Publications by year
In Press
Steinberg, G. Schliwa, M. (In Press). A molecular motor for microtubule-dependent organelle transport in Neurospora crassa. In Jockusch BM, Mandelkow E, Weber K (Eds.) 45. Colloquium Mosbach: the Cytoskeleton, Berlin, Heidelberg: Springer, 23-28.
Steinberg G, Kilaru S, Schuster M, Ma W (In Press). Fluorescent markers of various organelles in the wheat pathogen Zymoseptoria tritici. Fungal Genetics and Biology
2022
Cannon S, Kay W, Kilaru S, Schuster M, Gurr SJ, Steinberg G (2022). Multi-site fungicides suppress banana Panama disease, caused by Fusarium oxysporum f. sp. cubense Tropical Race 4.
PLOS Pathogens,
18(10), e1010860-e1010860.
Abstract:
Multi-site fungicides suppress banana Panama disease, caused by Fusarium oxysporum f. sp. cubense Tropical Race 4
Global banana production is currently challenged by Panama disease, caused by Fusarium oxysporum f.sp. cubense Tropical Race 4 (FocTR4). There are no effective fungicide-based strategies to control this soil-borne pathogen. This could be due to insensitivity of the pathogen to fungicides and/or soil application per se. Here, we test the effect of 12 single-site and 9 multi-site fungicides against FocTR4 and Foc Race1 (FocR1) in quantitative colony growth, and cell survival assays in purified FocTR4 macroconidia, microconidia and chlamydospores. We demonstrate that these FocTR4 morphotypes all cause Panama disease in bananas. These experiments reveal innate resistance of FocTR4 to all single-site fungicides, with neither azoles, nor succinate dehydrogenase inhibitors (SDHIs), strobilurins or benzimidazoles killing these spore forms. We show in fungicide-treated hyphae that this innate resistance occurs in a subpopulation of "persister" cells and is not genetically inherited. FocTR4 persisters respond to 3 μg ml-1 azoles or 1000 μg ml-1 strobilurins or SDHIs by strong up-regulation of genes encoding target enzymes (up to 660-fold), genes for putative efflux pumps and transporters (up to 230-fold) and xenobiotic detoxification enzymes (up to 200-fold). Comparison of gene expression in FocTR4 and Zymoseptoria tritici, grown under identical conditions, reveals that this response is only observed in FocTR4. In contrast, FocTR4 shows little innate resistance to most multi-site fungicides. However, quantitative virulence assays, in soil-grown bananas, reveals that only captan (20 μg ml-1) and all lipophilic cations (200 μg ml-1) suppress Panama disease effectively. These fungicides could help protect bananas from future yield losses by FocTR4.
Abstract.
Kilaru S, Fantozzi E, Cannon S, Schuster M, Chaloner TM, Guiu-Aragones C, Gurr SJ, Steinberg G (2022). Zymoseptoria tritici white-collar complex integrates light, temperature and plant cues to initiate dimorphism and pathogenesis.
Nature Communications,
13(1).
Abstract:
Zymoseptoria tritici white-collar complex integrates light, temperature and plant cues to initiate dimorphism and pathogenesis
AbstractTransitioning from spores to hyphae is pivotal to host invasion by the plant pathogenic fungus Zymoseptoria tritici. This dimorphic switch can be initiated by high temperature in vitro (~27 °C); however, such a condition may induce cellular heat stress, questioning its relevance to field infections. Here, we study the regulation of the dimorphic switch by temperature and other factors. Climate data from wheat-growing areas indicate that the pathogen sporadically experiences high temperatures such as 27 °C during summer months. However, using a fluorescent dimorphic switch reporter (FDR1) in four wild-type strains, we show that dimorphic switching already initiates at 15–18 °C, and is enhanced by wheat leaf surface compounds. Transcriptomics reveals 1261 genes that are up- or down-regulated in hyphae of all strains. These pan-strain core dimorphism genes (PCDGs) encode known effectors, dimorphism and transcription factors, and light-responsive proteins (velvet factors, opsins, putative blue light receptors). An FDR1-based genetic screen reveals a crucial role for the white-collar complex (WCC) in dimorphism and virulence, mediated by control of PCDG expression. Thus, WCC integrates light with biotic and abiotic cues to orchestrate Z. tritici infection.
Abstract.
2021
Fantozzi E, Kilaru S, Gurr SJ, Steinberg G (2021). Asynchronous development of Zymoseptoria tritici infection in wheat.
Fungal Genet Biol,
146Abstract:
Asynchronous development of Zymoseptoria tritici infection in wheat.
The fungus Zymoseptoria tritici causes Septoria tritici blotch of wheat. Pathogenicity begins with spore germination, followed by stomata invasion by hyphae, mesophyll colonization and fruiting body formation. It was previously found that entry into the plant via stomata occurs in a non-synchronized way over several days, while later developmental steps, such as early and late fruiting body formation, were reported to follow each other in time. This suggests synchronization of the pathogen population in planta prior to sporulation. Here, we image a fluorescent Z. tritici IPO323-derived strain during infection. We describe 6 morphologically distinct developmental stages, and determine their abundance in infected leaves, with time post inoculation. This demonstrates that 3-5 stages co-exist in infected tissues at any given time. Thus, later stages of pathogen development also occur asynchronously amongst the population of infecting cells. This merits consideration when interpreting transcriptomics or proteomics data gathered from infected plants.
Abstract.
Author URL.
Fantozzi E, Kilaru S, Cannon S, Schuster M, Gurr SJ, Steinberg G (2021). Conditional promoters to investigate gene function during wheat infection by Zymoseptoria tritici.
Fungal Genet Biol,
146Abstract:
Conditional promoters to investigate gene function during wheat infection by Zymoseptoria tritici.
