where they fluctuated between benign and severe conditions, was rapid. Adaption to warming was linked to major phenotypic changes in metabolism and elemental composition. Whole genome re-sequencing identified significant genetic divergence both among populations adapted to the different warming regimes and between the evolved and ancestral lineages. Consistent with the phenotypic changes, the most rapidly evolving genes were associated with transcriptional regulation, cellular response to oxidative stress and redox homeostasis. These results demonstrate that evolution of thermal tolerance in marine diatoms can be rapid, particularly in fluctuating environments, and is underpinned by major genomic and phenotypic divergence.
Abstract.
Yemataw Z, Mekonen A, Chala A, Tesfaye K, Mekonen K, Studholme DJ, Sharma K (2017). Farmers' knowledge and perception of enset Xanthomonas wilt in southern Ethiopia.
Agriculture and Food Security,
6(1).
Abstract:
Farmers' knowledge and perception of enset Xanthomonas wilt in southern Ethiopia
Background: Enset Xanthomonas wilt (EXW) was first reported in 1939 and continues to threaten the sustainability of farming systems in south and southwestern parts of Ethiopia. The present study was conducted in the central zones of southern Ethiopia to assess farmers' knowledge and perception about EXW, its etiology and mode of transmission, and its implications for the management of EXW. Methods: a survey was conducted in 240 households across Hadiya, Kembata-Tembaro and Wolaita zones of southern Ethiopia using focus group discussions and a structured questionnaire to assess farmers' perceptions of causes and modes of EXW transmission, and their knowledge on symptom identification. In addition, EXW prevalence, incidence and severity were determined for each zone. Data were analyzed through descriptive statistics. Results: the results showed that a significant number of farmers are aware of EXW, its symptoms, etiology and transmission and spread, but they are not able to readily relate modes of spread to control methods. Since 2002, EXW became prominent in Hadiya, with the highest EXW incidence and severity, followed by Wolaita, and Kembata-Tembaro. Farmers identified EXW as the major cause for declining production and productivity of enset in the region. Conclusion: EXW has spread widely and rapidly in southern Ethiopia, with significant socioeconomic impacts in smallholders' livelihoods. There is a need for developing knowledge-based strategies and awareness-raising campaign for EXW management.
Abstract.
Sollars ESA, Harper AL, Kelly LJ, Sambles CM, Ramirez-Gonzalez RH, Swarbreck D, Kaithakottil G, Cooper ED, Uauy C, Havlickova L, et al (2017). Genome sequence and genetic diversity of European ash trees.
Nature,
541(7636), 212-216.
Abstract:
Genome sequence and genetic diversity of European ash trees.
Ash trees (genus Fraxinus, family Oleaceae) are widespread throughout the Northern Hemisphere, but are being devastated in Europe by the fungus Hymenoscyphus fraxineus, causing ash dieback, and in North America by the herbivorous beetle Agrilus planipennis. Here we sequence the genome of a low-heterozygosity Fraxinus excelsior tree from Gloucestershire, UK, annotating 38,852 protein-coding genes of which 25% appear ash specific when compared with the genomes of ten other plant species. Analyses of paralogous genes suggest a whole-genome duplication shared with olive (Olea europaea, Oleaceae). We also re-sequence 37 F. excelsior trees from Europe, finding evidence for apparent long-term decline in effective population size. Using our reference sequence, we re-analyse association transcriptomic data, yielding improved markers for reduced susceptibility to ash dieback. Surveys of these markers in British populations suggest that reduced susceptibility to ash dieback may be more widespread in Great Britain than in Denmark. We also present evidence that susceptibility of trees to H. fraxineus is associated with their iridoid glycoside levels. This rapid, integrated, multidisciplinary research response to an emerging health threat in a non-model organism opens the way for mitigation of the epidemic.
Abstract.
Author URL.
Turner J, O'Neill P, Grant M, Mumford RA, Thwaites R, Studholme DJ (2017). Genome sequences of 12 isolates of the EU1 lineage of Phytophthora ramorum , a fungus-like pathogen that causes extensive damage and mortality to a wide range of trees and other plants. Genomics Data, 12, 17-21.
Stulberg MJ, Kasiborski B, Studholme D, Munkvold GP, Block CC, Arias S, Rascoe J, Nakhla MK (2017). Genomics-informed molecular detection of <i>Xanthomonas vasicola</i> causing bacterial leaf streak of corn in the United States.
Author URL.
Sambles C, Moore K, Lux TM, Jones K, Littlejohn GR, Gouveia JD, Aves SJ, Studholme DJ, Lee R, Love J, et al (2017). Metagenomic analysis of the complex microbial consortium associated with cultures of the oil‐rich alga <i>Botryococcus braunii</i>.
MicrobiologyOpen,
6(4).
Abstract:
Metagenomic analysis of the complex microbial consortium associated with cultures of the oil‐rich alga Botryococcus braunii
AbstractMicroalgae are widely viewed as a promising and sustainable source of renewable chemicals and biofuels. Botryococcus braunii synthesizes and secretes significant amounts of long‐chain (C30‐C40) hydrocarbons that can be subsequently converted into gasoline, diesel, and aviation fuel. B. braunii cultures are not axenic and the effects of co‐cultured microorganisms on B. braunii growth and hydrocarbon yield are important, but sometimes contradictory. To understand the composition of the B. braunii microbial consortium, we used high throughput Illumina sequencing of metagenomic DNA to profile the microbiota within a well established, stable B. braunii culture and characterized the demographic changes in the microcosm following modification to the culture conditions. DNA sequences attributed to B. braunii were present in equal quantities in all treatments, whereas sequences assigned to the associated microbial community were dramatically altered. Bacterial species least affected by treatments, and more robustly associated with the algal cells, included members of Rhizobiales, comprising Bradyrhizobium and Methylobacterium, and representatives of Dyadobacter, Achromobacter and Asticcacaulis. The presence of bacterial species identified by metagenomics was confirmed by additional 16S rDNA analysis of bacterial isolates. Our study demonstrates the advantages of high throughput sequencing and robust metagenomic analyses to define microcosms and further our understanding of microbial ecology.
Abstract.
Yemataw Z, Chala A, Ambachew D, Studholme DJ, Grant MR, Tesfaye K (2017). Morphological Variation and Inter-Relationships of Quantitative Traits in Enset (Ensete ventricosum (welw.) Cheesman) Germplasm from South and South-Western Ethiopia.
Plants (Basel),
6(4).
Abstract:
Morphological Variation and Inter-Relationships of Quantitative Traits in Enset (Ensete ventricosum (welw.) Cheesman) Germplasm from South and South-Western Ethiopia.
Enset (Ensete ventricosum (Welw.) Cheesman) is Ethiopia's most important root crop. A total of 387 accessions collected from nine different regions of Ethiopia were evaluated for 15 quantitative traits at Areka Agricultural Research Centre to determine the extent and pattern of distribution of morphological variation. The variations among the accessions and regions were significant (p ≤ 0.01) for all the 15 traits studied. Mean for plant height, central shoot weight before grating, and fermented squeezed kocho yield per hectare per year showed regional variation along an altitude gradient and across cultural differences related to the origin of the collection. Furthermore, there were significant correlations among most of the characters. This included the correlation among agronomic characteristics of primary interest in enset breeding such as plant height, pseudostem height, and fermented squeezed kocho yield per hectare per year. Altitude of the collection sites also significantly impacted the various characteristics studied. These results reveal the existence of significant phenotypic variations among the 387 accessions as a whole. Regional differentiations were also evident among the accessions. The implication of the current results for plant breeding, germplasm collection, and in situ and ex situ genetic resource conservation are discussed.
Abstract.
Author URL.
Vicente JG, Rothwell S, Holub EB, Studholme DJ (2017). Pathogenic, phenotypic and molecular characterisation of Xanthomonas nasturtii sp. nov. and Xanthomonas floridensis sp. nov. new species of Xanthomonas associated with watercress production in Florida.
Int J Syst Evol Microbiol,
67(9), 3645-3654.
Abstract:
Pathogenic, phenotypic and molecular characterisation of Xanthomonas nasturtii sp. nov. and Xanthomonas floridensis sp. nov. new species of Xanthomonas associated with watercress production in Florida.
We describe two new species of the genus Xanthomonas, represented by yellow mucoid bacterial strains isolated from diseased leaves of watercress (Nasturtium officinale) produced in Florida, USA. One strain was pathogenic on watercress, but not in other species including a range of brassicas; other strains were not pathogenic in any of the tested plants. Data from Biolog carbon source utilization tests and nucleotide sequence data from 16S and gyrB loci suggested that both pathogenic and non-pathogenic strains were related to, yet distinct from, previously described Xanthomonas species. Multilocus sequence analysis and whole genome-wide comparisons of the average nucleotide identity (ANI) of genomes of two strains from watercress showed that these are distinct and share less than 95 % ANI with all other known species; the non-pathogenic strain WHRI 8848 is close to Xanthomonascassavae (ANI of 93.72 %) whilst the pathogenic strain WHRI 8853 is close to a large clade of species that includes Xanthomonasvesicatoria (ANI ≤90.25 %). Based on these results, we propose that both strains represent new Xanthomonas species named Xanthomonas floridensis sp. nov. (type strain WHRI 8848=ATCC TSD-60=ICMP 21312=LMG 29665=NCPPB 4601) and Xanthomonas nasturtii sp. nov. (type strain WHRI 8853=ATCC TSD-61=ICMP 21313=LMG 29666=NCPPB 4600), respectively. The presence of non-pathogenic Xanthomonas strains in watercress and their interaction with pathogenic strains needs to be further investigated. Although the importance of the new pathogenic species is yet to be determined, the bacterial disease that it causes constitutes a threat to watercress production and its distribution should be monitored.
Abstract.
Author URL.
Galhano R, Illana A, Ryder LS, Rodríguez-Romero J, Demuez M, Badaruddin M, Martinez-Rocha AL, Soanes DM, Studholme DJ, Talbot NJ, et al (2017). Tpc1 is an important Zn(II)2Cys6 transcriptional regulator required for polarized growth and virulence in the rice blast fungus.
PLoS Pathog,
13(7).
Abstract:
Tpc1 is an important Zn(II)2Cys6 transcriptional regulator required for polarized growth and virulence in the rice blast fungus.
The establishment of polarity is a critical process in pathogenic fungi, mediating infection-related morphogenesis and host tissue invasion. Here, we report the identification of TPC1 (Transcription factor for Polarity Control 1), which regulates invasive polarized growth in the rice blast fungus Magnaporthe oryzae. TPC1 encodes a putative transcription factor of the fungal Zn(II)2Cys6 family, exclusive to filamentous fungi. Tpc1-deficient mutants show severe defects in conidiogenesis, infection-associated autophagy, glycogen and lipid metabolism, and plant tissue colonisation. By tracking actin-binding proteins, septin-5 and autophagosome components, we show that Tpc1 regulates cytoskeletal dynamics and infection-associated autophagy during appressorium-mediated plant penetration. We found that Tpc1 interacts with Mst12 and modulates its DNA-binding activity, while Tpc1 nuclear localisation also depends on the MAP kinase Pmk1, consistent with the involvement of Tpc1 in this signalling pathway, which is critical for appressorium development. Importantly, Tpc1 directly regulates NOXD expression, the p22phox subunit of the fungal NADPH oxidase complex via an interaction with Mst12. Tpc1 therefore controls spatial and temporal regulation of cortical F-actin through regulation of the NADPH oxidase complex during appressorium re-polarisation. Consequently, Tpc1 is a core developmental regulator in filamentous fungi, linking the regulated synthesis of reactive oxygen species and the Pmk1 pathway, with polarity control during host invasion.
Abstract.
Author URL.
2016
Asif H, Studholme DJ, Khan A, Aurongzeb M, Khan IA, Azim MK (2016). Comparative genomics of an endophytic Pseudomonas putida isolated from mango orchard.
Genet Mol Biol,
0Abstract:
Comparative genomics of an endophytic Pseudomonas putida isolated from mango orchard.
We analyzed the genome sequence of an endophytic bacterial strain Pseudomonas putida TJI51 isolated from mango bark tissues. Next generation DNA sequencing and short read de novo assembly generated the 5,805,096 bp draft genome of P. putida TJI51. Out of 6,036 protein coding genes in P. putida TJI51 sequences, 4,367 (72%) were annotated with functional specifications, while the remaining encoded hypothetical proteins. Comparative genome sequence analysis revealed that the P. putida TJI51genome contains several regions, not identified in so far sequenced P. putida genomes. Some of these regions were predicted to encode enzymes, including acetylornithine deacetylase, betaine aldehyde dehydrogenase, aldehyde dehydrogenase, benzoylformate decarboxylase, hydroxyacylglutathione hydrolase, and uroporphyrinogen decarboxylase. The genome of P. putida TJI51 contained three nonribosomal peptide synthetase gene clusters. Genome sequence analysis of P. putidaTJI51 identified this bacterium as an endophytic resident. The endophytic fitness might be linked with alginate, which facilitates bacterial colonization in plant tissues. Genome sequence analysis shed light on the presence of a diverse spectrum of metabolic activities and adaptation of this isolate to various niches.
Abstract.
Author URL.
Asif H, Studholme DJ, Khan A, Aurongzeb M, Khan IA, Azim MK (2016). Comparative genomics of an endophytic Pseudomonas putida isolated from mango orchard.
Genet Mol Biol,
39(3), 465-473.
Abstract:
Comparative genomics of an endophytic Pseudomonas putida isolated from mango orchard.
We analyzed the genome sequence of an endophytic bacterial strain Pseudomonas putida TJI51 isolated from mango bark tissues. Next generation DNA sequencing and short read de novo assembly generated the 5,805,096 bp draft genome of P. putida TJI51. Out of 6,036 protein coding genes in P. putida TJI51 sequences, 4,367 (72%) were annotated with functional specifications, while the remaining encoded hypothetical proteins. Comparative genome sequence analysis revealed that the P. putida TJI51genome contains several regions, not identified in so far sequenced P. putida genomes. Some of these regions were predicted to encode enzymes, including acetylornithine deacetylase, betaine aldehyde dehydrogenase, aldehyde dehydrogenase, benzoylformate decarboxylase, hydroxyacylglutathione hydrolase, and uroporphyrinogen decarboxylase. The genome of P. putida TJI51 contained three nonribosomal peptide synthetase gene clusters. Genome sequence analysis of P. putidaTJI51 identified this bacterium as an endophytic resident. The endophytic fitness might be linked with alginate, which facilitates bacterial colonization in plant tissues. Genome sequence analysis shed light on the presence of a diverse spectrum of metabolic activities and adaptation of this isolate to various niches.
Abstract.
Author URL.
Harrison J, Dornbusch MR, Samac D, Studholme DJ (2016). Draft Genome Sequence of Pseudomonas syringae pv. syringae ALF3 Isolated from Alfalfa.
Genome Announc,
4(1).
Abstract:
Draft Genome Sequence of Pseudomonas syringae pv. syringae ALF3 Isolated from Alfalfa.
We report here the annotated draft genome sequence of Pseudomonas syringae pv. syringae strain ALF3, isolated in Wyoming. A comparison of this genome sequence with those of closely related strains of P. syringae adapted to other hosts will facilitate research into interactions between this pathogen and alfalfa.
Abstract.
Author URL.
Jones KJ, Moore K, Sambles C, Love J, Studholme DJ, Aves SJ (2016). Draft Genome Sequences of Achromobacter piechaudii GCS2, Agrobacterium sp. Strain SUL3, Microbacterium sp. Strain GCS4, Shinella sp. Strain GWS1, and Shinella sp. Strain SUS2 Isolated from Consortium with the Hydrocarbon-Producing Alga Botryococcus braunii.
Genome Announc,
4(1).
Abstract:
Draft Genome Sequences of Achromobacter piechaudii GCS2, Agrobacterium sp. Strain SUL3, Microbacterium sp. Strain GCS4, Shinella sp. Strain GWS1, and Shinella sp. Strain SUS2 Isolated from Consortium with the Hydrocarbon-Producing Alga Botryococcus braunii.
A variety of bacteria associate with the hydrocarbon-producing microalga Botryococcus braunii, some of which may influence its growth. We report here the genome sequences for Achromobacter piechaudii GCS2, Agrobacterium sp. strain SUL3, Microbacterium sp. strain GCS4, and Shinella sp. strains GWS1 and SUS2, isolated from a laboratory culture of B. braunii, race B, strain Guadeloupe.
Abstract.
Author URL.
Harrison J, Grant MR, Studholme DJ (2016). Draft Genome Sequences of Two Strains of Xanthomonas arboricola pv. celebensis Isolated from Banana Plants.
Genome Announc,
4(1).
Abstract:
Draft Genome Sequences of Two Strains of Xanthomonas arboricola pv. celebensis Isolated from Banana Plants.
We report here the annotated draft genome sequences of strains Xanthomonas arboricola pv. celebensis NCPPB 1832 and NCPPB 1630 (NCPPB, National Collection of Plant Pathogenic Bacteria), both isolated from Musa species in New Zealand. This will allow the comparison of genomes between phylogenetically distant xanthomonads that have independently converged with the ability to colonize banana plants.
Abstract.
Author URL.
Studholme DJ (2016). Genome Update. Let the consumer beware: Streptomyces genome sequence quality.
Microbial Biotechnology,
9(1), 3-7.
Abstract:
Genome Update. Let the consumer beware: Streptomyces genome sequence quality
A genome sequence assembly represents a model of a genome. This article explores some tools and methods for assessing the quality of an assembly, using publicly available data for Streptomyces species as the example. There is great variability in quality of assemblies deposited in GenBank. Only in a small minority of these assemblies are the raw data available, enabling full appraisal of the assembly quality.
Abstract.
Studholme DJ, McDougal RL, Sambles C, Hansen E, Hardy G, Grant M, Ganley RJ, Williams NM (2016). Genome sequences of six Phytophthora species associated with forests in New Zealand.
Genomics Data,
7, 54-56.
Abstract:
Genome sequences of six Phytophthora species associated with forests in New Zealand
In New Zealand there has been a long association of Phytophthora diseases in forests, nurseries, remnant plantings and horticultural crops. However, new Phytophthora diseases of trees have recently emerged. Genome sequencing has been performed for 12 Phytophthora isolates, from six species: Phytophthora pluvialis, Phytophthora kernoviae, Phytophthora cinnamomi, Phytophthora agathidicida, Phytophthora multivora and Phytophthora taxon Totara. These sequences will enable comparative analyses to identify potential virulence strategies and ultimately facilitate better control strategies. This Whole Genome Shotgun data have been deposited in DDBJ/ENA/GenBank under the accession numbers LGTT00000000, LGTU00000000, JPWV00000000, JPWU00000000, LGSK00000000, LGSJ00000000, LGTR00000000, LGTS00000000, LGSM00000000, LGSL00000000, LGSO00000000, and LGSN00000000.
Abstract.
Cairns TC, Studholme DJ, Talbot NJ, Haynes K (2016). New and Improved Techniques for the Study of Pathogenic Fungi.
Trends in Microbiology,
24(1), 35-50.
Abstract:
New and Improved Techniques for the Study of Pathogenic Fungi
Fungal pathogens pose serious threats to human, plant, and ecosystem health. Improved diagnostics and antifungal strategies are therefore urgently required. Here, we review recent developments in online bioinformatic tools and associated interactive data archives, which enable sophisticated comparative genomics and functional analysis of fungal pathogens in silico. Additionally, we highlight cutting-edge experimental techniques, including conditional expression systems, recyclable markers, RNA interference, genome editing, compound screens, infection models, and robotic automation, which are promising to revolutionize the study of both human and plant pathogenic fungi. These novel techniques will allow vital knowledge gaps to be addressed with regard to the evolution of virulence, host-pathogen interactions and antifungal drug therapies in both the clinic and agriculture. This, in turn, will enable delivery of improved diagnosis and durable disease-control strategies.
Abstract.
Cairns TC, Studholme DJ, Talbot NJ, Haynes K (2016). New and Improved Techniques for the Study of Pathogenic Fungi.
Trends Microbiol,
24(1), 35-50.
Abstract:
New and Improved Techniques for the Study of Pathogenic Fungi.