The fungus Zymoseptoria tritici causes Septoria tritici leaf blotch, which poses a serious threat to temperate-grown wheat. Recently, we described a raft of molecular tools to study the biology of this fungus in vitro. Amongst these are 5 conditional promoters (Pnar1, Pex1A, Picl1, Pgal7, PlaraB), which allow controlled over-expression or repression of target genes in cells grown in liquid culture. However, their use in the host-pathogen interaction in planta was not tested. Here, we investigate the behaviour of these promoters by quantitative live cell imaging of green-fluorescent protein-expressing cells during 6 stages of the plant infection process. We show that Pnar1 and Picl1 are repressed in planta and demonstrate their suitability for studying essential gene expression and function in plant colonisation. The promoters Pgal7 and Pex1A are not fully-repressed in planta, but are induced during pycnidiation. This indicates the presence of inducing galactose or xylose and/or arabinose, released from the plant cell wall by the activity of fungal hydrolases. In contrast, the PlaraB promoter, which normally controls expression of an α-l-arabinofuranosidase B, is strongly induced inside the leaf. This suggests that the fungus is exposed to L-arabinose in the mesophyll apoplast. Taken together, this study establishes 2 repressible promoters (Pnar1 and Picl1) and three inducible promoters (Pgal7, Pex1A, PlaraB) for molecular studies in planta. Moreover, we provide circumstantial evidence for plant cell wall degradation during the biotrophic phase of Z. tritici infection.
Abstract.
Author URL.
Lin C, Ashwin P, Steinberg G (2021). Modelling the motion of organelles in an elongated cell
via the coordination of heterogeneous drift–diffusion and
long-range transport. The European Physical Journal E: soft Matter and Biological Physics
2020
Steinberg G, Schuster M, Gurr SJ, Schrader TA, Schrader M, Wood M, Early A, Kilaru S (2020). A lipophilic cation protects crops against fungal pathogens by multiple modes of action.
Nat Commun,
11(1).
Abstract:
A lipophilic cation protects crops against fungal pathogens by multiple modes of action.
The emerging resistance of crop pathogens to fungicides poses a challenge to food security and compels discovery of new antifungal compounds. Here, we show that mono-alkyl lipophilic cations (MALCs) inhibit oxidative phosphorylation by affecting NADH oxidation in the plant pathogens Zymoseptoria tritici, Ustilago maydis and Magnaporthe oryzae. One of these MALCs, consisting of a dimethylsulfonium moiety and a long alkyl chain (C18-SMe2+), also induces production of reactive oxygen species at the level of respiratory complex I, thus triggering fungal apoptosis. In addition, C18-SMe2+ activates innate plant defense. This multiple activity effectively protects cereals against Septoria tritici blotch and rice blast disease. C18-SMe2+ has low toxicity in Daphnia magna, and is not mutagenic or phytotoxic. Thus, MALCs hold potential as effective and non-toxic crop fungicides.
Abstract.
Author URL.
Wu H, Zhang W, Schuster M, Moch M, Windoffer R, Steinberg G, Staiger CJ, Panstruga R (2020). Alloxan Disintegrates the Plant Cytoskeleton and Suppresses mlo-Mediated Powdery Mildew Resistance.
Plant Cell Physiol,
61(3), 505-518.
Abstract:
Alloxan Disintegrates the Plant Cytoskeleton and Suppresses mlo-Mediated Powdery Mildew Resistance.
Recessively inherited mutant alleles of Mlo genes (mlo) confer broad-spectrum penetration resistance to powdery mildew pathogens in angiosperm plants. Although a few components are known to be required for mlo resistance, the detailed molecular mechanism underlying this type of immunity remains elusive. In this study, we identified alloxan (5,5-dihydroxyl pyrimidine-2,4,6-trione) and some of its structural analogs as chemical suppressors of mlo-mediated resistance in monocotyledonous barley (Hordeum vulgare) and dicotyledonous Arabidopsis thaliana. Apart from mlo resistance, alloxan impairs nonhost resistance in Arabidopsis. Histological analysis revealed that the chemical reduces callose deposition and hydrogen peroxide accumulation at attempted fungal penetration sites. Fluorescence microscopy revealed that alloxan interferes with the motility of cellular organelles (peroxisomes, endosomes and the endoplasmic reticulum) and the pathogen-triggered redistribution of the PEN1/SYP121 t-SNARE protein. These cellular defects are likely the consequence of disassembly of actin filaments and microtubules upon alloxan treatment. Similar to the situation in animal cells, alloxan elicited the temporary accumulation of reactive oxygen species (ROS) in cotyledons and rosette leaves of Arabidopsis plants. Our results suggest that alloxan may destabilize cytoskeletal architecture via induction of an early transient ROS burst, further leading to the failure of molecular and cellular processes that are critical for plant immunity.
Abstract.
Author URL.
Fones HN, Bebber DP, Chaloner TM, Kay WT, Steinberg G, Gurr SJ (2020). Author Correction: Threats to global food security from emerging fungal and oomycete crop pathogens. Nature Food, 1(7), 455-456.
Schuster M, Guiu-Aragones C, Steinberg G (2020). Class V chitin synthase and β(1,3)-glucan synthase co-travel in the same vesicle in Zymoseptoria tritici.
Fungal Genet Biol,
135Abstract:
Class V chitin synthase and β(1,3)-glucan synthase co-travel in the same vesicle in Zymoseptoria tritici.
The fungal cell wall consists of proteins and polysaccharides, formed by the co-ordinated activity of enzymes, such as chitin or glucan synthases. These enzymes are delivered via secretory vesicles to the hyphal tip. In the ascomycete Neurospora crassa, chitin synthases and β(1,3)-glucan synthase are transported in different vesicles, whereas they co-travel along microtubules in the basidiomycete Ustilago maydis. This suggests fundamental differences in wall synthesis between taxa. Here, we visualize the class V chitin synthase ZtChs5 and the β(1,3)-glucan synthase ZtGcs1 in the ascomycete Zymoseptoria tritici. Live cell imaging demonstrate that both enzymes co-locate to the apical plasma membrane, but are not concentrated in the Spitzenkörper. Delivery involves co-transport along microtubules of the chitin and glucan synthase. Live cell imaging and electron microscopy suggest that both cell wall synthases locate in the same vesicle. Thus, microtubule-dependent co-delivery of cell wall synthases in the same vesicle is found in asco- and basidiomycetes.