Fungal pathogens pose serious threats to human, plant, and ecosystem health. Improved diagnostics and antifungal strategies are therefore urgently required. Here, we review recent developments in online bioinformatic tools and associated interactive data archives, which enable sophisticated comparative genomics and functional analysis of fungal pathogens in silico. Additionally, we highlight cutting-edge experimental techniques, including conditional expression systems, recyclable markers, RNA interference, genome editing, compound screens, infection models, and robotic automation, which are promising to revolutionize the study of both human and plant pathogenic fungi. These novel techniques will allow vital knowledge gaps to be addressed with regard to the evolution of virulence, host-pathogen interactions and antifungal drug therapies in both the clinic and agriculture. This, in turn, will enable delivery of improved diagnosis and durable disease-control strategies.
Abstract.
Author URL.
2015
Kilaru S, Schuster M, Studholme D, Soanes D, Lin C, Talbot NJ, Steinberg G (2015). A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici.
Fungal Genet Biol,
79, 125-131.
Abstract:
A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici.
Fluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein localization. Cytoplasmic expression of FPs in fungal pathogens allows greater insight into invasion strategies and the host-pathogen interaction. Detection of their fluorescent signal depends on the right combination of microscopic setup and signal brightness. Slow rates of photo-bleaching are pivotal for in vivo observation of FPs over longer periods of time. Here, we test green-fluorescent proteins, including Aequorea coerulescens GFP (AcGFP), enhanced GFP (eGFP) from Aequorea victoria and a novel Zymoseptoria tritici codon-optimized eGFP (ZtGFP), for their usage in conventional and laser-enhanced epi-fluorescence, and confocal laser-scanning microscopy. We show that eGFP, expressed cytoplasmically in Z. tritici, is significantly brighter and more photo-stable than AcGFP. The codon-optimized ZtGFP performed even better than eGFP, showing significantly slower bleaching and a 20-30% further increase in signal intensity. Heterologous expression of all GFP variants did not affect pathogenicity of Z. tritici. Our data establish ZtGFP as the GFP of choice to investigate intracellular protein dynamics in Z. tritici, but also infection stages of this wheat pathogen inside host tissue.
Abstract.
Author URL.
Laver T, Harrison J, O'Neill PA, Moore K, Farbos A, Paszkiewicz K, Studholme DJ (2015). Assessing the performance of the Oxford Nanopore Technologies MinION.
Biomolecular Detection and Quantification,
3, 1-8.
Abstract:
Assessing the performance of the Oxford Nanopore Technologies MinION
The Oxford Nanopore Technologies (ONT) MinION is a new sequencing technology that potentially offers read lengths of tens of kilobases (kb) limited only by the length of DNA molecules presented to it. The device has a low capital cost, is by far the most portable DNA sequencer available, and can produce data in real-time. It has numerous prospective applications including improving genome sequence assemblies and resolution of repeat-rich regions. Before such a technology is widely adopted, it is important to assess its performance and limitations in respect of throughput and accuracy. In this study we assessed the performance of the MinION by re-sequencing three bacterial genomes, with very different nucleotide compositions ranging from 28.6% to 70.7%; the high G. +. C strain was underrepresented in the sequencing reads. We estimate the error rate of the MinION (after base calling) to be 38.2%. Mean and median read lengths were 2. kb and 1. kb respectively, while the longest single read was 98. kb. The whole length of a 5. kb rRNA operon was covered by a single read. As the first nanopore-based single molecule sequencer available to researchers, the MinION is an exciting prospect; however, the current error rate limits its ability to compete with existing sequencing technologies, though we do show that MinION sequence reads can enhance contiguity of de novo assembly when used in conjunction with Illumina MiSeq data.
Abstract.
Nandi T, Holden MTG, Didelot X, Mehershahi K, Boddey JA, Beacham I, Peak I, Harting J, Baybayan P, Guo Y, et al (2015). Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles.
Genome Res,
25(1), 129-141.
Abstract:
Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles.
Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity.
Abstract.
Author URL.
de Torres M, Littlejohn G, Jayaraman S, Studholme D, Bailey TC, Lawson T, Delfino L, Licht D, Truman W, Bölter B, et al (2015). Chloroplasts play a central role in plant defence
and are targeted by pathogen effectors. Nature Plants, 1, n/a-n/a.
Karamura G, Smith J, Studholme D, Kubiriba J, Karamura E (2015). Comparative pathogenicity studies of the Xanthomonas vasicola species on maize, sugarcane and banana. African Journal of Plant Science, 9(9), 385-400.
Cairns TC, Sidhu YS, Chaudhari YK, Talbot NJ, Studholme DJ, Haynes K (2015). Construction and high-throughput phenotypic screening ofZymoseptoria tritici over-expression strains.
Fungal Genet Biol,
79, 110-117.
Abstract:
Construction and high-throughput phenotypic screening ofZymoseptoria tritici over-expression strains.
Targeted gene deletion has been instrumental in elucidating many aspects of Zymoseptoria tritici pathogenicity. Gene over-expression is a complementary approach that is amenable to rapid strain construction and high-throughput screening, which has not been exploited to analyze Z. tritici, largely due to a lack of available techniques. Here we exploit the Gateway® cloning technology for rapid construction of over-expression vectors and improved homologous integration efficiency of a Z. tritici Δku70 strain to build a pilot over-expression library encompassing 32 genes encoding putative DNA binding proteins, GTPases or kinases. We developed a protocol using a Rotor-HDA robot for rapid and reproducible cell pinning for high-throughput in vitro screening. This screen identified an over-expression strain that demonstrated a marked reduction in hyphal production relative to the isogenic progenitor. This study provides a protocol for rapid generation of Z. tritici over-expression libraries and a technique for functional genomic screening in this important pathogen.
Abstract.
Author URL.
Hodgetts J, Karamura G, Johnson G, Hall J, Perkins K, Beed F, Nakato V, Grant M, Studholme DJ, Boonham N, et al (2015). Development of a lateral flow device for in-field detection and evaluation of PCR-based diagnostic methods for Xanthomonas campestris pv. musacearum, the causal agent of banana xanthomonas wilt.
Plant Pathology,
64(3), 559-567.
Abstract:
Development of a lateral flow device for in-field detection and evaluation of PCR-based diagnostic methods for Xanthomonas campestris pv. musacearum, the causal agent of banana xanthomonas wilt
Xanthomonas campestris pv. musacearum (Xcm) is the causal agent of banana xanthomonas wilt, a major threat to banana production in eastern and central Africa. The pathogen is present in very high levels within infected plants and can be transmitted by a broad range of mechanisms; therefore early specific detection is vital for effective disease management. In this study, a polyclonal antibody (pAb) was developed and deployed in a lateral flow device (LFD) format to allow rapid in-field detection of Xcm. Published Xcm PCR assays were also independently assessed: only two assays gave specific amplification of Xcm, whilst others cross-reacted with non-target Xanthomonas species. Pure cultures of Xcm were used to immunize a rabbit, the IgG antibodies purified from the serum and the resulting polyclonal antibodies tested using ELISA and LFD. Testing against a wide range of bacterial species showed the pAb detected all strains of Xcm, representing isolates from seven countries and the known genetic diversity of Xcm. The pAb also detected the closely related Xanthomonas axonopodis pv. vasculorum (Xav), primarily a sugarcane pathogen. Detection was successful in both naturally and experimentally infected banana plants, and the LFD limit of detection was 105 cells mL-1. Whilst the pAb is not fully specific for Xcm, Xav has never been found in banana. Therefore the LFD can be used as a first-line screening tool to detect Xcm in the field. Testing by LFD requires no equipment, can be performed by non-scientists and is cost-effective. Therefore this LFD provides a vital tool to aid in the management and control of Xcm.
Abstract.
Devran Z, Kahveci E, Özkaynak E, Studholme DJ, Tör M (2015). Development of molecular markers tightly linked to Pvr4 gene in pepper using next-generation sequencing.
Mol Breed,
35(4).
Abstract:
Development of molecular markers tightly linked to Pvr4 gene in pepper using next-generation sequencing.
It is imperative to identify highly polymorphic and tightly linked markers of a known trait for molecular marker-assisted selection. Potyvirus resistance 4 (Pvr4) locus in pepper confers resistance to three pathotypes of potato virus Y and to pepper mottle virus. We describe the use of next-generation sequencing technology to generate molecular markers tightly linked to Pvr4. Initially, comparative genomics was carried out, and a syntenic region of tomato on chromosome ten was used to generate PCR-based markers and map Pvr4. Subsequently, the genomic sequence of pepper was used, and more than 5000 single-nucleotide variants (SNVs) were identified within the interval. In addition, we identified nucleotide binding site-leucine-rich repeat-type disease resistance genes within the interval. Several of these SNVs were converted to molecular markers desirable for large-scale molecular breeding programmes.
Abstract.
Author URL.
Siddiqui FM, Ibrahim M, Noureen N, Noreen Z, Titball RW, Champion OL, Wren BW, Studholme D, Bokhari H (2015). Draft Genome Sequence of the Enteropathogenic Bacterium Campylobacter jejuni Strain cj255.
Genome Announc,
3(5).
Abstract:
Draft Genome Sequence of the Enteropathogenic Bacterium Campylobacter jejuni Strain cj255.
The enteropathogen Campylobacter jejuni is a global health disaster, being one of the leading causes of bacterial gastroenteritis. Here, we present the draft genome sequence of C. jejuni strain cj255, isolated from a chicken source in Islamabad, Pakistan. The draft genome sequence will aid in epidemiological studies and quarantine of this broad-host-range pathogen.
Abstract.
Author URL.
Sambles C, Schlenzig A, O'Neill P, Grant M, Studholme DJ (2015). Draft genome sequences of Phytophthora kernoviae and Phytophthora ramorum lineage EU2 from Scotland. Genomics Data, 6, 193-194.
Mata Saez LDL, McCracken AR, Cooke LR, O'Neill P, Grant M, Studholme DJ (2015). Draft genome sequences of seven isolates of Phytophthora ramorum EU2 from Northern Ireland.
Genomics Data,
6, 191-192.
Abstract:
Draft genome sequences of seven isolates of Phytophthora ramorum EU2 from Northern Ireland
Here we present draft-quality genome sequence assemblies for the oomycete Phytophthora ramorum genetic lineage EU2. We sequenced genomes of seven isolates collected in Northern Ireland between 2010 and 2012. Multiple genome sequences from P. ramorum EU2 will be valuable for identifying genetic variation within the clonal lineage that can be useful for tracking its spread.
Abstract.
Taylor TB, Mulley G, Dills AH, Alsohim AS, McGuffin LJ, Studholme DJ, Silby MW, Brockhurst MA, Johnson LJ, Jackson RW, et al (2015). Evolution. Evolutionary resurrection of flagellar motility via rewiring of the nitrogen regulation system.
Science,
347(6225), 1014-1017.
Abstract:
Evolution. Evolutionary resurrection of flagellar motility via rewiring of the nitrogen regulation system.
A central process in evolution is the recruitment of genes to regulatory networks. We engineered immotile strains of the bacterium Pseudomonas fluorescens that lack flagella due to deletion of the regulatory gene fleQ. Under strong selection for motility, these bacteria consistently regained flagella within 96 hours via a two-step evolutionary pathway. Step 1 mutations increase intracellular levels of phosphorylated NtrC, a distant homolog of FleQ, which begins to commandeer control of the fleQ regulon at the cost of disrupting nitrogen uptake and assimilation. Step 2 is a switch-of-function mutation that redirects NtrC away from nitrogen uptake and toward its novel function as a flagellar regulator. Our results demonstrate that natural selection can rapidly rewire regulatory networks in very few, repeatable mutational steps.
Abstract.
Author URL.
Sidhu YS, Cairns TC, Chaudhari YK, Usher J, Talbot NJ, Studholme DJ, Csukai M, Haynes K (2015). Exploitation of sulfonylurea resistance marker and non-homologous end joining mutants for functional analysis in Zymoseptoria tritici.
Fungal Genet Biol,
79, 102-109.
Abstract:
Exploitation of sulfonylurea resistance marker and non-homologous end joining mutants for functional analysis in Zymoseptoria tritici.
The lack of techniques for rapid assembly of gene deletion vectors, paucity of selectable marker genes available for genetic manipulation and low frequency of homologous recombination are major constraints in construction of gene deletion mutants in Zymoseptoria tritici. To address these issues, we have constructed ternary vectors for Agrobacterium tumefaciens mediated transformation of Z. tritici, which enable the single step assembly of multiple fragments via yeast recombinational cloning. The sulfonylurea resistance gene, which is a mutated allele of the Magnaporthe oryzae ILV2 gene, was established as a new dominant selectable marker for Z. tritici. To increase the frequency of homologous recombination, we have constructed Z. tritici strains deficient in the non-homologous end joining pathway of DNA double stranded break repair by inactivating the KU70 and KU80 genes. Targeted gene deletion frequency increased to more than 85% in both Z. tritici ku70 and ku80 null strains, compared to ⩽10% seen in the wild type parental strain IPO323. The in vitro growth and in planta pathogenicity of the Z. tritici ku70 and ku80 null strains were comparable to strain IPO323. Together these molecular tools add significantly to the platform available for genomic analysis through targeted gene deletion or promoter replacements and will facilitate large-scale functional characterization projects in Z. tritici.
Abstract.
Author URL.
Sharma R, Xia X, Cano LM, Evangelisti E, Kemen E, Judelson H, Oome S, Sambles C, van den Hoogen DJ, Kitner M, et al (2015). Genome analyses of the sunflower pathogen Plasmopara halstedii provide insights into effector evolution in downy mildews and Phytophthora.
BMC Genomics,
16Abstract:
Genome analyses of the sunflower pathogen Plasmopara halstedii provide insights into effector evolution in downy mildews and Phytophthora.
BACKGROUND: Downy mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily. RESULTS: Here a high-quality draft genome of Plasmopara halstedii is reported and analysed with respect to various aspects, including genome organisation, secondary metabolism, effector proteins and comparative genomics with other sequenced oomycetes. Interestingly, the present analyses revealed further variation of the RxLR motif, suggesting an important role of the conservation of the dEER-motif. Orthology analyses revealed the conservation of 28 RxLR-like core effectors among Phytophthora species. Only six putative RxLR-like effectors were shared by the two sequenced downy mildews, highlighting the fast and largely independent evolution of two of the three major downy mildew lineages. This is seemingly supported by phylogenomic results, in which downy mildews did not appear to be monophyletic. CONCLUSIONS: the genome resource will be useful for developing markers for monitoring the pathogen population and might provide the basis for new approaches to fight Phytophthora and downy mildew pathogens by targeting core pathogenicity effectors.
Abstract.
Author URL.
Aritua V, Harrison J, Sapp M, Buruchara R, Smith J, Studholme DJ (2015). Genome sequencing reveals a new lineage associated with lablab bean and genetic exchange between Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans.
Front Microbiol,
6Abstract:
Genome sequencing reveals a new lineage associated with lablab bean and genetic exchange between Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans.
Common bacterial blight is a devastating seed-borne disease of common beans that also occurs on other legume species including lablab and Lima beans. We sequenced and analyzed the genomes of 26 strains of Xanthomonas axonopodis pv. phaseoli and X. fuscans subsp. fuscans, the causative agents of this disease, collected over four decades and six continents. This revealed considerable genetic variation within both taxa, encompassing both single-nucleotide variants and differences in gene content, that could be exploited for tracking pathogen spread. The bacterial strain from Lima bean fell within the previously described Genetic Lineage 1, along with the pathovar type strain (NCPPB 3035). The strains from lablab represent a new, previously unknown genetic lineage closely related to strains of X. axonopodis pv. glycines. Finally, we identified more than 100 genes that appear to have been recently acquired by Xanthomonas axonopodis pv. phaseoli from X. fuscans subsp. fuscans.
Abstract.
Author URL.
Clarke CR, Studholme DJ, Hayes B, Runde B, Weisberg A, Cai R, Wroblewski T, Daunay M-C, Wicker E, Castillo JA, et al (2015). Genome-Enabled Phylogeographic Investigation of the Quarantine Pathogen Ralstonia solanacearum Race 3 Biovar 2 and Screening for Sources of Resistance Against its Core Effectors.
Phytopathology,
105(5), 597-607.
Abstract:
Genome-Enabled Phylogeographic Investigation of the Quarantine Pathogen Ralstonia solanacearum Race 3 Biovar 2 and Screening for Sources of Resistance Against its Core Effectors.
Phylogeographic studies inform about routes of pathogen dissemination and are instrumental for improving import/export controls. Genomes of 17 isolates of the bacterial wilt and potato brown rot pathogen Ralstonia solanacearum race 3 biovar 2 (R3bv2), a Select Agent in the United States, were thus analyzed to get insight into the phylogeography of this pathogen. Thirteen of fourteen isolates from Europe, Africa, and Asia were found to belong to a single clonal lineage while isolates from South America were genetically diverse and tended to carry ancestral alleles at the analyzed genomic loci consistent with a South American origin of R3bv2. The R3bv2 isolates share a core repertoire of 31 type III-secreted effector genes representing excellent candidates to be targeted with resistance genes in breeding programs to develop durable disease resistance. Toward this goal, 27 R3bv2 effectors were tested in eggplant, tomato, pepper, tobacco, and lettuce for induction of a hypersensitive-like response indicative of recognition by cognate resistance receptors. Fifteen effectors, eight of them core effectors, triggered a response in one or more plant species. These genotypes may harbor resistance genes that could be identified and mapped, cloned, and expressed in tomato or potato, for which sources of genetic resistance to R3bv2 are extremely limited.
Abstract.
Author URL.
Siddiqui F, Champion O, Akram M, Studholme D, Eqani SAMAS, Wren BW, Titball R, Bokhari H (2015). Molecular detection identified a type six secretion system in Campylobacter jejuni from various sources but not from human cases.
J Appl Microbiol,
118(5), 1191-1198.
Abstract:
Molecular detection identified a type six secretion system in Campylobacter jejuni from various sources but not from human cases.
AIM: to determine the presence of the T6SS in Campylobacter jejuni from diverse sources. METHODS AND RESULTS: the recently identified type VI secretion system (T6SS) is a bacterial injection machinery that plays a role in virulence, symbiosis, bacterial interactions and environmental stress responses. This system has been recently discovered in the major enteric pathogen Camp. jejuni. In this study, we used multiplex PCR (mPCR), based on conserved genetic markers of the T6SS, to screen 366 Pakistani Camp. jejuni isolates from humans, poultry, cattle, wildlife or waste-water sources. We identified the T6SS in isolates from all of these sources except humans. The overall prevalence of the T6SS among the isolates was 17/366 (4·6%) and the T6SS positive isolates clustered into four different groups. Transcription of the T6SS genes, determined using RT-PCR, was observed in bacteria cultured at 37 or 42°C but not in 37°C cultures adjusted to pH3. CONCLUSIONS: Campylobacter jejuni isolates harbouring T6SS markers genes were identified in livestock and non-livestock sources but in this study we did not identify human diarrhoeal isolates which possessed the T6SS. We demonstrated down-regulation of T6SS in an acidic environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study questions the role of the T6SS in human diarrhoeal disease. Moreover this study did not identify a clear association of Camp. jejuni isolates harbouring T6SS with any of the niches tested. Our study highlights the need to establish the role of the T6SS in environmental survival or virulence.
Abstract.
Author URL.
Hodgetts J, Hall J, Karamura G, Grant M, Studholme DJ, Boonham N, Karamura E, Smith JJ (2015). Rapid, specific, simple, in-field detection of Xanthomonas campestris pathovar musacearum by loop-mediated isothermal amplification.
J Appl Microbiol,
119(6), 1651-1658.
Abstract:
Rapid, specific, simple, in-field detection of Xanthomonas campestris pathovar musacearum by loop-mediated isothermal amplification.
AIMS: to develop and evaluate a loop-mediated isothermal amplification (LAMP) assay for Xanthomonas campestris pathovar musacearum (Xcm), the causal agent of banana Xanthomonas wilt, a major disease of banana in Africa. METHODS AND RESULTS: LAMP primers were designed to the general secretion pathway protein D gene and tested against 17 isolates of Xcm encompassing the known genetic and geographic diversity of the bacterium and all isolates were detected. Seventeen other Xanthomonas isolates, including closely related Xanthomonas vasicola, other bacterial pathogens/endophytes of Musa and two healthy Musa varieties gave negative results with the LAMP assay. The assay showed good sensitivity, detecting as little as 51 fg of Xcm DNA, a greater level of sensitivity than that of an Xcm PCR assay. Amplification with the LAMP assay was very rapid, typically within 9 min from bacterial cultures. Symptomatic field samples of Musa from Uganda were tested and all produced amplification in less than 13 min. CONCLUSIONS: the LAMP assay provides rapid, sensitive detection of the pathogen that is ideally suited for deployment in laboratories with basic facilities and in-field situations. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first LAMP assay for Xcm which provides a significant improvement compared to existing diagnostics.
Abstract.
Author URL.
Studholme DJ (2015). Some (bacilli) like it hot: Genomics of Geobacillus species. Microbial Biotechnology, 8(1), 40-48.
Aritua V, Musoni A, Kabeja A, Butare L, Mukamuhirwa F, Gahakwa D, Kato F, Abang MM, Buruchara R, Sapp M, et al (2015). The draft genome sequence of Xanthomonas species strain Nyagatare, isolated from diseased bean in Rwanda.
FEMS Microbiol Lett,
362(4).
Abstract:
The draft genome sequence of Xanthomonas species strain Nyagatare, isolated from diseased bean in Rwanda.
We announce the genome sequence for Xanthomonas species strain Nyagatare, isolated from beans showing unusual disease symptoms in Rwanda. This strain represents the first sequenced genome belonging to an as-yet undescribed Xanthomonas species known as species-level clade 1. It has at least 100 kb of genomic sequence that shows little or no sequence similarity to other xanthomonads, including a unique lipopolysaccharide synthesis gene cluster. At least one genomic region appears to have been acquired from relatives of Agrobacterium or Rhizobium species. The genome encodes homologues of only three known type-three secretion system effectors: AvrBs2, XopF1 and AvrXv4. Availability of the genome sequence will facilitate development of molecular tools for detection and diagnostics for this newly discovered pathogen of beans and facilitate epidemiological investigations of a potential causal link between this pathogen and the disease outbreak.
Abstract.
Author URL.
2014
Harrison J, Moore K, Paszkiewicz K, Jones T, Grant M, Ambacheew D, Muzemil S, Studholme D (2014). A Draft Genome Sequence for Ensete ventricosum, the Drought-Tolerant “Tree Against Hunger”.
Agronomy,
4, 13-33.
Abstract:
A Draft Genome Sequence for Ensete ventricosum, the Drought-Tolerant “Tree Against Hunger”
We present a draft genome sequence for enset (Ensete ventricosum) available via the Sequence Read Archive (accession number SRX202265) and GenBank (accession number AMZH01. Enset feeds 15 million people in Ethiopia, but is arguably the least studied African crop. Our sequence data suggest a genome size of approximately 547 megabases, similar to the 523-megabase genome of the closely related banana (Musa acuminata). At least 1.8% of the annotated M. acuminata genes are not conserved in E. ventricosum. Furthermore, enset contains genes not present in banana, including reverse transcriptases and virus-like sequences as well as a homolog of the RPP8-like resistance gene. We hope that availability of genome-wide sequence data will stimulate and accelerate research on this important but neglected crop.
Abstract.
Harrison J, Moore K, Paszkiewicz K, Jones T, Grant M, Ambacheew D, Muzemil S, Studholme D (2014). A Draft Genome Sequence for Ensete ventricosum, the Drought-Tolerant “Tree Against Hunger”.
Agronomy,
4, 13-33.
Abstract:
A Draft Genome Sequence for Ensete ventricosum, the Drought-Tolerant “Tree Against Hunger”
We present a draft genome sequence for enset (Ensete ventricosum) available via the Sequence Read Archive (accession number SRX202265) and GenBank (accession number AMZH01. Enset feeds 15 million people in Ethiopia, but is arguably the least studied African crop. Our sequence data suggest a genome size of approximately 547 megabases, similar to the 523-megabase genome of the closely related banana (Musa acuminata). At least 1.8% of the annotated M. acuminata genes are not conserved in E. ventricosum. Furthermore, enset contains genes not present in banana, including reverse transcriptases and virus-like sequences as well as a homolog of the RPP8-like resistance gene. We hope that availability of genome-wide sequence data will stimulate and accelerate research on this important but neglected crop.
Abstract.
O'Sullivan DM, Laver T, Temisak S, Redshaw N, Harris KA, Foy CA, Studholme DJ, Huggett JF (2014). Assessing the accuracy of quantitative molecular microbial profiling.
Int J Mol Sci,
15(11), 21476-21491.
Abstract:
Assessing the accuracy of quantitative molecular microbial profiling.
The application of high-throughput sequencing in profiling microbial communities is providing an unprecedented ability to investigate microbiomes. Such studies typically apply one of two methods: amplicon sequencing using PCR to target a conserved orthologous sequence (typically the 16S ribosomal RNA gene) or whole (meta)genome sequencing (WGS). Both methods have been used to catalog the microbial taxa present in a sample and quantify their respective abundances. However, a comparison of the inherent precision or bias of the different sequencing approaches has not been performed. We previously developed a metagenomic control material (MCM) to investigate error when performing different sequencing strategies. Amplicon sequencing using four different primer strategies and two 16S rRNA regions was examined (Roche 454 Junior) and compared to WGS (Illumina HiSeq). All sequencing methods generally performed comparably and in good agreement with organism specific digital PCR (dPCR); WGS notably demonstrated very high precision. Where discrepancies between relative abundances occurred they tended to differ by less than twofold. Our findings suggest that when alternative sequencing approaches are used for microbial molecular profiling they can perform with good reproducibility, but care should be taken when comparing small differences between distinct methods. This work provides a foundation for future work comparing relative differences between samples and the impact of extraction methods. We also highlight the value of control materials when conducting microbial profiling studies to benchmark methods and set appropriate thresholds.
Abstract.
Author URL.
Samac D, Studholme DJ, Ao S (2014). Characterization of the bacterial stem blight pathogen of alfalfa, <i>Pseudomonas syringae</i> pv. <i>syringae</i> ALF3.
Author URL.
Saunders D, Yoshida K, Sambles C, Glover R, Clavijo B, Corpas M, Bunting D, Dong S, Rallapalli G, Clark MD, et al (2014). Crowdsourced analysis of ash and ash dieback through the Open Ash Dieback project: a year 1 report on datasets and analyses contributed by a self-organising community.
Wagley S, Newcombe J, Laing E, Yusuf E, Sambles CM, Studholme DJ, La Ragione RM, Titball RW, Champion OL (2014). Differences in carbon source utilisation distinguish Campylobacter jejuni from Campylobacter coli.
BMC Microbiol,
14Abstract:
Differences in carbon source utilisation distinguish Campylobacter jejuni from Campylobacter coli.
BACKGROUND: Campylobacter jejuni and C. coli are human intestinal pathogens that are the most frequent causes of bacterial foodborne gastroenteritis in humans in the UK. In this study, we aimed to characterise the metabolic diversity of both C. jejuni and C. coli using a diverse panel of clinical strains isolated from the UK, Pakistan and Thailand, thereby representing both the developed and developing world. Our aim was to apply multi genome analysis and Biolog phenotyping to determine differences in carbon source utilisation by C. jejuni and C. coli strains. RESULTS: We have identified a core set of carbon sources (utilised by all strains tested) and a set that are differentially utilised for a diverse panel of thirteen C. jejuni and two C. coli strains. This study used multi genome analysis to show that propionic acid is utilised only by C. coli strains tested. A broader PCR screen of 16 C. coli strains and 42 C. jejuni confirmed the absence of the genes needed for propanoate metabolism. CONCLUSIONS: from our analysis we have identified a phenotypic method and two genotypic methods based on propionic utilisation that might be applicable for distinguishing between C. jejuni and C. coli.
Abstract.
Author URL.
Uzelac G, Bertani I, Kojic M, Paszkiewicz KH, Studholme DJ, Passos da Silva D, Venturi V (2014). Draft Genome Sequence of Beneficial Rice Rhizosphere Isolate Pseudomonas aeruginosa PUPa3.
Genome Announc,
2(4).
Abstract:
Draft Genome Sequence of Beneficial Rice Rhizosphere Isolate Pseudomonas aeruginosa PUPa3.
Pseudomonas aeruginosa PUPa3 is a rhizosphere-colonizing and plant growth-promoting strain isolated from the rhizosphere of rice. This strain has, however, been shown to be pathogenic in two nonmammalian infection models. Here we report the draft genome sequence of P. aeruginosa PUPa3.
Abstract.
Author URL.
Harrison J, Studholme DJ (2014). Draft genome sequence of Xanthomonas axonopodis pathovar vasculorum NCPPB 900.
FEMS Microbiology Letters,
360(2), 113-116.
Abstract:
Draft genome sequence of Xanthomonas axonopodis pathovar vasculorum NCPPB 900
Xanthomonas axonopodis pathovar vasculorum strain NCPPB 900 was isolated from sugarcane on Reunion island in 1960. Consistent with its belonging to fatty-acid type D, multi-locus sequence analysis confirmed that NCPPB 900 falls within the species X. axonopodis. This genome harbours sequences similar to plasmids pXCV183 from X. campestris pv. vesicatoria 85-10 and pPHB194 from Burkholderia pseudomallei. Its repertoire of predicted effectors includes homologues of XopAA, XopAD, XopAE, XopB, XopD, XopV, XopZ, XopC and XopI and transcriptional activator-like effectors and it is predicted to encode a novel phosphonate natural product also encoded by the genome of the phylogenetically distant X. vasicola pv. vasculorum. Availability of this novel genome sequence may facilitate the study of interactions between xanthomonads and sugarcane, a host-pathogen system that appears to have evolved several times independently within the genus Xanthomonas and may also provide a source of target sequences for molecular detection and diagnostics. We present the genome sequence of a bacterial agent of gumming disease of sugarcane.
Abstract.
Wasukira A, Coulter M, Al-Sowayeh N, Thwaites R, Paszkiewicz K, Kubiriba J, Smith J, Grant M, Studholme D (2014). Genome Sequencing of Xanthomonas vasicola Pathovar vasculorum Reveals Variation in Plasmids and Genes Encoding Lipopolysaccharide Synthesis, Type-IV Pilus and Type-III Secretion Effectors.
Pathogens,
3, 211-237.
Abstract:
Genome Sequencing of Xanthomonas vasicola Pathovar vasculorum Reveals Variation in Plasmids and Genes Encoding Lipopolysaccharide Synthesis, Type-IV Pilus and Type-III Secretion Effectors
Xanthomonas vasicola pathovar vasculorum (Xvv) is the bacterial agent causing gumming disease in sugarcane. Here, we compare complete genome sequences for five isolates of Xvv originating from sugarcane and one from maize. This identified two distinct types of lipopolysaccharide synthesis gene clusters among Xvv isolates: one is similar to that of Xanthomonas axonopodis pathovar citri (Xac) and is probably the ancestral type, while the other is similar to those of the sugarcane-inhabiting species, Xanthomonas sacchari. Four of six Xvv isolates harboured sequences similar to the Xac plasmid, pXAC47, and showed a distinct Type-IV pilus (T4P) sequence type, whereas the T4P locus of the other two isolates resembled that of the closely related banana pathogen, Xanthomonas campestris pathovar musacearum (Xcm). The Xvv isolate from maize has lost a gene encoding a homologue of the virulence effector, xopAF, which was present in all five of the sugarcane isolates, while xopL contained a premature stop codon in four out of six isolates. These findings shed new light on evolutionary events since the divergence of Xvv and Xcm, as well as further elucidating the relationships between the two closely related pathogens.
Abstract.
Harrison JW, Dung TTN, Siddiqui F, Korbrisate S, Bukhari H, Tra MPV, Hoang NVM, Carrique-Mas J, Bryant J, Campbell JI, et al (2014). Identification of possible virulence marker from Campylobacter jejuni isolates.
Emerg Infect Dis,
20(6), 1026-1029.
Abstract:
Identification of possible virulence marker from Campylobacter jejuni isolates.
A novel protein translocation system, the type-6 secretion system (T6SS), may play a role in virulence of Campylobacter jejuni. We investigated 181 C. jejuni isolates from humans, chickens, and environmental sources in Vietnam, Thailand, Pakistan, and the United Kingdom for T6SS. The marker was most prevalent in human and chicken isolates from Vietnam.
Abstract.
Author URL.
Harrison J, Studholme DJ (2014). Recently published Streptomyces genome sequences.
Microb Biotechnol,
7(5), 373-380.
Author URL.
Rhodes G, Bosma H, Studholme D, Arnold DL, Jackson RW, Pickup RW (2014). The rulB gene of plasmid pWW0 is a hotspot for the site-specific insertion of integron-like elements found in the chromosomes of environmental Pseudomonas fluorescens group bacteria.
Environmental Microbiology,
16(8), 2374-2388.
Abstract:
The rulB gene of plasmid pWW0 is a hotspot for the site-specific insertion of integron-like elements found in the chromosomes of environmental Pseudomonas fluorescens group bacteria
Summary: the rulAB operon of Pseudomonas spp. confers fitness traits on the host and has been suggested to be a hotspot for insertion of mobile elements that carry avirulence genes. Here, for the first time, we show that rulB on plasmid pWW0 is a hotspot for the active site-specific integration of related integron-like elements (ILEs) found in six environmental pseudomonads (strains FH1-FH6). Integration into rulB on pWW0 occurred at position 6488 generating a 3bp direct repeat. ILEs from FH1 and FH5 were 9403bp in length and contained eight open reading frames (ORFs), while the ILE from FH4 was 16233bp in length and contained 16 ORFs. In all three ILEs, the first 5.1kb (containing ORFs 1-4) were structurally conserved and contained three predicted site-specific recombinases/integrases and a tetR homologue. Downstream of these resided ORFs of the 'variable side' with structural and sequence similarity to those encoding survival traits on the fitness enhancing plasmid pGRT1 (ILEFH1 and ILEFH5) and the NR-II virulence region of genomic island PAGI-5 (ILEFH4). Collectively, these ILEs share features with the previously described type III protein secretion system effector ILEs and are considered important to host survival and transfer of fitness enhancing and (a)virulence genes between bacteria. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.
Abstract.
2013
Huggett JF, Laver T, Tamisak S, Nixon G, O'Sullivan DM, Elaswarapu R, Studholme DJ, Foy CA (2013). Considerations for the development and application of control materials to improve metagenomic microbial community profiling.
Accreditation and Quality Assurance,
18(2), 77-83.
Abstract:
Considerations for the development and application of control materials to improve metagenomic microbial community profiling
Advances in DNA sequencing technology provide the possibility to analyse and characterize the genetic material from microbial populations (the microbiome) as a whole. Such comprehensive analysis of a microbiome using these 'metagenomic' approaches offers the potential to understand industrial, clinical and environmental microbiology to a level of detail that is unfeasible using conventional molecular or culture-based methods. However, the complexity offered by metagenomic analysis is also the weakness of this method and poses considerable challenges during analytical standardisation. In this manuscript, we discuss options for developing control materials for metagenomic analysis and describe our preliminary work investigating how such materials can be used to assist metagenomic measurements. The control materials we have developed demonstrate that, when performing 16S rDNA sequencing, different library preparation methods (incorporating adapters before and after the PCR) and small primer mismatches can alter the reported metagenomic profile. These findings illustrate that metagenomic analysis can be heavily biased by the choice of method and underpin the need for control materials that can provide a useful tool in informing choice of protocol for accurate analysis. © 2012 the Author(s).
Abstract.
Passos da Silva D, Devescovi G, Paszkiewicz K, Moretti C, Buonaurio R, Studholme DJ, Venturi V (2013). Draft Genome Sequence of Erwinia toletana, a Bacterium Associated with Olive Knots Caused by Pseudomonas savastanoi pv. Savastanoi.
Genome Announc,
1(3).
Abstract:
Draft Genome Sequence of Erwinia toletana, a Bacterium Associated with Olive Knots Caused by Pseudomonas savastanoi pv. Savastanoi.
Erwinia toletana was first reported in 2004 as a bacterial species isolated from olive knots caused by the plant bacterium Pseudomonas savastanoi pv. savastanoi. Recent studies have shown that the presence of this bacterium in the olive knot environment increases the virulence of the disease, indicating possible interspecies interactions with P. savastanoi pv. savastanoi. Here, we report the first draft genome sequence of an E. toletana strain.
Abstract.
Author URL.
Khan A, Asif H, Studholme DJ, Khan IA, Azim MK (2013). Genome characterization of a novel Burkholderia cepacia complex genomovar isolated from dieback affected mango orchards.
World J Microbiol Biotechnol,
29(11), 2033-2044.
Abstract:
Genome characterization of a novel Burkholderia cepacia complex genomovar isolated from dieback affected mango orchards.
We characterized the genome of the antibiotic resistant, caseinolytic and non-hemolytic Burkholderia sp. strain TJI49, isolated from mango trees (Mangifera indica L.) with dieback disease. This isolate produced severe disease symptoms on the indicator plants. Next generation DNA sequencing and short-read assembly generated the 60X deep 7,631,934 nucleotide draft genome of Burkholderia sp. TJI49 which comprised three chromosomes and at least one mega plasmid. Genome annotation studies revealed a total 8,992 genes, out of which 8,940 were protein coding genes. Comparative genomics and phylogenetics identified Burkholderia sp. TJI49 as a distinct species of Burkholderia cepacia complex (BCC), closely related to B. multivorans ATCC17616. Genome-wide sequence alignment of this isolate with replicons of BCC members showed conservation of core function genes but considerable variations in accessory genes. Subsystem-based gene annotation identified the active presence of wide spread colonization island and type VI secretion system in Burkholderia sp. TJI49. Sequence comparisons revealed (a) 28 novel ORFs that have no database matches and (b) 23 ORFs with orthologues in species other than Burkholderia, indicating horizontal gene transfer events. Fold recognition of novel ORFs identified genes encoding pertactin autotransporter-like proteins (a constituent of type V secretion system) and Hap adhesion-like proteins (involved in cell-cell adhesion) in the genome of Burkholderia sp. TJI49. The genomic characterization of this isolate provided additional information related to the 'pan-genome' of Burkholderia species.
Abstract.
Author URL.
Quinn L, O'Neill PA, Harrison J, Paskiewicz KH, Mccracken AR, Cooke LR, Grant MR, Studholme DJ (2013). Genome-wide sequencing of Phytophthora lateralis reveals genetic variation among isolates from Lawson cypress (Chamaecyparis lawsoniana) in Northern Ireland.
FEMS Microbiology Letters,
344(2), 179-185.
Abstract:
Genome-wide sequencing of Phytophthora lateralis reveals genetic variation among isolates from Lawson cypress (Chamaecyparis lawsoniana) in Northern Ireland
Phytophthora lateralis is a fungus-like (oomycete) pathogen of trees in the family Cupressaceae, including Chamaecyparis lawsoniana (Lawson cypress or Port Orford cedar). Known in North America since the 1920s, presumably having been accidentally introduced from its assumed East Asian centre of origin, until recently, this pathogen has not been identified causing disease in Europe except for a few isolated outbreaks. However, since 2010, there have been several reports of infection of C. lawsoniana by P. lateralis in the United Kingdom, including Northern Ireland. We sequenced the genomes of four isolates of P. lateralis from two sites in Northern Ireland in 2011. Comparison with the closely related tree and shrub pathogen P. ramorum (cause of ramorum disease of larch and other species in the UK) shows that P. lateralis shares 91.47% nucleotide sequence identity over the core conserved compartments of the genome. The genomes of the four Northern Ireland isolates are almost identical, but we identified several single-nucleotide polymorphisms (SNPs) that distinguish between isolates, thereby presenting potential molecular markers of use for tracking routes of spread and in epidemiological studies. Our data reveal very low rates of heterozygosity (compared with P. ramorum), consistent with inbreeding within this P. lateralis population. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd.
Abstract.
Quinn L, O'Neill PA, Harrison J, Paskiewicz KH, McCracken AR, Cooke LR, Grant MR, Studholme DJ (2013). Genome-wide sequencing of Phytophthora lateralis reveals genetic variation among isolates from Lawson cypress (Chamaecyparis lawsoniana) in Northern Ireland.
FEMS Microbiol Lett,
344(2), 179-185.