Abstract.
Author URL.
Steinberg G, Gurr SJ (2020). Fungi, fungicide discovery and global food security.
Fungal Genet Biol,
144Abstract:
Fungi, fungicide discovery and global food security.
Securing sufficient food for a growing world population is of paramount importance for social stability and the well-being of mankind. Recently, it has become evident that fungal pathogens pose the greatest biotic challenge to our calorie crops. Moreover, the loss of commodity crops to fungal disease destabilises the economies of developing nations, thereby increasing the dimension of the threat. Our best weapon to control these pathogens is fungicides, but increasing resistance puts us in an arms race against them. New anti-fungal compounds need to be discovered, such as mono-alky lipophilic cations (MALCs) described herein. Collaborations between academia and industry are imperative to establish new and efficient ways to develop these new fungicides and to bring them to the market-place.
Abstract.
Author URL.
Kilaru S, Schuster M, Murray R, Steinberg G (2020). Optimal timing for Agrobacterium-mediated DNA transformation of Trichoderma reesei conidia revealed by live cell imaging.
Fungal Genet Biol,
142Abstract:
Optimal timing for Agrobacterium-mediated DNA transformation of Trichoderma reesei conidia revealed by live cell imaging.
Trichoderma reesei is the foremost fungal producer of enzymes for industrial processes. Here, we use fluorescent live cell imaging of germinating conidia to improve Agrobacterium tumefaciens-mediated transformation (ATMT) efficiency. We define the timing of (a) morphological changes and (b) nuclear reorganisation during initial conidia germination. This reveals that conidia swell for 7 h, during which nuclei undergo 2 non-synchronised mitotic divisions. Histones are recruited to the nucleus during the first 2 h, suggesting that conidia enter S-phase immediately after activation. This correlates with a significantly increased ATMT efficiency at 2 h after germination initiation. This finding promises to improve genetic manipulation efficiency in T. reesei.
Abstract.
Author URL.
Kilaru S, Schuster M, Cannon S, Steinberg G (2020). Optimised red- and green-fluorescent proteins for live cell imaging in the industrial enzyme-producing fungus Trichoderma reesei.
Fungal Genet Biol,
138Abstract:
Optimised red- and green-fluorescent proteins for live cell imaging in the industrial enzyme-producing fungus Trichoderma reesei.
The filamentous fungus Trichoderma reesei is a major source of cellulolytic enzymes in biofuel production. Despite its economic relevance, our understanding of its secretory pathways is fragmentary. A major challenge is to visualise the dynamic behaviour of secretory vesicles in living cells. To this end, we establish a location juxtaposing the succinate dehydrogenase locus as a "soft-landing" site for controlled expression of 4 green-fluorescent and 5 red-fluorescent protein-encoding genes (GFPs, RFPs). Quantitative and comparative analysis of their fluorescent signals in living cells demonstrates that codon-optimised monomeric superfolder GFP (TrmsGFP) and codon-optimised mCherry (TrmCherry) combine highest signal intensity with significantly improved signal-to-noise ratios. Finally, we show that integration of plasmid near the sdi1 locus does not affect secretion of cellulase activity in RUT-C30. The molecular and live cell imaging tools generated in this study will help our understanding the secretory pathway in the industrial fungus T. reesei.
Abstract.
Author URL.
Schuster M, Steinberg G (2020). The fungicide dodine primarily inhibits mitochondrial respiration in Ustilago maydis, but also affects plasma membrane integrity and endocytosis, which is not found in Zymoseptoria tritici.
Fungal Genet Biol,
142Abstract:
The fungicide dodine primarily inhibits mitochondrial respiration in Ustilago maydis, but also affects plasma membrane integrity and endocytosis, which is not found in Zymoseptoria tritici.
Early reports in the fungus Ustilago maydis suggest that the amphipathic fungicide dodine disrupts the fungal plasma membrane (PM), thereby killing this corn smut pathogen. However, a recent study in the wheat pathogen Zymoseptoria tritici does not support such mode of action (MoA). Instead, dodine inhibits mitochondrial ATP-synthesis, both in Z. tritici and U. maydis. This casts doubt on an fungicidal activity of dodine at the PM. Here, we use a cell biological approach and investigate further the effect of dodine on the plasma membrane in both fungi. We show that dodine indeed breaks the integrity of the PM in U. maydis, indicated by a concentration-dependent cell depolarization. In addition, the fungicide reduces PM fluidity and arrests endocytosis by inhibiting the internalization of endocytic vesicles at the PM. This is likely due to impaired recruitment of the actin-crosslinker fimbrin to endocytic actin patches. However, quantitative data reveal that the effect on mitochondria represents the primary MoA in U. maydis. None of these plasma membrane-associated effects were found in dodine-treated Z. tritici cells. Thus, the physiological effect of an anti-fungal chemistry can differ between pathogens. This merits consideration when characterizing a given fungicide.
Abstract.
Author URL.
Fones HN, Bebber DP, Chaloner TM, Kay WT, Steinberg G, Gurr SJ (2020). Threats to global food security from emerging fungal and oomycete crop pathogens. Nature Food, 1(6), 332-342.
2019
Derbyshire MC, Gohari AM, Mehrabi R, Kilaru S, Steinberg G, Ali S, Bailey A, Hammond-Kosack K, Kema GHJ, Rudd JJ, et al (2019). Phosphopantetheinyl transferase (Ppt)-mediated biosynthesis of lysine, but not siderophores or DHN melanin, is required for virulence of Zymoseptoria tritici on wheat (vol 8, 17069, 2018).