Abstract:
Genome-wide sequencing of Phytophthora lateralis reveals genetic variation among isolates from Lawson cypress (Chamaecyparis lawsoniana) in Northern Ireland.
Phytophthora lateralis is a fungus-like (oomycete) pathogen of trees in the family Cupressaceae, including Chamaecyparis lawsoniana (Lawson cypress or Port Orford cedar). Known in North America since the 1920s, presumably having been accidentally introduced from its assumed East Asian centre of origin, until recently, this pathogen has not been identified causing disease in Europe except for a few isolated outbreaks. However, since 2010, there have been several reports of infection of C. lawsoniana by P. lateralis in the United Kingdom, including Northern Ireland. We sequenced the genomes of four isolates of P. lateralis from two sites in Northern Ireland in 2011. Comparison with the closely related tree and shrub pathogen P. ramorum (cause of ramorum disease of larch and other species in the UK) shows that P. lateralis shares 91.47% nucleotide sequence identity over the core conserved compartments of the genome. The genomes of the four Northern Ireland isolates are almost identical, but we identified several single-nucleotide polymorphisms (SNPs) that distinguish between isolates, thereby presenting potential molecular markers of use for tracking routes of spread and in epidemiological studies. Our data reveal very low rates of heterozygosity (compared with P. ramorum), consistent with inbreeding within this P. lateralis population.
Abstract.
Author URL.
Studholme D, Harris B, Le Cocq K, Winsbury R, Perera V, Ryder L, Beale M, Ward J, Thornton CR, Grant M, et al (2013). Investigating the Beneficial Traits of Trichoderma hamatum GD12 for Sustainable Agriculture - Insights from Genomics.
Frontiers in Plant-Microbe Interactions,
4, 1-13.
Abstract:
Investigating the Beneficial Traits of Trichoderma hamatum GD12 for Sustainable Agriculture - Insights from Genomics
Trichoderma hamatum strain GD12 is unique in that it can promote plant growth, activate biocontrol against pre- and post-emergence soil pathogens and can induce systemic resistance to foliar pathogens. This study extends previous work in lettuce to demonstrate that GD12 can confer beneficial agronomic traits to other plants, providing examples of plant growth promotion in the model dicot, Arabidopsis thaliana and induced foliar resistance to Magnaporthe oryzae in the model monocot rice. We further characterize the lettuce-T. hamatum interaction to show that bran extracts from GD12 and a N-acetyl-β-D-glucosamindase-deficient mutant differentially promote growth in a concentration dependent manner, and these differences correlate with differences in the small molecule secretome. We show that GD12 mycoparasitises a range of isolates of the pre-emergent soil pathogen Sclerotinia sclerotiorum and that this interaction induces a further increase in plant growth promotion above that conferred by GD12. To understand the genetic potential encoded by T. hamatum GD12 and to facilitate its use as a model beneficial to study plant growth promotion, induced systemic resistance and mycoparasitism we present de novo genome sequence data. We compare GD12 with other published Trichoderma genomes and show that T. hamatum GD12 contains unique genomic regions with the potential to encode novel bioactive metabolites that may contribute to GD12’s agrochemically important traits.
Abstract.
Martinez-Argudo I, Veenendaal AKJ, Liu X, Roehrich AD, Ronessen MC, Franzoni G, van Rietschoten KN, Morimoto YV, Saijo-Hamano Y, Avison MB, et al (2013). Isolation of Salmonella mutants resistant to the inhibitory effect of Salicylidene acylhydrazides on flagella-mediated motility.
PLoS One,
8(1).
Abstract:
Isolation of Salmonella mutants resistant to the inhibitory effect of Salicylidene acylhydrazides on flagella-mediated motility.
Salicylidene acylhydrazides identified as inhibitors of virulence-mediating type III secretion systems (T3SSs) potentially target their inner membrane export apparatus. They also lead to inhibition of flagellar T3SS-mediated swimming motility in Salmonella enterica serovar. Typhimurium. We show that INP0404 and INP0405 act by reducing the number of flagella/cell. These molecules still inhibit motility of a Salmonella ΔfliH-fliI-fliJ/flhB((P28T)) strain, which lacks three soluble components of the flagellar T3S apparatus, suggesting that they are not the target of this drug family. We implemented a genetic screen to search for the inhibitors' molecular target(s) using motility assays in the ΔfliH-fliI/flhB((P28T)) background. Both mutants identified were more motile than the background strain in the absence of the drugs, although HM18 was considerably more so. HM18 was more motile than its parent strain in the presence of both drugs while DI15 was only insensitive to INP0405. HM18 was hypermotile due to hyperflagellation, whereas DI15 was not hyperflagellated. HM18 was also resistant to a growth defect induced by high concentrations of the drugs. Whole-genome resequencing of HM18 indicated two alterations within protein coding regions, including one within atpB, which encodes the inner membrane a-subunit of the F(O)F(1)-ATP synthase. Reverse genetics indicated that the alteration in atpB was responsible for all of HM18's phenotypes. Genome sequencing of DI15 uncovered a single A562P mutation within a gene encoding the flagellar inner membrane protein FlhA, the direct role of which in mediating drug insensitivity could not be confirmed. We discuss the implications of these findings in terms of T3SS export apparatus function and drug target identification.
Abstract.
Author URL.
Monteil CL, Cai R, Liu H, Llontop MEM, Leman S, Studholme DJ, Morris CE, Vinatzer BA (2013). Nonagricultural reservoirs contribute to emergence and evolution of Pseudomonas syringae crop pathogens.
New Phytol,
199(3), 800-811.
Abstract:
Nonagricultural reservoirs contribute to emergence and evolution of Pseudomonas syringae crop pathogens.
While the existence of environmental reservoirs of human pathogens is well established, less is known about the role of nonagricultural environments in emergence, evolution, and spread of crop pathogens. Here, we analyzed phylogeny, virulence genes, host range, and aggressiveness of Pseudomonas syringae strains closely related to the tomato pathogen P. syringae pv. tomato (Pto), including strains isolated from snowpack and streams. The population of Pto relatives in nonagricultural environments was estimated to be large and its diversity to be higher than that of the population of Pto and its relatives on crops. Ancestors of environmental strains, Pto, and other genetically monomorphic crop pathogens were inferred to have frequently recombined, suggesting an epidemic population structure for P. syringae. Some environmental strains have repertoires of type III-secreted effectors very similar to Pto, are almost as aggressive on tomato as Pto, but have a wider host range than typical Pto strains. We conclude that crop pathogens may have evolved through a small number of evolutionary events from a population of less aggressive ancestors with a wider host range present in nonagricultural environments.
Abstract.
Author URL.
Huggett JF, Studholme DJ, Laver T, Foy CA (2013). Progress in metagenomics requires a balanced appraisal of the available technologies. European Journal of Clinical Microbiology and Infectious Diseases, 32(8), 1097-1098.
Huggett JF, Studholme DJ, Laver T, Foy CA (2013). Progress in metagenomics requires a balanced appraisal of the available technologies.
Eur J Clin Microbiol Infect Dis,
32(8), 1097-1098.
Author URL.
Burlinson P, Studholme D, Cambray-Young J, Heavens D, Rathjen J, Hodgkin J, Preston GM (2013). Pseudomonas fluorescens NZI7 repels grazing by C. elegans, a natural predator.
ISME J,
7(6), 1126-1138.
Abstract:
Pseudomonas fluorescens NZI7 repels grazing by C. elegans, a natural predator.
The bacteriovorous nematode Caenorhabditis elegans has been used to investigate many aspects of animal biology, including interactions with pathogenic bacteria. However, studies examining C. elegans interactions with bacteria isolated from environments in which it is found naturally are relatively scarce. C. elegans is frequently associated with cultivation of the edible mushroom Agaricus bisporus, and has been reported to increase the severity of bacterial blotch of mushrooms, a disease caused by bacteria from the Pseudomonas fluorescens complex. We observed that pseudomonads isolated from mushroom farms showed differential resistance to nematode predation. Under nutrient poor conditions, in which most pseudomonads were consumed, the mushroom pathogenic isolate P. fluorescens NZI7 was able to repel C. elegans without causing nematode death. A draft genome sequence of NZI7 showed it to be related to the biocontrol strain P. protegens Pf-5. To identify the genetic basis of nematode repellence in NZI7, we developed a grid-based screen for mutants that lacked the ability to repel C. elegans. The mutants isolated in this screen included strains with insertions in the global regulator GacS and in a previously undescribed GacS-regulated gene cluster, 'EDB' ('edible'). Our results suggest that the product of the EDB cluster is a poorly diffusible or cell-associated factor that acts together with other features of NZI7 to provide a novel mechanism to deter nematode grazing. As nematodes interact with NZI7 colonies before being repelled, the EDB factor may enable NZI7 to come into contact with and be disseminated by C. elegans without being subject to intensive predation.
Abstract.
Author URL.
Van Immerseel F, Studholme DJ, Eeckhaut V, Heyndrickx M, Dewulf J, Dewaele I, Van Hoorebeke S, Haesebrouck F, Van Meirhaeghe H, Ducatelle R, et al (2013). Salmonella Gallinarum field isolates from laying hens are related to the vaccine strain SG9R.
Vaccine,
31(43), 4940-4945.
Abstract:
Salmonella Gallinarum field isolates from laying hens are related to the vaccine strain SG9R.
Salmonella enterica subspecies enterica serotype Gallinarum can cause severe systemic disease in chickens and a live Salmonella Gallinarum 9R vaccine (SG9R) has been used widely to control disease. Using whole-genome sequencing we found point mutations in the pyruvate dehydrogenase (aceE) and/or lipopolysaccharide 1,2-glucosyltransferase (rfaJ) genes that likely explain the attenuation of the SG9R vaccine strain. Molecular typing using Pulsed Field Gel Electrophoresis and Multiple-Locus Variable number of tandem repeat Analysis showed that strains isolated from different layer flocks in multiple countries and the SG9R vaccine strain were similar. The genome of one Salmonella Gallinarum field strain, isolated from a flock with a mortality peak and selected on the basis of identical PFGE and MLVA patterns with SG9R, was sequenced. We found 9 non-silent single-nucleotide differences distinguishing the field strain from the SG9R vaccine strain. Our data show that a Salmonella Gallinarum field strain isolated from laying hens is almost identical to the SG9R vaccine. Mutations in the aceE and rfaJ genes could explain the reversion to a more virulent phenotype. Our results highlight the importance of using well defined gene deletion mutants as vaccines.
Abstract.
Author URL.
Farrer RA, Henk DA, MacLean D, Studholme DJ, Fisher MC (2013). Using false discovery rates to benchmark SNP-callers in next-generation sequencing projects.
Sci Rep,
3Abstract:
Using false discovery rates to benchmark SNP-callers in next-generation sequencing projects.
Sequence alignments form the basis for many comparative and population genomic studies. Alignment tools provide a range of accuracies dependent on the divergence between the sequences and the alignment methods. Despite widespread use, there is no standard method for assessing the accuracy of a dataset and alignment strategy after resequencing. We present a framework and tool for determining the overall accuracies of an input read dataset, alignment and SNP-calling method providing an isolate in that dataset has a corresponding, or closely related reference sequence available. In addition to this tool for comparing False Discovery Rates (FDR), we include a method for determining homozygous and heterozygous positions from an alignment using binomial probabilities for an expected error rate. We benchmark this method against other SNP callers using our FDR method with three fungal genomes, finding that it was able achieve a high level of accuracy. These tools are available at http://cfdr.sourceforge.net/.
Abstract.
Author URL.
2012
Azam S, Thakur V, Ruperao P, Shah T, Balaji J, Amindala B, Farmer AD, Studholme DJ, May GD, Edwards D, et al (2012). Coverage-based consensus calling (CbCC) of short sequence reads and comparison of CbCC results to identify SNPs in chickpea (Cicer arietinum; Fabaceae), a crop species without a reference genome.
Am J Bot,
99(2), 186-192.
Abstract:
Coverage-based consensus calling (CbCC) of short sequence reads and comparison of CbCC results to identify SNPs in chickpea (Cicer arietinum; Fabaceae), a crop species without a reference genome.
PREMISE OF THE STUDY: Next-generation sequencing (NGS) technologies are frequently used for resequencing and mining of single nucleotide polymorphisms (SNPs) by comparison to a reference genome. In crop species such as chickpea (Cicer arietinum) that lack a reference genome sequence, NGS-based SNP discovery is a challenge. Therefore, unlike probability-based statistical approaches for consensus calling and by comparison with a reference sequence, a coverage-based consensus calling (CbCC) approach was applied and two genotypes were compared for SNP identification. METHODS: a CbCC approach is used in this study with four commonly used short read alignment tools (Maq, Bowtie, Novoalign, and SOAP2) and 15.7 and 22.1 million Illumina reads for chickpea genotypes ICC4958 and ICC1882, together with the chickpea trancriptome assembly (CaTA). KEY RESULTS: a nonredundant set of 4543 SNPs was identified between two chickpea genotypes. Experimental validation of 224 randomly selected SNPs showed superiority of Maq among individual tools, as 50.0% of SNPs predicted by Maq were true SNPs. For combinations of two tools, greatest accuracy (55.7%) was reported for Maq and Bowtie, with a combination of Bowtie, Maq, and Novoalign identifying 61.5% true SNPs. SNP prediction accuracy generally increased with increasing reads depth. CONCLUSIONS: This study provides a benchmark comparison of tools as well as read depths for four commonly used tools for NGS SNP discovery in a crop species without a reference genome sequence. In addition, a large number of SNPs have been identified in chickpea that would be useful for molecular breeding.
Abstract.
Author URL.
Studholme DJ (2012). Deep sequencing of small RNAs in plants: applied bioinformatics.
Brief Funct Genomics,
11(1), 71-85.
Abstract:
Deep sequencing of small RNAs in plants: applied bioinformatics.
Small RNAs, including microRNA and short-interfering RNAs, play important roles in plants. In recent years, developments in sequencing technology have enabled the large-scale discovery of sRNAs in various cells, tissues and developmental stages and in response to various stresses. This review describes the bioinformatics challenges to analysing these large datasets of short-RNA sequences and some of the solutions to those challenges.
Abstract.
Author URL.
Patel HK, da Silva DP, Devescovi G, Maraite H, Paszkiewicz K, Studholme DJ, Venturi V (2012). Draft genome sequence of Pseudomonas fuscovaginae, a broad-host-range pathogen of plants.
J Bacteriol,
194(10), 2765-2766.
Abstract:
Draft genome sequence of Pseudomonas fuscovaginae, a broad-host-range pathogen of plants.
Pseudomonas fuscovaginae was first reported as a pathogen of rice causing sheath rot in plants grown at high altitudes. P. fuscovaginae is now considered a broad-host-range plant pathogen causing disease in several economically important plants. We report what is, to our knowledge, the first draft genome sequence of a P. fuscovaginae strain.
Abstract.
Author URL.
Sohn KH, Jones JDG, Studholme DJ (2012). Draft genome sequence of Pseudomonas syringae pathovar syringae strain FF5, causal agent of stem tip dieback disease on ornamental pear.
J Bacteriol,
194(14), 3733-3734.
Abstract:
Draft genome sequence of Pseudomonas syringae pathovar syringae strain FF5, causal agent of stem tip dieback disease on ornamental pear.
Pseudomonas syringae FF5 causes stem tip dieback disease on ornamental pear (Pyrus calleryana). Its genome encodes a complete type III secretion system (T3SS) and HopAC1, HopM1, AvrE1, HopI1, HopAA1, HopJ1, HopAH2, HopAH1, HopAG1, and HopAZ1. Lacking detectable homologues of other T3SS effectors, it may encode novel, undiscovered effectors.
Abstract.
Author URL.
Wasukira A, Tayebwa J, Thwaites R, Paszkiewicz K, Aritua V, Kubiriba J, Smith J, Grant M, Studholme DJ (2012). Genome-wide sequencing reveals two major sub-lineages in the genetically monomorphic pathogen Xanthomonas campestris pathovar musacearum.
Genes,
3(3), 361-377.
Abstract:
Genome-wide sequencing reveals two major sub-lineages in the genetically monomorphic pathogen Xanthomonas campestris pathovar musacearum
The bacterium Xanthomonas campestris pathovar musacearum (Xcm) is the causal agent of banana Xanthomonas wilt (BXW). This disease has devastated economies based on banana and plantain crops (Musa species) in East Africa. Here we use genome-wide sequencing to discover a set of single-nucleotide polymorphisms (SNPs) among East African isolates of Xcm. These SNPs have potential as molecular markers for phylogeographic studies of the epidemiology and spread of the pathogen. Our analysis reveals two major sub-lineages of the pathogen, suggesting that the current outbreaks of BXW on Musa species in the region may have more than one introductory event, perhaps from Ethiopia. Also, based on comparisons of genome-wide sequence data from multiple isolates of Xcm and multiple strains of X. vasicola pathovar vasculorum, we identify genes specific to Xcm that could be used to specifically detect Xcm by PCR-based methods. © 2012 by the authors; licensee MDPI, Basel, Switzerland.
Abstract.
Paszkiewicz K, Studholme DJ (2012). High-throughput sequencing data analysis software: Current state and future developments. In (Ed)
Bioinformatics for High Throughput Sequencing, 231-248.
Abstract:
High-throughput sequencing data analysis software: Current state and future developments
Abstract.
Mazzaglia A, Studholme DJ, Taratufolo MC, Cai R, Almeida NF, Goodman T, Guttman DS, Vinatzer BA, Balestra GM (2012). Pseudomonas syringae pv. actinidiae (PSA) isolates from recent bacterial canker of kiwifruit outbreaks belong to the same genetic lineage.
PLoS One,
7(5).
Abstract:
Pseudomonas syringae pv. actinidiae (PSA) isolates from recent bacterial canker of kiwifruit outbreaks belong to the same genetic lineage.
Intercontinental spread of emerging plant diseases is one of the most serious threats to world agriculture. One emerging disease is bacterial canker of kiwi fruit (Actinidia deliciosa and A. chinensis) caused by Pseudomonas syringae pv. actinidiae (PSA). The disease first occurred in China and Japan in the 1980s and in Korea and Italy in the 1990s. A more severe form of the disease broke out in Italy in 2008 and in additional countries in 2010 and 2011 threatening the viability of the global kiwi fruit industry. To start investigating the source and routes of international transmission of PSA, genomes of strains from China (the country of origin of the genus Actinidia), Japan, Korea, Italy and Portugal have been sequenced. Strains from China, Italy, and Portugal have been found to belong to the same clonal lineage with only 6 single nucleotide polymorphisms (SNPs) in 3,453,192 bp and one genomic island distinguishing the Chinese strains from the European strains. Not more than two SNPs distinguish each of the Italian and Portuguese strains from each other. The Japanese and Korean strains belong to a separate genetic lineage as previously reported. Analysis of additional European isolates and of New Zealand isolates exploiting genome-derived markers showed that these strains belong to the same lineage as the Italian and Chinese strains. Interestingly, the analyzed New Zealand strains are identical to European strains at the tested SNP loci but test positive for the genomic island present in the sequenced Chinese strains and negative for the genomic island present in the European strains. Results are interpreted in regard to the possible direction of movement of the pathogen between countries and suggest a possible Chinese origin of the European and New Zealand outbreaks.
Abstract.
Author URL.
Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, Pang N, Forslund K, Ceric G, Clements J, et al (2012). The Pfam protein families database.
NUCLEIC ACIDS RESEARCH,
40(D1), D290-D301.
Author URL.
2011
Studholme DJ, Glover RH, Boonham N (2011). Application of high-throughput DNA sequencing in phytopathology.
Annu Rev Phytopathol,
49, 87-105.
Abstract:
Application of high-throughput DNA sequencing in phytopathology.
The new sequencing technologies are already making a big impact in academic research on medically important microbes and may soon revolutionize diagnostics, epidemiology, and infection control. Plant pathology also stands to gain from exploiting these opportunities. This manuscript reviews some applications of these high-throughput sequencing methods that are relevant to phytopathology, with emphasis on the associated computational and bioinformatics challenges and their solutions. Second-generation sequencing technologies have recently been exploited in genomics of both prokaryotic and eukaryotic plant pathogens. They are also proving to be useful in diagnostics, especially with respect to viruses.
Abstract.
Author URL.
Studholme DJ (2011). Application of high-throughput genome sequencing to intrapathovar variation in Pseudomonas syringae.
Mol Plant Pathol,
12(8), 829-838.
Abstract:
Application of high-throughput genome sequencing to intrapathovar variation in Pseudomonas syringae.
One reason for the success of Pseudomonas syringae as a model pathogen has been the availability of three complete genome sequences since 2005. Now, at the beginning of 2011, more than 25 strains of P. syringae have been sequenced and many more will soon be released. To date, published analyses of P. syringae have been largely descriptive, focusing on catalogues of genetic differences among strains and between species. Numerous powerful statistical tools are now available that have yet to be applied to P. syringae genomic data for robust and quantitative reconstruction of evolutionary events. The aim of this review is to provide a snapshot of the current status of P. syringae genome sequence data resources, including very recent and unpublished studies, and thereby demonstrate the richness of resources available for this species. Furthermore, certain specific opportunities and challenges in making the best use of these data resources are highlighted.
Abstract.
Author URL.
Studholme DJ, Wasukira A, Paszkiewicz K, Aritua V, Thwaites R, Smith J, Grant M (2011). Draft genome sequences of xanthomonas sacchari and two banana-associated xanthomonads reveal insights into the xanthomonas group 1 clade.
Genes,
2(4), 1050-1065.
Abstract:
Draft genome sequences of xanthomonas sacchari and two banana-associated xanthomonads reveal insights into the xanthomonas group 1 clade
We present draft genome sequences for three strains of Xanthomonas species, each of which was associated with banana plants (Musa species) but is not closely related to the previously sequenced banana-pathogen Xanthomonas campestris pathovar musacearum. Strain NCPPB4393 had been deposited as Xanthomonas campestris pathovar musacearum but in fact falls within the species Xanthomonas sacchari. Strain NCPPB1132 is more distantly rel ted to Xanthomonas sacchari whilst strain NCPPB 1131 grouped in a distinct species-level clade related to X. sacchari, along with strains from ginger, rice, cotton and sugarcane. These three newly sequenced strains share many genomic features with the previously sequenced Xanthomonas albilineans, for example possessing an unsual metE allele and lacking the Hrp type III secretion system. However, they are distinct from Xanthomonas albilineans in many respects, for example showing little evidence of genome reduction. They also lack the SPI-1 type III secretion system found in Xanthomonas albilineans. Unlike X. albilineans, all three strains possess a gum gene cluster. The data reported here provide the first genome-wide survey of non-Hrp Xanthomonas species other than Xanthomonas albilineans, which is an atypical member of this group. We hope that the availability of complete sequence data for this group of organisms is the first step towards understanding their interactions with plants and identifying potential virulence factors. © 2011 by the authors; licensee MDPI, Basel, Switzerland.
Abstract.
Kemen E, Gardiner A, Schultz-Larsen T, Kemen AC, Balmuth AL, Robert-Seilaniantz A, Bailey K, Holub E, Studholme DJ, Maclean D, et al (2011). Gene gain and loss during evolution of obligate parasitism in the white rust pathogen of Arabidopsis thaliana.
PLoS Biol,
9(7).
Abstract:
Gene gain and loss during evolution of obligate parasitism in the white rust pathogen of Arabidopsis thaliana.
Biotrophic eukaryotic plant pathogens require a living host for their growth and form an intimate haustorial interface with parasitized cells. Evolution to biotrophy occurred independently in fungal rusts and powdery mildews, and in oomycete white rusts and downy mildews. Biotroph evolution and molecular mechanisms of biotrophy are poorly understood. It has been proposed, but not shown, that obligate biotrophy results from (i) reduced selection for maintenance of biosynthetic pathways and (ii) gain of mechanisms to evade host recognition or suppress host defence. Here we use Illumina sequencing to define the genome, transcriptome, and gene models for the obligate biotroph oomycete and Arabidopsis parasite, Albugo laibachii. A. laibachii is a member of the Chromalveolata, which incorporates Heterokonts (containing the oomycetes), Apicomplexa (which includes human parasites like Plasmodium falciparum and Toxoplasma gondii), and four other taxa. From comparisons with other oomycete plant pathogens and other chromalveolates, we reveal independent loss of molybdenum-cofactor-requiring enzymes in downy mildews, white rusts, and the malaria parasite P. falciparum. Biotrophy also requires "effectors" to suppress host defence; we reveal RXLR and Crinkler effectors shared with other oomycetes, and also discover and verify a novel class of effectors, the "CHXCs", by showing effector delivery and effector functionality. Our findings suggest that evolution to progressively more intimate association between host and parasite results in reduced selection for retention of certain biosynthetic pathways, and particularly reduced selection for retention of molybdopterin-requiring biosynthetic pathways. These mechanisms are not only relevant to plant pathogenic oomycetes but also to human pathogens within the Chromalveolata.
Abstract.
Author URL.
Paszkiewicz K, Studholme DJ (2011). High-Throughput Sequencing Data Analysis Software: Current State and Future Developments. In (Ed) Bioinformatics for High Throughput Sequencing, Springer New York, 231-248.
Fabro G, Steinbrenner J, Coates M, Ishaque N, Baxter L, Studholme DJ, Körner E, Allen RL, Piquerez SJM, Rougon-Cardoso A, et al (2011). Multiple candidate effectors from the oomycete pathogen Hyaloperonospora arabidopsidis suppress host plant immunity.
PLoS Pathog,
7(11).
Abstract:
Multiple candidate effectors from the oomycete pathogen Hyaloperonospora arabidopsidis suppress host plant immunity.
Oomycete pathogens cause diverse plant diseases. To successfully colonize their hosts, they deliver a suite of effector proteins that can attenuate plant defenses. In the oomycete downy mildews, effectors carry a signal peptide and an RxLR motif. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on the model plant Arabidopsis thaliana (Arabidopsis). We investigated if candidate effectors predicted in the genome sequence of Hpa isolate Emoy2 (HaRxLs) were able to manipulate host defenses in different Arabidopsis accessions. We developed a rapid and sensitive screening method to test HaRxLs by delivering them via the bacterial type-three secretion system (TTSS) of Pseudomonas syringae pv tomato DC3000-LUX (Pst-LUX) and assessing changes in Pst-LUX growth in planta on 12 Arabidopsis accessions. The majority (~70%) of the 64 candidates tested positively contributed to Pst-LUX growth on more than one accession indicating that Hpa virulence likely involves multiple effectors with weak accession-specific effects. Further screening with a Pst mutant (ΔCEL) showed that HaRxLs that allow enhanced Pst-LUX growth usually suppress callose deposition, a hallmark of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We found that HaRxLs are rarely strong avirulence determinants. Although some decreased Pst-LUX growth in particular accessions, none activated macroscopic cell death. Fewer HaRxLs conferred enhanced Pst growth on turnip, a non-host for Hpa, while several reduced it, consistent with the idea that turnip's non-host resistance against Hpa could involve a combination of recognized HaRxLs and ineffective HaRxLs. We verified our results by constitutively expressing in Arabidopsis a sub-set of HaRxLs. Several transgenic lines showed increased susceptibility to Hpa and attenuation of Arabidopsis PTI responses, confirming the HaRxLs' role in Hpa virulence. This study shows TTSS screening system provides a useful tool to test whether candidate effectors from eukaryotic pathogens can suppress/trigger plant defense mechanisms and to rank their effectiveness prior to subsequent mechanistic investigation.
Abstract.
Author URL.
Cai R, Lewis J, Yan S, Liu H, Clarke CR, Campanile F, Almeida NF, Studholme DJ, Lindeberg M, Schneider D, et al (2011). The plant pathogen Pseudomonas syringae pv. tomato is genetically monomorphic and under strong selection to evade tomato immunity.
PLoS Pathog,
7(8).
Abstract:
The plant pathogen Pseudomonas syringae pv. tomato is genetically monomorphic and under strong selection to evade tomato immunity.
Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today's Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain.
Abstract.
Author URL.
2010
MacLean D, Studholme DJ (2010). A Boolean model of the Pseudomonas syringae hrp regulon predicts a tightly regulated system.
PLoS One,
5(2).
Abstract:
A Boolean model of the Pseudomonas syringae hrp regulon predicts a tightly regulated system.
The Type III secretion system (TTSS) is a protein secretion machinery used by certain gram-negative bacterial pathogens of plants and animals to deliver effector molecules to the host and is at the core of the ability to cause disease. Extensive molecular and biochemical study has revealed the components and their interactions within this system but reductive approaches do not consider the dynamical properties of the system as a whole. In order to gain a better understanding of these dynamical behaviours and to create a basis for the refinement of the experimentally derived knowledge we created a Boolean model of the regulatory interactions within the hrp regulon of Pseudomonas syringae pathovar tomato strain DC3000 Pseudomonas syringae. We compared simulations of the model with experimental data and found them to be largely in accordance, though the hrpV node shows some differences in state changes to that expected. Our simulations also revealed interesting dynamical properties not previously predicted. The model predicts that the hrp regulon is a biologically stable two-state system, with each of the stable states being strongly attractive, a feature indicative of selection for a tightly regulated and responsive system. The model predicts that the state of the GacS/GacA node confers control, a prediction that is consistent with experimental observations that the protein has a role as master regulator. Simulated gene "knock out" experiments with the model predict that HrpL is a central information processing point within the network.
Abstract.
Author URL.
Green S, Studholme DJ, Laue BE, Dorati F, Lovell H, Arnold D, Cottrell JE, Bridgett S, Blaxter M, Huitema E, et al (2010). Comparative genome analysis provides insights into the evolution and adaptation of Pseudomonas syringae pv. aesculi on Aesculus hippocastanum.
PLoS One,
5(4).
Abstract:
Comparative genome analysis provides insights into the evolution and adaptation of Pseudomonas syringae pv. aesculi on Aesculus hippocastanum.
A recently emerging bleeding canker disease, caused by Pseudomonas syringae pathovar aesculi (Pae), is threatening European horse chestnut in northwest Europe. Very little is known about the origin and biology of this new disease. We used the nucleotide sequences of seven commonly used marker genes to investigate the phylogeny of three strains isolated recently from bleeding stem cankers on European horse chestnut in Britain (E-Pae). On the basis of these sequences alone, the E-Pae strains were identical to the Pae type-strain (I-Pae), isolated from leaf spots on Indian horse chestnut in India in 1969. The phylogenetic analyses also showed that Pae belongs to a distinct clade of P. syringae pathovars adapted to woody hosts. We generated genome-wide Illumina sequence data from the three E-Pae strains and one strain of I-Pae. Comparative genomic analyses revealed pathovar-specific genomic regions in Pae potentially implicated in virulence on a tree host, including genes for the catabolism of plant-derived aromatic compounds and enterobactin synthesis. Several gene clusters displayed intra-pathovar variation, including those encoding type IV secretion, a novel fatty acid biosynthesis pathway and a sucrose uptake pathway. Rates of single nucleotide polymorphisms in the four Pae genomes indicate that the three E-Pae strains diverged from each other much more recently than they diverged from I-Pae. The very low genetic diversity among the three geographically distinct E-Pae strains suggests that they originate from a single, recent introduction into Britain, thus highlighting the serious environmental risks posed by the spread of an exotic plant pathogenic bacterium to a new geographic location. The genomic regions in Pae that are absent from other P. syringae pathovars that infect herbaceous hosts may represent candidate genetic adaptations to infection of the woody parts of the tree.
Abstract.
Author URL.
Paszkiewicz K, Studholme DJ (2010). De novo assembly of short sequence reads.
Brief Bioinform,
11(5), 457-472.
Abstract:
De novo assembly of short sequence reads.
A new generation of sequencing technologies is revolutionizing molecular biology. Illumina's Solexa and Applied Biosystems' SOLiD generate gigabases of nucleotide sequence per week. However, a perceived limitation of these ultra-high-throughput technologies is their short read-lengths. De novo assembly of sequence reads generated by classical Sanger capillary sequencing is a mature field of research. Unfortunately, the existing sequence assembly programs were not effective for short sequence reads generated by Illumina and SOLiD platforms. Early studies suggested that, in principle, sequence reads as short as 20-30 nucleotides could be used to generate useful assemblies of both prokaryotic and eukaryotic genome sequences, albeit containing many gaps. The early feasibility studies and proofs of principle inspired several bioinformatics research groups to implement new algorithms as freely available software tools specifically aimed at assembling reads of 30-50 nucleotides in length. This has led to the generation of several draft genome sequences based exclusively on short sequence Illumina sequence reads, recently culminating in the assembly of the 2.25-Gb genome of the giant panda from Illumina sequence reads with an average length of just 52 nucleotides. As well as reviewing recent developments in the field, we discuss some practical aspects such as data filtering and submission of assembly data to public repositories.
Abstract.
Author URL.
MacLean D, Elina N, Havecker ER, Heimstaedt SB, Studholme DJ, Baulcombe DC (2010). Evidence for large complex networks of plant short silencing RNAs.
PLoS One,
5(3).
Abstract:
Evidence for large complex networks of plant short silencing RNAs.
BACKGROUND: in plants and animals there are many classes of short RNAs that carry out a wide range of functions within the cell; short silencing RNAs (ssRNAs) of 21-25 nucleotides in length are produced from double-stranded RNA precursors by the protein Dicer and guide nucleases and other proteins to their RNA targets through base pairing interactions. The consequence of this process is degradation of the targeted RNA, suppression of its translation or initiation of secondary ssRNA production. The secondary ssRNAs in turn could then initiate further layers of ssRNA production to form extensive cascades and networks of interacting RNA [1]. Previous empirical analysis in plants established the existence of small secondary ssRNA cascade [2], in which a single instance of this event occurred but it was not known whether there are other more extensive networks of secondary sRNA production. METHODOLOGY/PRINCIPAL FINDINGS: We generated a network by predicting targets of ssRNA populations obtained from high-throughput sequencing experiments. The topology of the network shows it to have power law connectivity distribution, to be dissortative, highly clustered and composed of multiple components. We also identify protein families, PPR and ULP1, that act as hubs within the network. Comparison of the repetition of genomic sub-sequences of ssRNA length between Arabidopsis and E.coli suggest that the network structure is made possible by the underlying repetitiveness in the genome sequence. CONCLUSIONS/SIGNIFICANCE: Together our results provide good evidence for the existence of a large, robust ssRNA interaction network with distinct regulatory function. Such a network could have a massive effect on the regulation of gene expression via mediation of transcript levels.
Abstract.
Author URL.
MacLean D, Moulton V, Studholme DJ (2010). Finding sRNA generative locales from high-throughput sequencing data with NiBLS.
BMC Bioinformatics,
11Abstract:
Finding sRNA generative locales from high-throughput sequencing data with NiBLS.
BACKGROUND: Next-generation sequencing technologies allow researchers to obtain millions of sequence reads in a single experiment. One important use of the technology is the sequencing of small non-coding regulatory RNAs and the identification of the genomic locales from which they originate. Currently, there is a paucity of methods for finding small RNA generative locales. RESULTS: We describe and implement an algorithm that can determine small RNA generative locales from high-throughput sequencing data. The algorithm creates a network, or graph, of the small RNAs by creating links between them depending on their proximity on the target genome. For each of the sub-networks in the resulting graph the clustering coefficient, a measure of the interconnectedness of the subnetwork, is used to identify the generative locales. We test the algorithm over a wide range of parameters using RFAM sequences as positive controls and demonstrate that the algorithm has good sensitivity and specificity in a range of Arabidopsis and mouse small RNA sequence sets and that the locales it generates are robust to differences in the choice of parameters. CONCLUSIONS: NiBLS is a fast, reliable and sensitive method for determining small RNA locales in high-throughput sequence data that is generally applicable to all classes of small RNA.
Abstract.
Author URL.
Raffaele S, Farrer RA, Cano LM, Studholme DJ, MacLean D, Thines M, Jiang RHY, Zody MC, Kunjeti SG, Donofrio NM, et al (2010). Genome evolution following host jumps in the Irish potato famine pathogen lineage.
Science (New York, N.Y.),
330(6010), 1540-1543.
Abstract:
Genome evolution following host jumps in the Irish potato famine pathogen lineage.
Many plant pathogens, including those in the lineage of the Irish potato famine organism Phytophthora infestans, evolve by host jumps followed by specialization. However, how host jumps affect genome evolution remains largely unknown. To determine the patterns of sequence variation in the P. infestans lineage, we resequenced six genomes of four sister species. This revealed uneven evolutionary rates across genomes with genes in repeat-rich regions showing higher rates of structural polymorphisms and positive selection. These loci are enriched in genes induced in planta, implicating host adaptation in genome evolution. Unexpectedly, genes involved in epigenetic processes formed another class of rapidly evolving residents of the gene-sparse regions. These results demonstrate that dynamic repeat-rich genome compartments underpin accelerated gene evolution following host jumps in this pathogen lineage.
Abstract.
Studholme DJ, Kemen E, MacLean D, Schornack S, Aritua V, Thwaites R, Grant M, Smith J, Jones JDG (2010). Genome-wide sequencing data reveals virulence factors implicated in banana Xanthomonas wilt.
FEMS Microbiol Lett,
310(2), 182-192.
Abstract:
Genome-wide sequencing data reveals virulence factors implicated in banana Xanthomonas wilt.
Banana Xanthomonas wilt is a newly emerging disease that is currently threatening the livelihoods of millions of farmers in East Africa. The causative agent is Xanthomonas campestris pathovar musacearum (Xcm), but previous work suggests that this pathogen is much more closely related to species Xanthomonas vasicola than to X. campestris. We have generated draft genome sequences for a banana-pathogenic strain of Xcm isolated in Uganda and for a very closely related strain of X. vasicola pathovar vasculorum, originally isolated from sugarcane, that is nonpathogenic on banana. The draft sequences revealed overlapping but distinct repertoires of candidate virulence effectors in the two strains. Both strains encode homologues of the Pseudomonas syringae effectors HopW, HopAF1 and RipT from Ralstonia solanacearum. The banana-pathogenic and non-banana-pathogenic strains also differed with respect to lipopolysaccharide synthesis and type-IV pili, and in at least several thousand single-nucleotide polymorphisms in the core conserved genome. We found evidence of horizontal transfer between X. vasicola and very distantly related bacteria, including members of other divisions of the Proteobacteria. The availability of these draft genomes will be an invaluable tool for further studies aimed at understanding and combating this important disease.
Abstract.
Author URL.
Nemri A, Atwell S, Tarone AM, Huang YS, Zhao K, Studholme DJ, Nordborg M, Jones JDG (2010). Genome-wide survey of Arabidopsis natural variation in downy mildew resistance using combined association and linkage mapping.
Proc Natl Acad Sci U S A,
107(22), 10302-10307.
Abstract:
Genome-wide survey of Arabidopsis natural variation in downy mildew resistance using combined association and linkage mapping.
The model plant Arabidopsis thaliana exhibits extensive natural variation in resistance to parasites. Immunity is often conferred by resistance (R) genes that permit recognition of specific races of a disease. The number of such R genes and their distribution are poorly understood. In this study, we investigated the basis for resistance to the downy mildew agent Hyaloperonospora arabidopsidis ex parasitica (Hpa) in a global sample of A. thaliana. We implemented a combined genome-wide mapping of resistance using populations of recombinant inbred lines and a collection of wild A. thaliana accessions. We tested the interaction between 96 host genotypes collected worldwide and five strains of Hpa. Then, a fraction of the species-wide resistance was genetically dissected using six recently constructed populations of recombinant inbred lines. We found that resistance is usually governed by single dominant R genes that are concentrated in four genomic regions only. We show that association genetics of resistance to diseases such as downy mildew enables increased mapping resolution from quantitative trait loci interval to candidate gene level. Association patterns in quantitative trait loci intervals indicate that the pool of A. thaliana resistance sources against the tested Hpa isolates may be predominantly confined to six RPP (Resistance to Hpa) loci isolated in previous studies. Our results suggest that combining association and linkage mapping could accelerate resistance gene discovery in plants.
Abstract.
Author URL.
Clarke CR, Cai R, Studholme DJ, Guttman DS, Vinatzer BA (2010). Pseudomonas syringae strains naturally lacking the classical P. syringae hrp/hrc Locus are common leaf colonizers equipped with an atypical type III secretion system.
Mol Plant Microbe Interact,
23(2), 198-210.
Abstract:
Pseudomonas syringae strains naturally lacking the classical P. syringae hrp/hrc Locus are common leaf colonizers equipped with an atypical type III secretion system.