SCIENTIFIC REPORTS,
9 Author URL.
2018
Derbyshire MC, Gohari AM, Mehrabi R, Kilaru S, Steinberg G, Ali S, Bailey A, Hammond-Kosack K, Kema GHJ, Rudd JJ, et al (2018). Phosphopantetheinyl transferase (Ppt)-mediated biosynthesis of lysine, but not siderophores or DHN melanin, is required for virulence of Zymoseptoria tritici on wheat.
Scientific Reports,
8(1).
Abstract:
Phosphopantetheinyl transferase (Ppt)-mediated biosynthesis of lysine, but not siderophores or DHN melanin, is required for virulence of Zymoseptoria tritici on wheat
Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB) disease of wheat. Z. tritici is an apoplastic fungal pathogen, which does not penetrate plant cells at any stage of infection, and has a long initial period of symptomless leaf colonisation. During this phase it is unclear to what extent the fungus can access host plant nutrients or communicate with plant cells. Several important primary and secondary metabolite pathways in fungi are regulated by the post-translational activator phosphopantetheinyl transferase (Ppt) which provides an essential co-factor for lysine biosynthesis and the activities of non-ribosomal peptide synthases (NRPS) and polyketide synthases (PKS). To investigate the relative importance of lysine biosynthesis, NRPS-based siderophore production and PKS-based DHN melanin biosynthesis, we generated deletion mutants of ZtPpt. The ∆ZtPpt strains were auxotrophic for lysine and iron, non-melanised and non-pathogenic on wheat. Deletion of the three target genes likely affected by ZtPpt loss of function (Aar- lysine; Nrps1-siderophore and Pks1- melanin), highlighted that lysine auxotrophy was the main contributing factor for loss of virulence, with no reduction caused by loss of siderophore production or melanisation. This reveals Ppt, and the lysine biosynthesis pathway, as potential targets for fungicides effective against Z. tritici.
Abstract.
Duan YB, Yang Y, Wang JX, Chen CJ, Steinberg G, Fraaije BA, Zhou MG (2018). Simultaneous Detection of Multiple Benzimidazole-Resistant β-Tubulin Variants of Botrytis cinerea using Loop-Mediated Isothermal Amplification.
Plant Dis,
102(10), 2016-2024.
Abstract:
Simultaneous Detection of Multiple Benzimidazole-Resistant β-Tubulin Variants of Botrytis cinerea using Loop-Mediated Isothermal Amplification.
Optimal disease management depends on the ability to monitor the development of fungicide resistance in plant pathogen populations. Benzimidazole resistance is caused by the point mutations of the β-tubulin gene in Botrytis cinerea, and three mutations (E198A, E198K, and E198V) at codon 198 account for more than 98% of all resistant strains. Although traditional methods remain a cornerstone in monitoring fungicide resistance, molecular methods that do not require the isolation of pathogens can detect resistance alleles present at low frequencies, and require less time and labor than traditional methods. In this study, we present an efficient, rapid, and highly specific method for detecting highly benzimidazole-resistant B. cinerea isolates based on loop-mediated isothermal amplification (LAMP). By using specific primers, we could simultaneously detect all three resistance-conferring mutations at codon 198. The LAMP reaction components and conditions were optimized, and the best reaction temperatures and times were 60 to 62°C and 45 min, respectively. When B. cinerea field isolates were assessed for benzimidazole resistance, similar results were obtained with LAMP, minimal inhibition concentration, and sequencing. The LAMP assay developed in the current study was highly suitable for detection of highly benzimidazole-resistant field isolates of B. cinerea.
Abstract.
Author URL.
2017
Steinberg G, Schuster M, Hacker C, Kilaru S, Correia A (2017). ATP prevents Woronin bodies from sealing septal pores in unwounded cells of the fungus Zymoseptoria tritici.
Cell Microbiol,
19(11).
Abstract:
ATP prevents Woronin bodies from sealing septal pores in unwounded cells of the fungus Zymoseptoria tritici.
Septa of filamentous ascomycetes are perforated by septal pores that allow communication between individual hyphal compartments. Upon injury, septal pores are plugged rapidly by Woronin bodies (WBs), thereby preventing extensive cytoplasmic bleeding. The mechanism by which WBs translocate into the pore is not known, but it has been suggested that wound-induced cytoplasmic bleeding "flushes" WBs into the septal opening. Alternatively, contraction of septum-associated tethering proteins may pull WBs into the septal pore. Here, we investigate WB dynamics in the wheat pathogen Zymoseptoria tritici. Ultrastructural studies showed that 3.4 ± 0.2 WBs reside on each side of a septum and that single WBs of 128.5 ± 3.6 nm in diameter seal the septal pore (41 ± 1.5 nm). Live cell imaging of green fluorescent ZtHex1, a major protein in WBs, and the integral plasma membrane protein ZtSso1 confirms WB translocation into the septal pore. This was associated with the occasional formation of a plasma membrane "balloon," extruding into the dead cell, suggesting that the plasma membrane rapidly seals the wounded septal pore wound. Minor amounts of fluorescent ZtHex1-enhanced green fluorescent protein (eGFP) appeared associated with the "ballooning" plasma membrane, indicating that cytoplasmic ZtHex1-eGFP is recruited to the extending plasma membrane. Surprisingly, in ~15% of all cases, WBs moved from the ruptured cell into the septal pore. This translocation against the cytoplasmic flow suggests that an active mechanism drives WB plugging. Indeed, treatment of unwounded and intact cells with the respiration inhibitor carbonyl cyanide m-chlorophenyl hydrazone induced WB translocation into the pores. Moreover, carbonyl cyanide m-chlorophenyl hydrazone treatment recruited cytoplasmic ZtHex1-eGFP to the lateral plasma membrane of the cells. Thus, keeping the WBs out of the septal pores, in Z. tritici, is an ATP-dependent process.