Pseudomonas syringae is best known as a plant pathogen that causes disease by translocating immune-suppressing effector proteins into plant cells through a type III secretion system (T3SS). However, P. syringae strains belonging to a newly described phylogenetic subgroup (group 2c) are missing the canonical P. syringae hrp/hrc cluster coding for a T3SS, flanking effector loci, and any close orthologue of known P. syringae effectors. Nonetheless, P. syringae group 2c strains are common leaf colonizers and grow on some tested plant species to population densities higher than those obtained by other P. syringae strains on nonhost species. Moreover, group 2c strains have genes necessary for the production of phytotoxins, have an ice nucleation gene, and, most interestingly, contain a novel hrp/hrc cluster, which is only distantly related to the canonical P. syringae hrp/hrc cluster. This hrp/hrc cluster appears to encode a functional T3SS although the genes hrpK and hrpS, present in the classical P. syringae hrp/hrc cluster, are missing. The genome sequence of a representative group 2c strain also revealed distant orthologues of the P. syringae effector genes avrE1 and hopM1 and the P. aeruginosa effector genes exoU and exoY. A putative life cycle for group 2c P. syringae is discussed.
Abstract.
Author URL.
2009
Studholme DJ, Ibanez SG, MacLean D, Dangl JL, Chang JH, Rathjen JP (2009). A draft genome sequence and functional screen reveals the repertoire of type III secreted proteins of Pseudomonas syringae pathovar tabaci 11528.
BMC Genomics,
10Abstract:
A draft genome sequence and functional screen reveals the repertoire of type III secreted proteins of Pseudomonas syringae pathovar tabaci 11528.
BACKGROUND: Pseudomonas syringae is a widespread bacterial pathogen that causes disease on a broad range of economically important plant species. Pathogenicity of P. syringae strains is dependent on the type III secretion system, which secretes a suite of up to about thirty virulence 'effector' proteins into the host cytoplasm where they subvert the eukaryotic cell physiology and disrupt host defences. P. syringae pathovar tabaci naturally causes disease on wild tobacco, the model member of the Solanaceae, a family that includes many crop species as well as on soybean. RESULTS: We used the 'next-generation' Illumina sequencing platform and the Velvet short-read assembly program to generate a 145X deep 6,077,921 nucleotide draft genome sequence for P. syringae pathovar tabaci strain 11528. From our draft assembly, we predicted 5,300 potential genes encoding proteins of at least 100 amino acids long, of which 303 (5.72%) had no significant sequence similarity to those encoded by the three previously fully sequenced P. syringae genomes. of the core set of Hrp Outer Proteins that are conserved in three previously fully sequenced P. syringae strains, most were also conserved in strain 11528, including AvrE1, HopAH2, HopAJ2, HopAK1, HopAN1, HopI, HopJ1, HopX1, HrpK1 and HrpW1. However, the hrpZ1 gene is partially deleted and hopAF1 is completely absent in 11528. The draft genome of strain 11528 also encodes close homologues of HopO1, HopT1, HopAH1, HopR1, HopV1, HopAG1, HopAS1, HopAE1, HopAR1, HopF1, and HopW1 and a degenerate HopM1'. Using a functional screen, we confirmed that hopO1, hopT1, hopAH1, hopM1', hopAE1, hopAR1, and hopAI1' are part of the virulence-associated HrpL regulon, though the hopAI1' and hopM1' sequences were degenerate with premature stop codons. We also discovered two additional HrpL-regulated effector candidates and an HrpL-regulated distant homologue of avrPto1. CONCLUSION: the draft genome sequence facilitates the continued development of P. syringae pathovar tabaci on wild tobacco as an attractive model system for studying bacterial disease on plants. The catalogue of effectors sheds further light on the evolution of pathogenicity and host-specificity as well as providing a set of molecular tools for the study of plant defence mechanisms. We also discovered several large genomic regions in Pta 11528 that do not share detectable nucleotide sequence similarity with previously sequenced Pseudomonas genomes. These regions may include horizontally acquired islands that possibly contribute to pathogenicity or epiphytic fitness of Pta 11528.
Abstract.
Author URL.
Almeida NF, Yan S, Lindeberg M, Studholme DJ, Schneider DJ, Condon B, Liu H, Viana CJ, Warren A, Evans C, et al (2009). A draft genome sequence of Pseudomonas syringae pv. tomato T1 reveals a type III effector repertoire significantly divergent from that of Pseudomonas syringae pv. tomato DC3000.
Mol Plant Microbe Interact,
22(1), 52-62.
Abstract:
A draft genome sequence of Pseudomonas syringae pv. tomato T1 reveals a type III effector repertoire significantly divergent from that of Pseudomonas syringae pv. tomato DC3000.
Diverse gene products including phytotoxins, pathogen-associated molecular patterns, and type III secreted effectors influence interactions between Pseudomonas syringae strains and plants, with additional yet uncharacterized factors likely contributing as well. of particular interest are those interactions governing pathogen-host specificity. Comparative genomics of closely related pathogens with different host specificity represents an excellent approach for identification of genes contributing to host-range determination. A draft genome sequence of Pseudomonas syringae pv. tomato T1, which is pathogenic on tomato but nonpathogenic on Arabidopsis thaliana, was obtained for this purpose and compared with the genome of the closely related A. thaliana and tomato model pathogen P. syringae pv. tomato DC3000. Although the overall genetic content of each of the two genomes appears to be highly similar, the repertoire of effectors was found to diverge significantly. Several P. syringae pv. tomato T1 effectors absent from strain DC3000 were confirmed to be translocated into plants, with the well-studied effector AvrRpt2 representing a likely candidate for host-range determination. However, the presence of avrRpt2 was not found sufficient to explain A. thaliana resistance to P. syringae pv. tomato T1, suggesting that other effectors and possibly type III secretion system-independent factors also play a role in this interaction.
Abstract.
Author URL.
MacLean D, Jones JDG, Studholme DJ (2009). Application of 'next-generation' sequencing technologies to microbial genetics.
NATURE REVIEWS MICROBIOLOGY,
7(4), 287-296.
Author URL.
MacLean D, Jones JDG, Studholme DJ (2009). Application of 'next-generation' sequencing technologies to microbial genetics.
Nat Rev Microbiol,
7(4), 287-296.
Abstract:
Application of 'next-generation' sequencing technologies to microbial genetics.
New sequencing methods generate data that can allow the assembly of microbial genome sequences in days. With such revolutionary advances in technology come new challenges in methodologies and informatics. In this article, we review the capabilities of high-throughput sequencing technologies and discuss the many options for getting useful information from the data.
Abstract.
Author URL.
MacLean D, Studholme DJ (2009). Bioinformatics Aspects of High-Throughput Sequencing Technology. In Jackson RW (Ed) Plant Pathogenic Bacteria: Genomics and Molecular Biology, Caister Academic Press.
Studholme DJ, Gimenez Ibanez S, Maclean D, Dangl JL, Chang JH, Rathjen JP (2009). Correction: a draft genome sequence and functional screen reveals the repertoire of type III secreted proteins of Pseudomonas syringae pathovar tabaci 11528.
BMC Genomics,
10(1), 569-569.
Abstract:
Correction: a draft genome sequence and functional screen reveals the repertoire of type III secreted proteins of Pseudomonas syringae pathovar tabaci 11528.
ABSTRACT: After the publication of this work [1], we became aware of several errors in our descriptions of proteins associated with the type III secretion system (T3SS) of P. syringae pathovar tabaci (Pta) strain 11528. As mentioned in the text of the article [1], Pta 11528 encodes a full-length homologue of HopAB2. Therefore, in Figure 1, HopAB2 should not have been marked with asterisks. A corrected summary of the Hop protein repertoire in Pta 11528 is shown in Figure 1 of this Correction. We confirmed the presence of a full-length hopAB2 using capillary sequencing. Unfortunately, in the draft assembly of Illumina sequence data presented in the paper [1], there was a mis-assembly error that resulted in deletion of 271 nucleotides from the 5' end of the hopAB2 gene. This type of error is, unfortunately, not uncommon in assemblies of short sequence reads, though recent versions of the Velvet assembly software seem to be less prone to such errors. We are currently generating 454 GS-FLX sequence data from Pta 11528 genomic DNA and hope to make public an improved genome assembly and annotation in due course. Pta 11528 encodes a full-length homologue of T3SS helper protein HrpA2 (Locus tag C1E_5326 in our annotation; RefSeq: ZP_05641290.1). Therefore hrpA2 should have been shown in boldface and underlined in Figure 1 of the manuscript [1]. This has been remedied in Figure 1 of this Correction Contrary to the original manuscript [1], HopR1 is degenerate in Pta 11528. In the Pta 11528 draft assembly, the hopR1 gene was split into at least two open reading frames (RefSeq: ZP_05639788.1, ZP_05639787.1; locus tags C1E_3889, C1E_3890) suggesting that is a degenerate pseudogene. We confirmed the presence of an internal stop codon in hopR1 using capillary sequencing. This degeneracy should have been indicated by marking hopR1 with a double asterisk (. ) in Figure 1. This has been remedied in Figure 1 of this Correction Pta 11528 encodes a full-length HopM1 homologue (RefSeq: ZP_05641297.1; locus tag C1E_5336; GenBank: ACR46722.1). The fact that HopM1 is intact and not degenerate should have been indicated in Figure 1 (by highlighting hopM1 in boldface and underlined) in the original manuscript [1]. This has been remedied in Figure 1 of this Correction. We regret any inconvenience caused by these errors and are grateful to Dr Magdalen Lindeberg for bringing them to our attention.
Abstract.
Farrer RA, Kemen E, Jones JDG, Studholme DJ (2009). De novo assembly of the Pseudomonas syringae pv. syringae B728a genome using Illumina/Solexa short sequence reads.
FEMS Microbiol Lett,
291(1), 103-111.
Abstract:
De novo assembly of the Pseudomonas syringae pv. syringae B728a genome using Illumina/Solexa short sequence reads.
Illumina's Genome Analyzer generates ultra-short sequence reads, typically 36 nucleotides in length, and is primarily intended for resequencing. We tested the potential of this technology for de novo sequence assembly on the 6 Mbp genome of Pseudomonas syringae pv. syringae B728a with several freely available assembly software packages. Using an unpaired data set, velvet assembled >96% of the genome into contigs with an N50 length of 8289 nucleotides and an error rate of 0.33%. EDENA generated smaller contigs (N50 was 4192 nucleotides) and comparable error rates. SSAKE and VCAKE yielded shorter contigs with very high error rates. Assembly of paired-end sequence data carrying 400 bp inserts produced longer contigs (N50 up to 15 628 nucleotides), but with increased error rates (0.5%). Contig length and error rate were very sensitive to the choice of parameter values. Noncoding RNA genes were poorly resolved in de novo assemblies, while >90% of the protein-coding genes were assembled with 100% accuracy over their full length. This study demonstrates that, in practice, de novo assembly of 36-nucleotide reads can generate reasonably accurate assemblies from about 40 x deep sequence data sets. These draft assemblies are useful for exploring an organism's proteomic potential, at a very economic low cost.
Abstract.
Author URL.
Haas BJ, Kamoun S, Zody MC, Jiang RHY, Handsaker RE, Cano LM, Grabherr M, Kodira CD, Raffaele S, Torto-Alalibo T, et al (2009). Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans.
Nature,
461(7262), 393-398.
Abstract:
Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans.
Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at approximately 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for approximately 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.
Abstract.
Author URL.
Setubal JC, dos Santos P, Goldman BS, Ertesvåg H, Espin G, Rubio LM, Valla S, Almeida NF, Balasubramanian D, Cromes L, et al (2009). Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes.
J Bacteriol,
191(14), 4534-4545.
Abstract:
Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes.
Azotobacter vinelandii is a soil bacterium related to the Pseudomonas genus that fixes nitrogen under aerobic conditions while simultaneously protecting nitrogenase from oxygen damage. In response to carbon availability, this organism undergoes a simple differentiation process to form cysts that are resistant to drought and other physical and chemical agents. Here we report the complete genome sequence of A. vinelandii DJ, which has a single circular genome of 5,365,318 bp. In order to reconcile an obligate aerobic lifestyle with exquisitely oxygen-sensitive processes, A. vinelandii is specialized in terms of its complement of respiratory proteins. It is able to produce alginate, a polymer that further protects the organism from excess exogenous oxygen, and it has multiple duplications of alginate modification genes, which may alter alginate composition in response to oxygen availability. The genome analysis identified the chromosomal locations of the genes coding for the three known oxygen-sensitive nitrogenases, as well as genes coding for other oxygen-sensitive enzymes, such as carbon monoxide dehydrogenase and formate dehydrogenase. These findings offer new prospects for the wider application of A. vinelandii as a host for the production and characterization of oxygen-sensitive proteins.
Abstract.
Author URL.
Jones AME, MacLean D, Studholme DJ, Serna-Sanz A, Andreasson E, Rathjen JP, Peck SC (2009). Phosphoproteomic analysis of nuclei-enriched fractions from Arabidopsis thaliana.
J Proteomics,
72(3), 439-451.
Abstract:
Phosphoproteomic analysis of nuclei-enriched fractions from Arabidopsis thaliana.
Phosphorylation is a ubiquitous regulatory mechanism, that governs the activity, subcellular localisation and molecular interactions of proteins. To identify a broad range of nuclear phosphoproteins from Arabidopsis thaliana, we enriched for nuclei from suspension cell cultures and seedlings before extensive fractionation and identification of phosphopeptides by mass spectrometry. We identified 416 phosphopeptides from 345 proteins with high confidence. Our data show that sub-cellular fractionation is an effective strategy for identifying nuclear phosphoproteins, two thirds of our dataset are known or predicted to be nuclear localised and one half of the nuclear localised proteins have novel phosphorylation sites. We identified novel phosphorylation sites on transcription factors, chromatin remodelling proteins, RNA silencing components and the spliceosome. Intriguingly, we also identified phosphorylation sites on several proteins associated with Golgi vesicle trafficking such as the exocyst complex, and speculate that these may be involved in cell plate formation during cytokinesis.
Abstract.
Author URL.
Mosher RA, Melnyk CW, Kelly KA, Dunn RM, Studholme DJ, Baulcombe DC (2009). Uniparental expression of PolIV-dependent siRNAs in developing endosperm of Arabidopsis.
Nature,
460(7252), 283-286.
Abstract:
Uniparental expression of PolIV-dependent siRNAs in developing endosperm of Arabidopsis.
Most eukaryotes produce small RNA (sRNA) mediators of gene silencing that bind to Argonaute proteins and guide them, by base pairing, to an RNA target. MicroRNAs (miRNAs) that normally target messenger RNAs for degradation or translational arrest are the best-understood class of sRNAs. However, in Arabidopsis thaliana flowers, miRNAs account for only 5% of the sRNA mass and less than 0.1% of the sequence complexity. The remaining sRNAs form a complex population of more than 100,000 different small interfering RNAs (siRNAs) transcribed from thousands of loci. The biogenesis of most of the siRNAs in Arabidopsis are dependent on RNA polymerase IV (PolIV), a homologue of DNA-dependent RNA polymerase II. A subset of these PolIV-dependent (p4)-siRNAs are involved in stress responses, and others are associated with epigenetic modifications to DNA or chromatin; however, the biological role is not known for most of them. Here we show that the predominant phase of p4-siRNA accumulation is initiated in the maternal gametophyte and continues during seed development. Expression of p4-siRNAs in developing endosperm is specifically from maternal chromosomes. Our results provide the first evidence for a link between genomic imprinting and RNA silencing in plants.
Abstract.
Author URL.
2008
Moxon S, Schwach F, Dalmay T, Maclean D, Studholme DJ, Moulton V (2008). A toolkit for analysing large-scale plant small RNA datasets.
Bioinformatics,
24(19), 2252-2253.
Abstract:
A toolkit for analysing large-scale plant small RNA datasets.
UNLABELLED: Recent developments in high-throughput sequencing technologies have generated considerable demand for tools to analyse large datasets of small RNA sequences. Here, we describe a suite of web-based tools for processing plant small RNA datasets. Our tools can be used to identify micro RNAs and their targets, compare expression levels in sRNA loci, and find putative trans-acting siRNA loci. AVAILABILITY: the tools are freely available for use at http://srna-tools.cmp.uea.ac.uk.
Abstract.
Author URL.
Thompson AH, Studholme DJ, Green EM, Leak DJ (2008). Heterologous expression of pyruvate decarboxylase in Geobacillus thermoglucosidasius.
Biotechnol Lett,
30(8), 1359-1365.
Abstract:
Heterologous expression of pyruvate decarboxylase in Geobacillus thermoglucosidasius.
Expression of a pyruvate decarboxylase (Pdc) pathway in metabolically versatile thermophilic bacteria could create novel ethanologenic organisms, but no suitable thermostable Pdc is available. We have demonstrated that Pdc from Zymomonas mobilis can be expressed in an active form in Geobacillus thermoglucosidasius at up to 52 degrees C, while expression of Pdc polypeptides up to 54 degrees C was evident from Western blotting. By using an unstable lactate dehydrogenase (ldh) mutant of G. thermoglucosidasius, indirect evidence of Pdc activity in vivo was also obtained.
Abstract.
Author URL.
MacLean D, Burrell MA, Studholme DJ, Jones AM (2008). PhosCalc: a tool for evaluating the sites of peptide phosphorylation from mass spectrometer data.
BMC Res Notes,
1Abstract:
PhosCalc: a tool for evaluating the sites of peptide phosphorylation from mass spectrometer data.
BACKGROUND: We have created a software implementation of a published and verified method for assigning probabilities to potential phosphorylation sites on peptides using mass spectrometric data. Our tool, named PhosCalc, determines the number of possible phosphorylation sites and calculates the theoretical masses for the b and y fragment ions of a user-provided peptide sequence. A corresponding user-provided mass spectrum is examined to determine which putative b and y ions have support in the spectrum and a probability score is calculated for each combination of phosphorylation sites. FINDINGS: We test the implementation using spectra of phosphopeptides from bovine beta-casein and we compare the results from the implementation to those from manually curated and verified phosphopeptides from our own experiments. We find that the PhosCalc scores are capable of helping a user to identify phosphorylated sites and can remove a bottleneck in high throughput proteomics analyses. CONCLUSION: PhosCalc is available as a web-based interface for examining up to 100 peptides and as a downloadable tool for examining larger numbers of peptides. PhosCalc can be used to speed up identification of phosphorylation sites and can be easily integrated into data handling pipelines making it a very useful tool for those involved in phosphoproteomic research.
Abstract.
Author URL.
Mosher RA, Schwach F, Studholme D, Baulcombe DC (2008). PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis.
Proc Natl Acad Sci U S A,
105(8), 3145-3150.
Abstract:
PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis.
DNA-dependent RNA polymerase (Pol)IV in Arabidopsis exists in two isoforms (PolIVa and PolIVb), with NRPD1a and NRPD1b as their respective largest subunits. Both isoforms are implicated in production and activity of siRNAs and in RNA-directed DNA methylation (RdDM). Deep sequence analysis of siRNAs in WT Arabidopsis flowers and in nrpd1a and nrpd1b mutants identified >4,200 loci producing siRNAs in a PolIV-dependent manner, with PolIVb reinforcing siRNA production by PolIVa. Transposable element identity and pericentromeric localization are both features that predispose a locus for siRNA production via PolIV proteins and determine the extent to which siRNA production relies on PolIVb. Detailed analysis of DNA methylation at PolIV-dependent loci revealed unexpected deviations from the previously noted association of PolIVb-dependent siRNA production and RdDM. Notably, PolIVb functions independently in DNA methylation and siRNA generation. Additionally, we have uncovered siRNA-directed loss of DNA methylation, a process requiring both PolIV isoforms. From these findings, we infer that the role of PolIVb in siRNA production is secondary to a role in chromatin modification and is influenced by chromatin context.
Abstract.
Author URL.
Bagnall A, Moxon S, Studholme D, Moulton V (2008). Time series data mining algorithms for identifying short RNA in Arabidopsis thaliana.
Proceedings of the 2008 International Conference on Bioinformatics and Computational Biology, BIOCOMP 2008, 182-188.
Abstract:
Time series data mining algorithms for identifying short RNA in Arabidopsis thaliana
The class of molecules called short RNAs (sRNAs) are known to play a key role in gene regulation. They are typically sequences of nucleotides between 21-25 nucleotides in length. The identification, clustering and classification of sRNA has recently become the focus of much research activity. The basic problem involves detecting regions of interest on the chromosome where the pattern of candidate matches is somehow unusual. In this paper we propose two generic approaches that place the specific biological problem in the wider context of time series data mining problems. Both methods are based on treating the occurrences on a chromosome, or "hit count" data, as a time series, then running a sliding window along a chromosome and measuring unusualness. This formulation means we can treat finding unusual areas of candidate RNA activity as a variety of time series anomaly detection problem. The first set of approaches is model based. We specify a null hypothesis distribution for not being a sRNA, then estimate the p-values along the chromosome. The second approach is instance based. We identify some typical shapes from known sRNA, then use dynamic time warping and fourier transform based distance to measure how closely the candidate series matches. We demonstrate that these methods can find known sRNA on Arabidopsis thaliana chromosomes and illustrate the benefits of the added information provided by these algorithms.
Abstract.
2007
Kamal N, Dorrell N, Jagannathan A, Turner SM, Constantinidou C, Studholme DJ, Marsden G, Hinds J, Laing KG, Wren BW, et al (2007). Deletion of a previously uncharacterized flagellar-hook-length control gene fliK modulates the sigma54-dependent regulon in Campylobacter jejuni.
Microbiology (Reading),
153(Pt 9), 3099-3111.
Abstract:
Deletion of a previously uncharacterized flagellar-hook-length control gene fliK modulates the sigma54-dependent regulon in Campylobacter jejuni.
A previously unannotated, putative fliK gene was identified in the Campylobacter jejuni genome based on sequence analysis; deletion mutants in this gene had a 'polyhook' phenotype characteristic of fliK mutants in other genera. The mutants greatly overexpressed the sigma(54)-dependent flagellar hook protein FlgE, to form unusual filamentous structures resembling straight flagella in addition to polyhooks. The genome sequence reveals only one gene predicted to encode an orthologue of the NtrC-family activator required for sigma(54)-dependent transcription. Hence, all sigma(54)-dependent genes in the genome would be overexpressed in the fliK mutant together with flgE. Microarray analysis of genome-wide transcription in the mutant showed increased transcription of a subset of genes, often downstream of sigma(54)-dependent promoters identified by a quality-predictive algorithm applied to the whole genome. Assessment of genome-wide transcription in deletion mutants in rpoN, encoding sigma(54), and in the sigma(54)-activator gene flgR, showed reciprocally reduced transcription of genes that were overexpressed in the fliK mutant. The fliA (sigma(28))-dependent regulon was also analysed. Together the data clearly define the roles of the alternative sigma factors RpoN and FliA in flagellar biogenesis in C. jejuni, and identify additional putative members of their respective regulons.
Abstract.
Author URL.
Jones J, Studholme DJ, Knight CG, Preston GM (2007). Integrated bioinformatic and phenotypic analysis of RpoN-dependent traits in the plant growth-promoting bacterium Pseudomonas fluorescens SBW25.
Environ Microbiol,
9(12), 3046-3064.
Abstract:
Integrated bioinformatic and phenotypic analysis of RpoN-dependent traits in the plant growth-promoting bacterium Pseudomonas fluorescens SBW25.
The alternative sigma factor RpoN is a key regulator in the acclimation of Pseudomonas to complex natural environments. In this study we show that RpoN is required for efficient colonization of sugar beet seedlings by the plant growth-promoting bacterium Pseudomonas fluorescens SBW25, and use phenotypic and bioinformatic approaches to profile the RpoN-dependent traits and genes of P. fluorescens SBW25. RpoN is required for flagellar biosynthesis and for assimilation of a wide variety of nutrient sources including inorganic nitrogen, amino acids, sugar alcohols and dicarboxylic acids. Chemosensitivity assays indicate that RpoN-regulated genes contribute to acid tolerance and resistance to some antibiotics, including tetracyclines and aminoglycosides. Gain of function changes associated with loss of RpoN included increased tolerance to hydroxyurea and Guanazole. Bioinformatic predictions of RpoN-regulated genes show a close correspondence with phenotypic analyses of RpoN-regulated traits and suggest novel functions for RpoN in P. fluorescens, including regulation of poly(A) polymerase. The reduced plant colonization ability observed for an rpoN mutant of P. fluorescens is therefore likely to be due to defects in multiple traits including nutrient assimilation, protein secretion and stress tolerance.
Abstract.
Author URL.
Nemri A, Neff MM, Burrell M, Jones JDG, Studholme DJ (2007). Marker development for the genetic study of natural variation in Arabidopsis thaliana.
Bioinformatics,
23(22), 3108-3109.
Abstract:
Marker development for the genetic study of natural variation in Arabidopsis thaliana.
We report AtPRIMER, an application that automates the discovery of new polymorphic markers between ecotypes of Arabidopsis thaliana. On specifying two ecotypes and the genomic region of interest, the script retrieves all corresponding single nucleotide polymorphisms (SNPs) and generates CAPS and/or dCAPS PCR primer sequences. We show that AtPRIMER accurately found specific polymorphic markers for our linkage mapping project. AtPRIMER will therefore be useful for efficient marker development with high density and specificity.
Abstract.
Author URL.
Maor R, Jones A, Nühse TS, Studholme DJ, Peck SC, Shirasu K (2007). Multidimensional protein identification technology (MudPIT) analysis of ubiquitinated proteins in plants.
Mol Cell Proteomics,
6(4), 601-610.
Abstract:
Multidimensional protein identification technology (MudPIT) analysis of ubiquitinated proteins in plants.
Protein conjugation with ubiquitin, known as ubiquitination, is a key regulatory mechanism to control protein abundance, localization, and activity in eukaryotic cells. To identify ubiquitin-dependent regulatory steps in plants, we developed a robust affinity purification/identification system for ubiquitinated proteins. Using GST-tagged ubiquitin binding domains, we performed a large scale affinity purification of ubiquitinated proteins from Arabidopsis cell suspension culture. High molecular weight ubiquitinated proteins were separated by SDS-PAGE, and the trypsin-digested samples were then analyzed by a multidimensional protein identification technology (MudPIT) system. A total of 294 proteins specifically bound by the GST-tagged ubiquitin binding domains were identified. From these we determined 85 ubiquitinated lysine residues in 56 proteins, confirming the enrichment of the target class of proteins. Our data provide the first view of the ubiquitinated proteome in plants. We also provide evidence that this technique can be broadly applied to the study of protein ubiquitination in diverse plant species.
Abstract.
Author URL.
Hernandez-Pinzon I, Yelina NE, Schwach F, Studholme DJ, Baulcombe D, Dalmay T (2007). SDE5, the putative homologue of a human mRNA export factor, is required for transgene silencing and accumulation of trans-acting endogenous siRNA.
Plant J,
50(1), 140-148.
Abstract:
SDE5, the putative homologue of a human mRNA export factor, is required for transgene silencing and accumulation of trans-acting endogenous siRNA.
Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation process conserved in fungi, plants and animals. The trigger of the mechanism is double-stranded RNA derived from transgenic or endogenous loci and formed by intra- or inter-molecular interactions of single-stranded RNAs or the action of RNA-dependent RNA polymerases (RDRs). Double-stranded RNA from various sources is processed by one of the four Dicer-like (DCL) proteins in Arabidopsis, and the resulting short RNAs enter into at least four different pathways, one of which involves the production of trans-acting short interfering RNAs (tasiRNAs). We report here a novel gene (SDE5) that is required for transgene silencing and the production of tasiRNAs. Mutation in SDE5 also results in hyper-susceptibility to cucumber mosaic virus but not turnip mosaic virus. However, like RDR6, SDE5 is not involved in inverted repeat-induced transgene silencing or the biogenesis of microRNAs and 24 nt siRNAs produced by DCL3. Based on these results, we propose that SDE5 acts together with RDR6 in generating double-stranded RNA from specific single-stranded RNAs. As the sequence of SDE5 has sequence features shared by TAP, a human mRNA export factor, we propose that its role could be in the transport of RNA molecules that are converted into a double-stranded form by RDR6.
Abstract.
Author URL.
Molnár A, Schwach F, Studholme DJ, Thuenemann EC, Baulcombe DC (2007). miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii.
Nature,
447(7148), 1126-1129.
Abstract:
miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii.
MicroRNAs (miRNAs) in eukaryotes guide post-transcriptional regulation by means of targeted RNA degradation and translational arrest. They are released by a Dicer nuclease as a 21-24-nucleotide RNA duplex from a precursor in which an imperfectly matched inverted repeat forms a partly double-stranded region. One of the two strands is then recruited by an Argonaute nuclease that is the effector protein of the silencing mechanism. Short interfering RNAs (siRNAs), which are similar to miRNAs, are also produced by Dicer but the precursors are perfectly double-stranded RNA. These siRNAs guide post-transcriptional regulation, as with miRNAs, and epigenetic genome modification. Diverse eukaryotes including fungi, plants, protozoans and metazoans produce siRNAs but, until now, miRNAs have not been described in unicellular organisms and it has been suggested that they evolved together with multicellularity in separate plant and animal lineages. Here we show that the unicellular alga Chlamydomonas reinhardtii contains miRNAs, putative evolutionary precursors of miRNAs and species of siRNAs resembling those in higher plants. The common features of miRNAs and siRNAs in an alga and in higher plants indicate that complex RNA-silencing systems evolved before multicellularity and were a feature of primitive eukaryotic cells.
Abstract.
Author URL.
2006
He Q-Y, Liu X-H, Li Q, Studholme DJ, Li X-W, Liang S-P (2006). G8: a novel domain associated with polycystic kidney disease and non-syndromic hearing loss.
Bioinformatics,
22(18), 2189-2191.
Abstract:
G8: a novel domain associated with polycystic kidney disease and non-syndromic hearing loss.
UNLABELLED: We report a novel protein domain-G8-which contains five repeated beta-strand pairs and is present in some disease-related proteins such as PKHD1, KIAA1199, TMEM2 as well as other uncharacterized proteins. Most G8-containing proteins are predicted to be membrane-integral or secreted. The G8 domain may be involved in extracellular ligand binding and catalysis. It has been reported that mis-sense mutations in the two G8 domains of human PKHD1 protein resulted in a less stable protein and are associated with autosomal-recessive polycystic kidney disease, indicating the importance of the domain structure. G8 is also present in the N-terminus of some non-syndromic hearing loss disease-related proteins such as KIAA1109 and TMEM2. Discovery of G8 domain will be important for the research of the structure/function of related proteins and beneficial for the development of novel therapeutics. CONTACT: liangsp@hunnu.edu.cn
Abstract.
Author URL.
2005
Mulder NJ, Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, Bradley P, Bork P, Bucher P, Cerutti L, et al (2005). InterPro, progress and status in 2005.
Nucleic Acids Res,
33(Database issue), D201-D205.
Abstract:
InterPro, progress and status in 2005.
InterPro, an integrated documentation resource of protein families, domains and functional sites, was created to integrate the major protein signature databases. Currently, it includes PROSITE, Pfam, PRINTS, ProDom, SMART, TIGRFAMs, PIRSF and SUPERFAMILY. Signatures are manually integrated into InterPro entries that are curated to provide biological and functional information. Annotation is provided in an abstract, Gene Ontology mapping and links to specialized databases. New features of InterPro include extended protein match views, taxonomic range information and protein 3D structure data. One of the new match views is the InterPro Domain Architecture view, which shows the domain composition of protein matches. Two new entry types were introduced to better describe InterPro entries: these are active site and binding site. PIRSF and the structure-based SUPERFAMILY are the latest member databases to join InterPro, and CATH and PANTHER are soon to be integrated. InterPro release 8.0 contains 11 007 entries, representing 2573 domains, 8166 families, 201 repeats, 26 active sites, 21 binding sites and 20 post-translational modification sites. InterPro covers over 78% of all proteins in the Swiss-Prot and TrEMBL components of UniProt. The database is available for text- and sequence-based searches via a webserver (http://www.ebi.ac.uk/interpro), and for download by anonymous FTP (ftp://ftp.ebi.ac.uk/pub/databases/interpro).
Abstract.
Author URL.
Fleck SC, Andreasson E, Merkouropolous G, Nuehse TS, Serna-Sanz A, Studholme D (2005). Phosphoproteomics in <i>Arabidopsis</i> from databases to signalling networks.
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY,
141(3), S250-S250.
Author URL.
Preston GM, Studholme DJ, Caldelari I (2005). Profiling the secretomes of plant pathogenic Proteobacteria. FEMS Microbiology Reviews, 29(2), 331-360.
Preston GM, Studholme DJ, Caldelari I (2005). Profiling the secretomes of plant pathogenic Proteobacteria.
FEMS Microbiol Rev,
29(2), 331-360.
Abstract:
Profiling the secretomes of plant pathogenic Proteobacteria.
Secreted proteins are central to the success of plant pathogenic bacteria. They are used by plant pathogens to adhere to and degrade plant cell walls, to suppress plant defence responses, and to deliver bacterial DNA and proteins into the cytoplasm of plant cells. However, experimental investigations into the identity and role of secreted proteins in plant pathogenesis have been hindered by the fact that many of these proteins are only expressed or secreted in planta, that knockout mutations of individual proteins frequently have little or no obvious phenotype, and that some obligate and fastidious plant pathogens remain recalcitrant to genetic manipulation. The availability of genome sequence data for a large number of agriculturally and scientifically important plant pathogens enables us to predict and compare the complete secretomes of these bacteria. In this paper we outline strategies that are currently being used to identify secretion systems and secreted proteins in Proteobacterial plant pathogens and discuss the implications of these analyses for future investigations into the molecular mechanisms of plant pathogenesis.
Abstract.
Author URL.
Studholme DJ, Downie JA, Preston GM (2005). Protein domains and architectural innovation in plant-associated Proteobacteria.
BMC Genomics,
6Abstract:
Protein domains and architectural innovation in plant-associated Proteobacteria.
BACKGROUND: Evolution of new complex biological behaviour tends to arise by novel combinations of existing building blocks. The functional and evolutionary building blocks of the proteome are protein domains, the function of a protein being dependent on its constituent domains. We clustered completely-sequenced proteomes of prokaryotes on the basis of their protein domain content, as defined by Pfam (release 16.0). This revealed that, although there was a correlation between phylogeny and domain content, other factors also have an influence. This observation motivated an investigation of the relationship between an organism's lifestyle and the complement of domains and domain architectures found within its proteome. RESULTS: We took a census of all protein domains and domain combinations (architectures) encoded in the completely-sequenced proteobacterial genomes. Nine protein domain families were identified that are found in phylogenetically disparate plant-associated bacteria but are absent from non-plant-associated bacteria. Most of these are known to play a role in the plant-associated lifestyle, but they also included domain of unknown function DUF1427, which is found in plant symbionts and pathogens of the alpha-, beta- and gamma-Proteobacteria, but not known in any other organism. Further, several domains were identified as being restricted to phytobacteria and Eukaryotes. One example is the RolB/RolC glucosidase family, which is found only in Agrobacterium species and in plants. We identified the 0.5% of Pfam protein domain families that were most significantly over-represented in the plant-associated Proteobacteria with respect to the background frequencies in the whole set of available proteobacterial proteomes. These included guanylate cyclase, domains implicated in aromatic catabolism, cellulase and several domains of unknown function. We identified 459 unique domain architectures found in phylogenetically diverse plant pathogens and symbionts that were absent from non-pathogenic and non-symbiotic relatives. The vast majority of these were restricted to a single species or several closely related species and so their distributions could be better explained by phylogeny than by lifestyle. However, several architectures were found in two or more very distantly related phytobacteria but absent from non-plant-associated bacteria. Many of the proteins with these unique architectures are predicted to be secreted. In Pseudomonas syringae pathovar tomato, those genes encoding genes with novel domain architectures tended to have atypical GC contents and were adjacent to insertion sequence elements and phage-like sequences, suggesting acquisition by horizontal transfer. CONCLUSIONS: By identifying domains and architectures unique to plant pathogens and symbionts, we highlighted candidate proteins for involvement in plant-associated bacterial lifestyles. Given that characterisation of novel gene products in vivo and in vitro is time-consuming and expensive, this computational approach may be useful for reducing experimental search space. Furthermore we discuss the biological significance of novel proteins highlighted by this study in the context of plant-associated lifestyles.
Abstract.
Author URL.
Guo JH, Huang Q, Studholme DJ, Wu CQ, Zhao Z (2005). Transcriptomic analyses support the similarity of gene expression between brain and testis in human as well as mouse.
Cytogenet Genome Res,
111(2), 107-109.
Abstract:
Transcriptomic analyses support the similarity of gene expression between brain and testis in human as well as mouse.
We previously revealed similarity in gene expression patterns between human brain and testis, based on digital differential display analyses of 760 human Unigenes. In the present work, we reanalyzed the gene expression data in many tissues of human and mouse for a large number of genes almost covering the respective whole genomes. The results indicated that both in human and in mouse, the gene expression profiles exhibited by brain, cerebellum and testis are most similar to each other compared with other tissues.
Abstract.
Author URL.
2004
Studholme DJ, Bentley SD, Kormanec J (2004). Bioinformatic identification of novel regulatory DNA sequence motifs in Streptomyces coelicolor.
BMC Microbiol,
4Abstract:
Bioinformatic identification of novel regulatory DNA sequence motifs in Streptomyces coelicolor.
BACKGROUND: Streptomyces coelicolor is a bacterium with a vast repertoire of metabolic functions and complex systems of cellular development. Its genome sequence is rich in genes that encode regulatory proteins to control these processes in response to its changing environment. We wished to apply a recently published bioinformatic method for identifying novel regulatory sequence signals to gain new insights into regulation in S. coelicolor. RESULTS: the method involved production of position-specific weight matrices from alignments of over-represented words of DNA sequence. We generated 2497 weight matrices, each representing a candidate regulatory DNA sequence motif. We scanned the genome sequence of S. coelicolor against each of these matrices. A DNA sequence motif represented by one of the matrices was found preferentially in non-coding sequences immediately upstream of genes involved in polysaccharide degradation, including several that encode chitinases. This motif (TGGTCTAGACCA) was also found upstream of genes encoding components of the phosphoenolpyruvate phosphotransfer system (PTS). We hypothesise that this DNA sequence motif represents a regulatory element that is responsive to availability of carbon-sources. Other motifs of potential biological significance were found upstream of genes implicated in secondary metabolism (TTAGGTtAGgCTaACCTAA), sigma factors (TGACN19TGAC), DNA replication and repair (ttgtCAGTGN13TGGA), nucleotide conversions (CTACgcNCGTAG), and ArsR (TCAGN12TCAG). A motif found upstream of genes involved in chromosome replication (TGTCagtgcN7Tagg) was similar to a previously described motif found in UV-responsive promoters. CONCLUSIONS: We successfully applied a recently published in silico method to identify conserved sequence motifs in S. coelicolor that may be biologically significant as regulatory elements. Our data are broadly consistent with and further extend data from previously published studies. We invite experimental testing of our hypotheses in vitro and in vivo.
Abstract.
Author URL.
Tucker NP, D'Autréaux B, Studholme DJ, Spiro S, Dixon R (2004). DNA binding activity of the Escherichia coli nitric oxide sensor NorR suggests a conserved target sequence in diverse proteobacteria.
J Bacteriol,
186(19), 6656-6660.
Abstract:
DNA binding activity of the Escherichia coli nitric oxide sensor NorR suggests a conserved target sequence in diverse proteobacteria.
The Escherichia coli nitric oxide sensor NorR was shown to bind to the promoter region of the norVW transcription unit, forming at least two distinct complexes detectable by gel retardation. Three binding sites for NorR and two integration host factor binding sites were identified in the norR-norV intergenic region. The derived consensus sequence for NorR binding sites was used to search for novel members of the E. coli NorR regulon and to show that NorR binding sites are partially conserved in other members of the proteobacteria.
Abstract.
Author URL.
Studholme DJ, Dixon R (2004). In silico analysis of the sigma54-dependent enhancer-binding proteins in Pirellula species strain 1.
FEMS Microbiol Lett,
230(2), 215-225.
Abstract:
In silico analysis of the sigma54-dependent enhancer-binding proteins in Pirellula species strain 1.