Abstract.
Author URL.
Steinberg G, Peñalva MA, Riquelme M, Wösten HA, Harris SD (2017). Cell Biology of Hyphal Growth.
Microbiol Spectr,
5(2).
Abstract:
Cell Biology of Hyphal Growth.
Filamentous fungi are a large and ancient clade of microorganisms that occupy a broad range of ecological niches. The success of filamentous fungi is largely due to their elongate hypha, a chain of cells, separated from each other by septa. Hyphae grow by polarized exocytosis at the apex, which allows the fungus to overcome long distances and invade many substrates, including soils and host tissues. Hyphal tip growth is initiated by establishment of a growth site and the subsequent maintenance of the growth axis, with transport of growth supplies, including membranes and proteins, delivered by motors along the cytoskeleton to the hyphal apex. Among the enzymes delivered are cell wall synthases that are exocytosed for local synthesis of the extracellular cell wall. Exocytosis is opposed by endocytic uptake of soluble and membrane-bound material into the cell. The first intracellular compartment in the endocytic pathway is the early endosomes, which emerge to perform essential additional functions as spatial organizers of the hyphal cell. Individual compartments within septated hyphae can communicate with each other via septal pores, which allow passage of cytoplasm or organelles to help differentiation within the mycelium. This article introduces the reader to more detailed aspects of hyphal growth in fungi.
Abstract.
Author URL.
Steinberg G, Penalva MA, Riquelme M, Wösten HA, Harris SD (2017). Cell biology of hyphal growth. In (Ed)
The Fungal Kingdom, 231-265.
Abstract:
Cell biology of hyphal growth
Abstract.
Lin C, Steinberg G (2017). Spatial organization of organelles in fungi: Insights from mathematical modelling.
Fungal Genet Biol,
103, 55-59.
Abstract:
Spatial organization of organelles in fungi: Insights from mathematical modelling.
Mathematical modelling in cellular systems aims to describe biological processes in a quantitative manner. Most accurate modelling is based on robust experimental data. Here we review recent progress in the theoretical description of motor behaviour, early endosome motility, ribosome distribution and peroxisome transport in the fungal model system Ustilago maydis and illustrate the power of modelling in our quest to understand molecular details and cellular roles of membrane trafficking in filamentous fungi.
Abstract.
Author URL.
Geoghegan I, steinberg, gurr (2017). The Role of the Fungal Cell Wall in the Infection of Plants. Trends in Microbiology
Steinberg G, Harmer NJ, Schuster M, Kilaru S (2017). Woronin body-based sealing of septal pores.
Fungal Genet Biol,
109, 53-55.
Abstract:
Woronin body-based sealing of septal pores.
In ascomycete fungi, hyphal cells are separated by perforate septa, which allow cell-to-cell communication. To protect against extensive wound-induced damage, septal pores are sealed by peroxisome-derived Woronin bodies (WBs). The mechanism underpinning WB movement is unknown, but cytoplasmic bulk flow may "flush" WBs into the pore. However, some studies suggest a controlled and active mechanism of WB movement. Indeed, in the wheat pathogen Zymoseptoria tritici cellular ATP prevents WBs from pore sealing in unwounded cells. Thus, cells appear to exert active control over WB closure. Here, we summarize our current understanding of WB-based pore sealing in ascomycete fungi.
Abstract.
Author URL.
2016
Lin C, Schuster M, Guimaraes SC, Ashwin P, Schrader M, Metz J, Hacker C, Gurr SJ, Steinberg G (2016). Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells.
Nat Commun,
7Abstract:
Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells.
Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes.
Abstract.
Author URL.
Schuster M, Martin-Urdiroz M, Higuchi Y, Hacker C, Kilaru S, Gurr SJ, Steinberg G (2016). Co-Delivery of Cell-Wall-Forming enzymes in the same vesicle for coordinated fungal cell wall formation.
Nature Microbiology,
1Abstract:
Co-Delivery of Cell-Wall-Forming enzymes in the same vesicle for coordinated fungal cell wall formation
Fungal cells are surrounded by an extracellular cell wall. This complex matrix of proteins and polysaccharides protects against adverse stresses and determines the shape of fungal cells. The polysaccharides of the fungal wall include 1,3-β-glucan and chitin, which are synthesized by membrane-bound synthases at the growing cell tip. A hallmark of filamentous fungi is the class V chitin synthase, which carries a myosin-motor domain. In the corn smut fungus Ustilago maydis, the myosin-chitin synthase Mcs1 moves to the plasma membrane in secretory vesicles, being delivered by kinesin-1 and myosin-5. The myosin domain of Mcs1 enhances polar secretion by tethering vesicles at the site of exocytosis. It remains elusive, however, how other cell-wall-forming enzymes are delivered and how their activity is coordinated post secretion. Here, we show that the U. maydis class VII chitin synthase and 1,3-β-glucan synthase travel in Mcs1-containing vesicles, and that their apical secretion depends on Mcs1. Once in the plasma membrane, anchorage requires enzyme activity, which suggests co-synthesis of chitin and 1,3-β-glucan polysaccharides at sites of exocytosis. Thus, delivery of cell-wall-forming enzymes in Mcs1 vesicles ensures local foci of fungal cell wall formation.
Abstract.
Steinberg G (2016). Editorial overview: Parasitic and fungal diseases.
Curr Opin Microbiol,
34, v-vi.
Author URL.
Steinberg G (2016). The mechanism of peroxisome motility in filamentous fungi.
Fungal Genet Biol,
97, 33-35.
Abstract:
The mechanism of peroxisome motility in filamentous fungi.