The planctomycetes are a phylogenetically distinct group of bacteria, widespread in aquatic and terrestrial environments. Their cell walls lack peptidoglycan and their compartmentalised cells undergo a yeast-like budding cell division process. Many bacteria regulate a subset of their genes by an enhancer-dependent mechanism involving the alternative sigma factor sigma54 (RpoN, sigmaN) in association with sigma54-dependent transcriptional activators known as enhancer-binding proteins (EBPs). The sigma54-dependent regulon has previously been studied in several groups of bacteria, but not in the planctomycetes. We wished to exploit the recently published complete genome sequence of Pirellula species strain 1 to predict and analyse the sigma54-dependent regulon in this interesting group of bacteria. The genome of Pirellula species strain 1 encodes one homologue of sigma54, and 16 sigma54-dependent EBPs, including 10 two-component response regulators and a homologue of Escherichia coli RtcR. Two EBPs contain forkhead-associated domains, representing a novel protein domain combination not previously observed in bacterial EBPs and suggesting a novel link between the enhancer-dependent regulon and 'eukaryotic-like' protein phosphorylation in bacterial signal transduction. We identified several potential sigma54-dependent promoters upstream of genes and operons including two homologues of csrA, which encodes the global regulator CsrA, and rtcBA, encoding a RNA 3'-terminal phosphate cyclase. Phylogenetic analysis of EBP sequences from a wide range of bacterial taxa suggested that planctomycete EBPs fall into several distinct clades. Also the phylogeny of the sigma54 factors is broadly consistent with that of the host organisms. These results are consistent with a very ancient origin of sigma54 within the bacterial lineage. The repertoire of functions predicted to be under the control of the sigma54-dependent regulon in Pirellula shares some similarities (e.g. rtcBA) as well as exhibiting differences with that in other taxonomic groups of bacteria, reinforcing the evolutionarily dynamic nature of this regulon.
Abstract.
Author URL.
Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, Bradley P, Bordoli L. Studholme DJ, Wu CH, et al (2004). InterPro -prediction of protein families, domains and functional sites. In Williams CR (Ed) Focus on Genome Research, New York: Nova Science Publishers Inc. 169-204.
Guo J, Chen S, Huang C, Chen L, Studholme DJ, Zhao S, Yu L (2004). MANSC: a seven-cysteine-containing domain present in animal membrane and extracellular proteins.
Trends Biochem Sci,
29(4), 172-174.
Author URL.
Liu XH, He QY, Studholme DJ, Wu Q, Liang SP, Yu L (2004). NCD3G: a novel nine-cysteine domain in family 3 GPCRs (vol 19, pg 458, 2004).
TRENDS IN BIOCHEMICAL SCIENCES,
29(11), 571-571.
Author URL.
Studholme DJ, Fuerst JA, Bateman A (2004). Novel protein domains and motifs in the marine planctomycete Rhodopirellula baltica. FEMS Microbiology Letters, 236(2), 333-340.
Studholme DJ, Fuerst JA, Bateman A (2004). Novel protein domains and motifs in the marine planctomycete Rhodopirellula baltica.
FEMS Microbiol Lett,
236(2), 333-340.
Abstract:
Novel protein domains and motifs in the marine planctomycete Rhodopirellula baltica.
The planctomycetes are a phylum of bacteria that have a unique cell compartmentalisation and yeast-like budding cell division and peptidoglycan-less proteinaceous cell walls. We wished to further our understanding of these unique organisms at the molecular level by searching for conserved amino acid sequence motifs and domains in the proteins encoded by Rhodopirellula baltica. Using BLAST and single-linkage clustering, we have discovered several new protein domains and sequence motifs in this planctomycete. R. baltica has multiple members of the newly discovered GEFGR protein family and the ASPIC C-terminal domain family, whilst most other organisms for which whole genome sequence is available have no more than one. Many of the domains and motifs appear to be restricted to the planctomycetes. It is possible that these protein domains and motifs may have been lost or replaced in other phyla, or they may have undergone multiple duplication events in the planctomycete lineage. One of the novel motifs probably represents a novel N-terminal export signal peptide. With their unique cell biology, it may be that the planctomycete cell compartmentalisation plan in particular needs special membrane transport mechanisms. The discovery of these new domains and motifs, many of which are associated with secretion and cell-surface functions, will help to stimulate experimental work and thus enhance further understanding of this fascinating group of organisms.
Abstract.
Author URL.
Scorpio DG, Caspersen K, Ogata H, Park J, Dumler JS (2004). Restricted changes in major surface protein-2 (<i>msp2</i>) transcription after prolonged in vitro passage of <i>Anaplasma phagocytophilum</i> -: art. no. 1.
BMC MICROBIOLOGY,
4 Author URL.
Bateman A, Coin L, Durbin R, Finn RD, Hollich V, Griffiths-Jones S, Khanna A, Marshall M, Moxon S, Sonnhammer ELL, et al (2004). The Pfam protein families database.
Nucleic Acids Res,
32(Database issue), D138-D141.
Abstract:
The Pfam protein families database.
Pfam is a large collection of protein families and domains. Over the past 2 years the number of families in Pfam has doubled and now stands at 6190 (version 10.0). Methodology improvements for searching the Pfam collection locally as well as via the web are described. Other recent innovations include modelling of discontinuous domains allowing Pfam domain definitions to be closer to those found in structure databases. Pfam is available on the web in the UK (http://www.sanger.ac.uk/Software/Pfam/), the USA (http://pfam.wustl.edu/), France (http://pfam.jouy.inra.fr/) and Sweden (http://Pfam.cgb.ki.se/).
Abstract.
Author URL.
2003
Studholme DJ, Pau RN (2003). A DNA element recognised by the molybdenum-responsive transcription factor ModE is conserved in Proteobacteria, green sulphur bacteria and Archaea.
BMC Microbiol,
3Abstract:
A DNA element recognised by the molybdenum-responsive transcription factor ModE is conserved in Proteobacteria, green sulphur bacteria and Archaea.
BACKGROUND: the transition metal molybdenum is essential for life. Escherichia coli imports this metal into the cell in the form of molybdate ions, which are taken up via an ABC transport system. In E. coli and other Proteobacteria molybdenum metabolism and homeostasis are regulated by the molybdate-responsive transcription factor ModE. RESULTS: Orthologues of ModE are widespread amongst diverse prokaryotes, but not ubiquitous. We identified probable ModE-binding sites upstream of genes implicated in molybdenum metabolism in green sulphur bacteria and methanogenic Archaea as well as in Proteobacteria. We also present evidence of horizontal transfer of nitrogen fixation genes between green sulphur bacteria and methanogenic Archaea. CONCLUSIONS: Whereas most of the archaeal helix-turn-helix-containing transcription factors belong to families that are Archaea-specific, ModE is unusual in that it is found in both Archaea and Bacteria. Moreover, its cognate upstream DNA recognition sequence is also conserved between Archaea and Bacteria, despite the fundamental differences in their core transcription machinery. ModE is the third example of a transcriptional regulator with a binding signal that is conserved in Bacteria and Archaea.
Abstract.
Author URL.
Studholme DJ, Rawlings ND, Barrett AJ, Bateman A (2003). A comparison of Pfam and MEROPS: two databases, one comprehensive, and one specialised.
BMC Bioinformatics,
4Abstract:
A comparison of Pfam and MEROPS: two databases, one comprehensive, and one specialised.
BACKGROUND: We wished to compare two databases based on sequence similarity: one that aims to be comprehensive in its coverage of known sequences, and one that specialises in a relatively small subset of known sequences. One of the motivations behind this study was quality control. Pfam is a comprehensive collection of alignments and hidden Markov models representing families of proteins and domains. MEROPS is a catalogue and classification of enzymes with proteolytic activity (peptidases or proteases). These secondary databases are used by researchers worldwide, yet their contents are not peer reviewed. Therefore, we hoped that a systematic comparison of the contents of Pfam and MEROPS would highlight missing members and false-positives leading to improvements in quality of both databases. An additional reason for carrying out this study was to explore the extent of consensus in the definition of a protein family. RESULTS: About half (89 out of 174) of the peptidase families in MEROPS overlapped single Pfam families. A further 32 MEROPS families overlapped multiple Pfam families. Where possible, new Pfam families were built to represent most of the MEROPS families that did not overlap Pfam. When comparing the numbers of sequences found in the overlap between a MEROPS family and its corresponding Pfam family, in most cases the overlap was substantial (52 pairs of MEROPS and Pfam families had an intersection size of greater than 75% of the union) but there were some differences in the sets of sequences included in the MEROPS families versus the overlapping Pfam families. CONCLUSIONS: a number of the discrepancies between MEROPS families and their corresponding Pfam families arose from differences in the aims and philosophies of the two databases. Examination of some of the discrepancies highlighted additional members of families, which have subsequently been added in both Pfam and MEROPS. This has led to improvements in the quality of both databases. Overall there was a great deal of consensus between the databases in definitions of a protein family.
Abstract.
Author URL.
Yost CK, Clark KT, Del Bel KL, Hynes MF (2003). Characterization of the nodulation plasmid encoded chemoreceptor gene <i>mcpG</i> from <i>Rhizobium leguminosarum</i> -: art. no. 1.
BMC MICROBIOLOGY,
3 Author URL.
Studholme DJ, Dixon R (2003). Domain architectures of sigma54-dependent transcriptional activators.
J Bacteriol,
185(6), 1757-1767.
Author URL.
2002
Studholme DJ (2002). Enhancer-dependent transcription in Salmonella enterica Typhimurium: new members of the sigmaN regulon inferred from protein sequence homology and predicted promoter sites.
J Mol Microbiol Biotechnol,
4(4), 367-374.
Abstract:
Enhancer-dependent transcription in Salmonella enterica Typhimurium: new members of the sigmaN regulon inferred from protein sequence homology and predicted promoter sites.
DNA-looping mediated by regulatory proteins is a ubiquitous mode of transcriptional control that allows interactions between genetic elements separated over long distances in DNA. In prokaryotes, one of the best-studied examples of regulatory proteins that use DNA-looping is the NtrC family of enhancer-binding proteins (EBPs), which activate transcription from sigmaN (sigma-N, sigma-54) dependent promoters. The completely sequenced genome of food-borne pathogen Salmonella enterica serovar Typhimurium LT2 contains seven novel EBPs of unknown function. Four of these EBPs have a similar domain organisation to NtrC whilst surprisingly the remaining three resemble LevR in Bacillus subtilis. Probable transcriptional targets are identified for each of the EBPs, including novel homologues of phosphotransferase system Enzyme II (EII) and several virulence-associated functions. Comparisons are made with the related enteric bacteria Salmonella Typhi, Escherichia coli and Yersinia pestis.
Abstract.
Author URL.
Elderkin S, Jones S, Schumacher J, Studholme D, Buck M (2002). Mechanism of action of the Escherichia coli phage shock protein PspA in repression of the AAA family transcription factor PspF.
J Mol Biol,
320(1), 23-37.
Abstract:
Mechanism of action of the Escherichia coli phage shock protein PspA in repression of the AAA family transcription factor PspF.
The PspA protein, a negative regulator of the Escherichia coli phage shock psp operon, is produced when virulence factors are exported through secretins in many Gram-negative pathogenic bacteria and its homologue in plants, VIPP1, plays a critical role in thylakoid biogenesis, essential for photosynthesis. Activation of transcription by the enhancer-dependent bacterial sigma(54) containing RNA polymerase occurs through ATP hydrolysis-driven protein conformational changes enabled by activator proteins that belong to the large AAA(+) mechanochemical protein family. We show that PspA directly and specifically acts upon and binds to the AAA(+) domain of the PspF transcription activator. Interactions involving PspF and nucleotide are changed by the action of PspA. These changes and the complexes that form between PspF and PspA can explain how PspA exerts its negative effects upon transcription activated by PspF, and are of significance when considering how activities of other AAA(+) proteins might be controlled.
Abstract.
Author URL.
2000
Studholme DJ, Wigneshwereraraj SR, Gallegos MT, Buck M (2000). Functionality of purified sigma(N) (sigma(54)) and a NifA-like protein from the hyperthermophile Aquifex aeolicus.
J Bacteriol,
182(6), 1616-1623.
Abstract:
Functionality of purified sigma(N) (sigma(54)) and a NifA-like protein from the hyperthermophile Aquifex aeolicus.
The genome sequence of the extremely thermophilic bacterium Aquifex aeolicus encodes alternative sigma factor sigma(N) (sigma(54), RpoN) and five potential sigma(N)-dependent transcriptional activators. Although A. aeolicus possesses no recognizable nitrogenase genes, two of the activators have a high degree of sequence similarity to NifA proteins from nitrogen-fixing proteobacteria. We identified five putative sigma(N)-dependent promoters upstream of operons implicated in functions including sulfur respiration, nitrogen assimilation, nitrate reductase, and nitrite reductase activity. We cloned, overexpressed (in Escherichia coli), and purified A. aeolicus sigma(N) and the NifA homologue, AQ_218. Purified A. aeolicus sigma(N) bound to E. coli core RNA polymerase and bound specifically to a DNA fragment containing E. coli promoter glnHp2 and to several A. aeolicus DNA fragments containing putative sigma(N)-dependent promoters. When combined with E. coli core RNA polymerase, A. aeolicus sigma(N) supported A. aeolicus NifA-dependent transcription from the glnHp2 promoter. The E. coli activator PspFDeltaHTH did not stimulate transcription. The NifA homologue, AQ_218, bound specifically to a DNA sequence centered about 100 bp upstream of the A. aeolicus glnBA operon and so is likely to be involved in the regulation of nitrogen assimilation in this organism. These results argue that the sigma(N) enhancer-dependent transcription system operates in at least one extreme environment, and that the activator and sigma(N) have coevolved.
Abstract.
Author URL.
Studholme DJ, Buck M, Nixon T (2000). Identification of potential sigma(N)-dependent promoters in bacterial genomes.
Microbiology (Reading),
146 Pt 12, 3021-3023.
Author URL.
Studholme DJ, Buck M (2000). Novel roles of sigmaN in small genomes.
Microbiology (Reading),
146 ( Pt 1), 4-5.
Author URL.
Bateman A, Birney E, Durbin R, Eddy SR, Howe KL, Sonnhammer ELL (2000). The Pfam protein families database.
NUCLEIC ACIDS RESEARCH,
28(1), 263-266.
Author URL.
Studholme DJ, Buck M (2000). The alternative sigma factor sigma(28) of the extreme thermophile Aquifex aeolicus restores motility to an Escherichia coli fliA mutant.
FEMS Microbiol Lett,
191(1), 103-107.
Abstract:
The alternative sigma factor sigma(28) of the extreme thermophile Aquifex aeolicus restores motility to an Escherichia coli fliA mutant.
Sigma factor sigma(28) (sigma(F), FliA, SigD) directs RNA polymerase to transcribe the genes required for flagellar biosynthesis and chemotaxis in many bacteria, including Bacillus subtilis, Legionella pneumophila, Salmonella typhimurium, Escherichia coli, Yersinia enterolytica, Treponema maltophilum and Pseudomonas aeruginosa. Remarkably the fliA gene from the extreme thermophile Aquifex aeolicus restored motility to the E. coli mutant at relatively low temperature, albeit partially. This clearly demonstrates that A. aeolicus sigma(28) is able to direct RNA polymerase to E. coli sigma(28)-dependent promoters and take part in the complex interactions required to support transcription of the flagellar apparatus in vivo. The ability of A. aeolicus sigma(28) to function with mesophilic components shows that critical functional interactions made by these sigma factors are well conserved, and are not dependent upon high temperature. We over-produced and purified the sigma(28) protein and demonstrated binding to E. coli core RNA polymerase in vitro. In common with SigD from B. subtilis, but unlike most sigma factors, A. aeolicus sigma(28) showed DNA binding activity in vitro but there was no evidence of sequence specificity. We note that A. aeolicus sigma(28) is a good candidate for structural studies.
Abstract.
Author URL.
Studholme DJ, Buck M (2000). The alternative sigma factor Ï<sup>28</sup>of the extreme thermophile<i>Aquifex aeolicus</i>restores motility to an<i>Escherichia coli fliA</i>mutant. FEMS Microbiology Letters, 191(1), 103-107.
Buck M, Gallegos MT, Studholme DJ, Guo Y, Gralla JD (2000). The bacterial enhancer-dependent sigma(54) (sigma(N)) transcription factor.
J Bacteriol,
182(15), 4129-4136.
Author URL.
Studholme DJ, Buck M (2000). The biology of enhancer-dependent transcriptional regulation in bacteria: insights from genome sequences. FEMS Microbiology Letters, 186(1), 1-9.
Studholme DJ, Buck M (2000). The biology of enhancer-dependent transcriptional regulation in bacteria: insights from genome sequences.
FEMS Microbiol Lett,
186(1), 1-9.
Abstract:
The biology of enhancer-dependent transcriptional regulation in bacteria: insights from genome sequences.
The bacterial transcription factor sigma(N) (sigma-N, sigma-54, RpoN) confers upon RNA polymerase (RNAP) properties distinct from those of the major house-keeping form of RNAP, which contains sigma(70) (sigma-70, RpoD). Transcription by RNAP containing sigma(N) is subject to enhancer-dependent regulation. Far from being an 'oddity' or 'exception to the rule', the occurrence of sigma(N) in the genome sequences of such diverse bacteria as Aquifex aeolicus, Bacillus subtilis, Chlamydia spp. and Borrelia burgdorferi argues for its biological importance. The availability of complete genome sequences of several (eu)bacteria offers an opportunity to extend our understanding of this special form of transcriptional regulation. By scanning their genome sequences, new functions have been predicted for enhancer-dependent transcription in A. aeolicus, Chlamydia trachomatis, Escherichia coli, Treponema pallidum and B. burgdorferi.
Abstract.
Author URL.
1999
Studholme DJ, Jackson RA, Leak DJ (1999). Phylogenetic analysis of transformable strains of thermophilic Bacillus species.
FEMS Microbiol Lett,
172(1), 85-90.
Abstract:
Phylogenetic analysis of transformable strains of thermophilic Bacillus species.
Few strains of thermophilic Bacillus spp are readily transformable with plasmid DNA. Given the considerable phylogenetic and phenotypic diversity amongst thermophilic bacilli, we have examined whether transformability is a trait associated with a particular phylogenetic group, by sequencing the 16S ribosomal RNA genes from transformable strains NUB3621, K1041, and NRRL1174. Although all of these strains were described in the literature as B. stearothermophilus, only NRRL1174 is closely related to the type strain of this species. Based on its 16S rDNA sequence and physiological data K1041 appeared to belong to the species B. thermodenitrificans, while NUB3621 showed a slightly closer relationship to B. thermoglucosidasius than to B. stearothermophilus. Therefore we conclude that the trait of transformability, though possibly strain-specific, is not limited to a single species of thermophilic Bacillus.
Abstract.
Author URL.
Studholme DJ, Jackson RA, Leak DJ (1999). Phylogenetic analysis of transformable strains of thermophilic<i>Bacillus</i>species. FEMS Microbiology Letters, 172(1), 85-90.
Studholme DJ, Finn RD, Chaney MK, Buck M (1999). The C-terminal 12 amino acids of sigma(N) are required for structure and function.
Arch Biochem Biophys,
371(2), 234-240.
Abstract:
The C-terminal 12 amino acids of sigma(N) are required for structure and function.
The sigma(N) protein is an alternative sigma subunit of bacterial RNA polymerase. We investigated the role of a 12-amino-acid "tail" at the C-terminus of Klebsiella pneumoniae sigma(N), which was predicted to be largely surface-exposed and to be mostly loop (that is not alpha-helical or beta-strand). Deletion of this tail from N-terminal hexahistidine-tagged sigma(N) led to loss of sigma(N)-dependent transcription activity in vivo. We overexpressed and purified this deletion-mutant protein for in vitro characterization. The purified deleted protein showed decreased RNA polymerase core- and DNA-binding activities compared to the full-length protein and transcription activity was greatly impaired. Furthermore, evidence from circular dichroism and protease digestion experiments together suggested that deletion of the C-terminus tail resulted in a loss of conformational constraint in the protein. We discuss a possible structural role for the 12 amino acids at the C-terminus of sigma(N).
Abstract.
Author URL.