Peroxisomes (POs) are an essential part of the fungal cell's inventory. They are involved in cellular lipid homeostasis, reactive oxygen metabolism and in the synthesis of secondary metabolites. These functions are thought to require frequent organelle-organelle interactions and the even-distribution of POs in fungal hypha. Recent work in the basidiomycete Ustilago maydis and the ascomycete Aspergillus nidulans reveals a crucial role of early endosomes (EEs) in the dynamic behavior of POs required for their cellular distribution and interaction. This article summarizes the main findings, which provided unexpected insight into the mechanism by which fungal cells organize themselves.
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.
Kilaru S, Schuster M, Latz M, Das Gupta S, Steinberg N, Fones H, Gurr SJ, Talbot NJ, Steinberg G (2015). A gene locus for targeted ectopic gene integration in Zymoseptoria tritici.
Fungal Genet Biol,
79, 118-124.
Abstract:
A gene locus for targeted ectopic gene integration in Zymoseptoria tritici.
Understanding the cellular organization and biology of fungal pathogens requires accurate methods for genomic integration of mutant alleles or fluorescent fusion-protein constructs. In Zymoseptoria tritici, this can be achieved by integrating of plasmid DNA randomly into the genome of this wheat pathogen. However, untargeted ectopic integration carries the risk of unwanted side effects, such as altered gene expression, due to targeting regulatory elements, or gene disruption following integration into protein-coding regions of the genome. Here, we establish the succinate dehydrogenase (sdi1) locus as a single "soft-landing" site for targeted ectopic integration of genetic constructs by using a carboxin-resistant sdi1(R) allele, carrying the point-mutation H267L. We use various green and red fluorescent fusion constructs and show that 97% of all transformants integrate correctly into the sdi1 locus as single copies. We also demonstrate that such integration does not affect the pathogenicity of Z. tritici, and thus the sdi1 locus is a useful tool for virulence analysis in genetically modified Z. tritici strains. Furthermore, we have developed a vector which facilitates yeast recombination cloning and thus allows assembly of multiple overlapping DNA fragments in a single cloning step for high throughput vector and strain generation.
Abstract.
Author URL.
Steinberg G (2015). Cell biology of Zymoseptoria tritici: Pathogen cell organization and wheat infection.
Fungal Genet Biol,
79, 17-23.
Abstract:
Cell biology of Zymoseptoria tritici: Pathogen cell organization and wheat infection.
Cell biological research in the wheat pathogen Zymoseptoria tritici (formerly Mycosphaerella graminicola) has led to a good understanding of the histology of the infection process. Expression profiling and bioinformatic approaches, combined with molecular studies on signaling pathways, effectors and potential necrosis factors provides first insight into the complex interplay between the host and the pathogen. Cell biological studies will help to further our understanding of the infection strategy of the fungus. The cellular organization and intracellular dynamics of the fungus itself is largely unexplored. Insight into essential cellular processes within the pathogen will expand our knowledge of the basic biology of Z. tritici, thereby providing putative new anti-fungal targets.
Abstract.
Author URL.
Kilaru S, Ma W, Schuster M, Courbot M, Steinberg G (2015). Conditional promoters for analysis of essential genes in Zymoseptoria tritici.
Fungal Genet Biol,
79, 166-173.
Abstract:
Conditional promoters for analysis of essential genes in Zymoseptoria tritici.
Development of new fungicides, needed for sustainable control of fungal plant pathogens, requires identification of novel anti-fungal targets. Essential fungal-specific proteins are good candidates, but due to their importance, gene deletion mutants are not viable. Consequently, their cellular role often remains elusive. This hindrance can be overcome by the use of conditional mutants, where expression is controlled by an inducible/repressible promoter. Here, we introduce 5 inducible/repressible promoter systems to study essential genes in the wheat pathogen Zymoseptoria tritici. We fused the gene for enhanced green-fluorescent protein (egfp) to the promoter region of Z. tritici nitrate reductase (Pnar1; induced by nitrogen and repressed by ammonium), 1,4-β-endoxylanase a (Pex1A; induced by xylose and repressed by maltodextrin), l-arabinofuranosidase B (PlaraB; induced by arabinose and repressed by glucose), galactose-1-phosphate uridylyltransferase 7 (Pgal7; induced by galactose and repressed by glucose) and isocitrate lyase (Picl1; induced by sodium acetate and repressed by glucose). This was followed by quantitative analysis of cytoplasmic reporter fluorescence under induced and repressed conditions. We show that Pnar1, PlaraB and Pex1A drive very little or no egfp expression when repressed, but induce moderate protein production when induced. In contrast, Pgal7 and Picl1 show considerable egfp expression when repressed, and were strongly induced in the presence of their inducers. Normalising the expression levels of all promoters to that of the α-tubulin promoter Ptub2 revealed that PlaraB was the weakest promoter (∼20% of Ptub2), whereas Picl1 strongly expressed the reporter (∼250% of Ptub2). The use of these tools promises a better understanding of essential genes, which will help developing novel control strategies that protect wheat from Z. tritici.
Abstract.
Author URL.
Higuchi Y, Steinberg G (2015). Early endosomes motility in filamentous fungi: How and why they move.
Fungal Biology Reviews,
29(1), 1-6.
Abstract:
Early endosomes motility in filamentous fungi: How and why they move
Elongate hyphae of filamentous fungi grow predominantly at their tips, whereas organelles are positioned in the subapical parts of the cell. Organelle positioning and long-distance intracellular communication involves active, energy-dependent transport along microtubules (MTs). This is mediated by specialized molecular motors, named kinesins and dynein, which utilize ATP hydrolysis to "walk" along the tubulin polymers. Work in the basidiomycete Ustilago maydis and the ascomycete Aspergillus nidulans has shown that early endosomes (EEs) are one of the major cargos of MT-dependent motors in fungi. EEs are part of the early endocytic pathway, and their motility behavior and the underlying transport machinery is well understood. However, the physiological role of constant bi-directional EE motility remains elusive. Recent reports, conducted in the corn smut fungus U. maydis, have provided novel insights into the cellular function of EE motility. They show that EE motility is crucial for the distribution of the protein synthesis machinery, and also that EEs transmit signals during plant infection that trigger the production of fungal effector proteins, required for successful invasion into host plants.
Abstract.
Mehrabi R, Mirzadi Gohari A, da Silva GF, Steinberg G, Kema GHJ, de Wit PJGM (2015). Flexible gateway constructs for functional analyses of genes in plant pathogenic fungi.
Fungal Genet Biol,
79, 186-192.
Abstract:
Flexible gateway constructs for functional analyses of genes in plant pathogenic fungi.
Genetic manipulation of fungi requires quick, low-cost, efficient, high-throughput and molecular tools. In this paper, we report 22 entry constructs as new molecular tools based on the Gateway technology facilitating rapid construction of binary vectors that can be used for functional analysis of genes in fungi. The entry vectors for single, double or triple gene-deletion mutants were developed using hygromycin, geneticin and nourseothricin resistance genes as selection markers. Furthermore, entry vectors containing green fluorescent (GFP) or red fluorescent (RFP) in combination with hygromycin, geneticin or nourseothricin selection markers were generated. The latter vectors provide the possibility of gene deletion and simultaneous labelling of the fungal transformants with GFP or RFP reporter genes. The applicability of a number of entry vectors was validated in Zymoseptoria tritici, an important fungal wheat pathogen.
Abstract.
Author URL.
Guo M, Kilaru S, Schuster M, Latz M, Steinberg G (2015). Fluorescent markers for the Spitzenkörper and exocytosis in Zymoseptoria tritici.
Fungal Genet Biol,
79, 158-165.
Abstract:
Fluorescent markers for the Spitzenkörper and exocytosis in Zymoseptoria tritici.
Fungal hyphae are highly polarized cells that invade their substrate by tip growth. In plant pathogenic fungi, hyphal growth is essential for host invasion. This makes polarity factors and secretion regulators potential new targets for novel fungicides. Polarization requires delivery of secretory vesicles to the apical Spitzenkörper, followed by polarized exocytosis at the expanding cell tip. Here, we introduce fluorescent markers to visualize the apical Spitzenkörper and the apical site of exocytosis in hyphae of the wheat pathogen Zymoseptoria tritici. We fused green fluorescent protein to the small GTPase ZtSec4, the myosin light chain ZtMlc1 and the small GTPase ZtRab11 and co-localize the fusion proteins with the dye FM4-64 in the hyphal apex, suggesting that the markers label the hyphal Spitzenkörper in Z. tritici. In addition, we localize GFP-fusions to the exocyst protein ZtExo70, the polarisome protein ZtSpa2. Consistent with results in the ascomycete Neurospora crassa, these markers did localize near the plasma membrane at the hyphal tip and only partially co-localize with FM4-64. Thus, these fluorescent markers are useful molecular tools that allow phenotypic analysis of mutants in Z. tritici. These tools will help develop new avenues of research in our quest to control STB infection in wheat.
Abstract.
Author URL.
Kilaru S, Schuster M, Latz M, Guo M, Steinberg G (2015). Fluorescent markers of the endocytic pathway in Zymoseptoria tritici.
Fungal Genet Biol,
79, 150-157.
Abstract:
Fluorescent markers of the endocytic pathway in Zymoseptoria tritici.
Hyphal growth in filamentous fungi is supported by the uptake (endocytosis) and recycling of membranes and associated proteins at the growing tip. An increasing body of published evidence in various fungi demonstrates that this process is of essential importance for fungal growth and pathogenicity. Here, we introduce fluorescent markers to visualize the endocytic pathway in the wheat pathogen Zymoseptoria tritici. We fused enhanced green-fluorescent protein (eGFP) to the actin-binding protein fimbrin (ZtFim1), which is located in actin patches that are formed at the plasma membrane and are participating in endocytic uptake at the cell surface. In addition, we tagged early endosomes by eGFP-labelling a Rab5-homologue (ZtRab5) and late endosomes and vacuoles by expressing eGFP-Rab7 homologue (ZtRab7). Using fluorescent dyes and live cell imaging we confirmed the dynamic behavior and localization of these markers. This set of molecular tools enables an in-depth phenotypic analysis of Z. tritici mutant strains thereby supporting new strategies towards the goal of controlling wheat against Z. tritici.
Abstract.
Author URL.
Schuster M, Kilaru S, Latz M, Steinberg G (2015). Fluorescent markers of the microtubule cytoskeleton in Zymoseptoria tritici.
Fungal Genet Biol,
79, 141-149.
Abstract:
Fluorescent markers of the microtubule cytoskeleton in Zymoseptoria tritici.
The microtubule cytoskeleton supports vital processes in fungal cells, including hyphal growth and mitosis. Consequently, it is a target for fungicides, such as benomyl. The use of fluorescent fusion proteins to illuminate microtubules and microtubule-associated proteins has led to a break-through in our understanding of their dynamics and function in fungal cells. Here, we introduce fluorescent markers to visualize microtubules and accessory proteins in the wheat pathogen Zymoseptoria tritici. We fused enhanced green-fluorescent protein to α-tubulin (ZtTub2), to ZtPeb1, a homologue of the mammalian plus-end binding protein EB1, and to ZtGrc1, a component of the minus-end located γ-tubulin ring complex, involved in the nucleation of microtubules. In vivo observation confirms the localization and dynamic behaviour of all three markers. These marker proteins are useful tools for understanding the organization and importance of the microtubule cytoskeleton in Z. tritici.
Abstract.
Author URL.
Steinberg G (2015). Kinesin-3 in the basidiomycete Ustilago maydis transports organelles along the entire microtubule array.
Fungal Genet Biol,
74, 59-61.
Abstract:
Kinesin-3 in the basidiomycete Ustilago maydis transports organelles along the entire microtubule array.
The molecular motor kinesin-3 transports early endosomes along microtubules in filamentous fungi. It was reported that kinesin-3 from the ascomycete fungi Aspergillus nidulans and Neurospora crassa use a subset of post-translationally modified and more stable microtubules. Here, I show that kinesin-3 from the basidiomycete Ustilago maydis moves along all hyphal microtubules. This difference is likely due to variation in cell cycle control and associated organization of the microtubule array.
Abstract.
Author URL.
Ma W, Kilaru S, Collins C, Courbot M, Steinberg G (2015). Libraries for two-hybrid screening of yeast and hyphal growth forms in Zymoseptoria tritici.
Fungal Genet Biol,
79, 94-101.
Abstract:
Libraries for two-hybrid screening of yeast and hyphal growth forms in Zymoseptoria tritici.
Pathogenic fungi are constantly emerging resistance to anti-fungal treatments. Therefore, identification of new fungicide targets is important. Good candidates are essential fungal proteins and their regulators. An efficient way to reveal the molecular environment of an essential protein is the search for interacting factors. Here, we establish three yeast two-hybrid libraries, covering yeast and hyphal stages of the wheat pathogen Zymoseptoria tritici. No detectable genomic DNA was present in any of the 3 libraries. Random amplification revealed that the libraries include cDNA fragments of up to 2000bp, suggesting that small-to-medium sized proteins are represented therein. Indeed, full-length cDNAs of five proteins were found in all libraries. The full-length cDNA of large chitin synthase gene mcs1 (5742bp with introns; 5568bp without introns) could not be amplified, but its 5' and 3' regions were represented, suggesting that even larger genes are covered in all libraries. Finally, we tested for the expected interaction of the autophagy proteins ZtAtg4 and ZtAtg8 in Z. tritici, and then used ZtAtg4 to screen one of the two-hybrid libraries. Indeed, we found ZtAtg8 as a positive interaction partner, confirming that interacting proteins can be identified. Thus, these molecular tools promise to be useful in identifying novel fungicide target proteins.
Abstract.
Author URL.
Fones HN, Steinberg G, Gurr SJ (2015). Measurement of virulence in Zymoseptoria tritici through low inoculum-density assays.
Fungal Genet Biol,
79, 89-93.
Abstract:
Measurement of virulence in Zymoseptoria tritici through low inoculum-density assays.
Hitherto, pathogenicity assays with mutants or wildtype variants of Zymoseptoria tritici have been based on pycnidial counts, following inoculation of host leaves with high density inoculum. Here, we present data which suggest that high inoculum densities may mask deficiencies in virulence due to symptom saturation. We describe a low inoculum-density method which obviates this problem. This method can also be used to (i) interrogate the process of lesion formation in Z. tritici (ii) determine whether individuals of the same or different genotypes co-operate or compete during the establishment of apoplastic infections (iii) dissect the determinants of virulence, by assessing a given strain's stomatal penetration efficiency (SPE), its ability to spread within the apoplast and its pycnidiation efficiency. Such methodology can thus be used to investigate the reasons underpinning attenuated virulence in mutant or avirulent wildtype strains.
Abstract.
Author URL.
Camões F, Islinger M, Guimarães SC, Kilaru S, Schuster M, Godinho LF, Steinberg G, Schrader M (2015). New insights into the peroxisomal protein inventory: Acyl-CoA oxidases and -dehydrogenases are an ancient feature of peroxisomes.
Biochim Biophys Acta,
1853(1), 111-125.
Abstract:
New insights into the peroxisomal protein inventory: Acyl-CoA oxidases and -dehydrogenases are an ancient feature of peroxisomes.
Peroxisomes are ubiquitous organelles which participate in a variety of essential biochemical pathways. An intimate interrelationship between peroxisomes and mitochondria is emerging in mammals, where both organelles cooperate in fatty acid β-oxidation and cellular lipid homeostasis. As mitochondrial fatty acid β-oxidation is lacking in yeast and plants, suitable genetically accessible model systems to study this interrelationship are scarce. Here, we propose the filamentous fungus Ustilago maydis as a suitable model for those studies. We combined molecular cell biology, bioinformatics and phylogenetic analyses and provide the first comprehensive inventory of U. maydis peroxisomal proteins and pathways. Studies with a peroxisome-deficient Δpex3 mutant revealed the existence of parallel and complex, cooperative β-oxidation pathways in peroxisomes and mitochondria, mimicking the situation in mammals. Furthermore, we provide evidence that acyl-CoA dehydrogenases (ACADs) are bona fide peroxisomal proteins in fungi and mammals and together with acyl-CoA oxidases (ACOX) belong to the basic enzymatic repertoire of peroxisomes. A genome comparison with baker's yeast and human gained new insights into the basic peroxisomal protein inventory shared by humans and fungi and revealed novel peroxisomal proteins and functions in U. maydis. The importance of our findings for the evolution and function of the complex interrelationship between peroxisomes and mitochondria in fatty acid β-oxidation is discussed.
Abstract.
Author URL.
Guimaraes SC, Schuster M, Bielska E, Dagdas G, Kilaru S, Meadows BRA, Schrader M, Steinberg G (2015). Peroxisomes, lipid droplets, and endoplasmic reticulum "hitchhike" on motile early endosomes.
J Cell Biol,
211(5), 945-954.
Abstract:
Peroxisomes, lipid droplets, and endoplasmic reticulum "hitchhike" on motile early endosomes.
Intracellular transport is mediated by molecular motors that bind cargo to be transported along the cytoskeleton. Here, we report, for the first time, that peroxisomes (POs), lipid droplets (LDs), and the endoplasmic reticulum (ER) rely on early endosomes (EEs) for intracellular movement in a fungal model system. We show that POs undergo kinesin-3- and dynein-dependent transport along microtubules. Surprisingly, kinesin-3 does not colocalize with POs. Instead, the motor moves EEs that drag the POs through the cell. PO motility is abolished when EE motility is blocked in various mutants. Most LD and ER motility also depends on EE motility, whereas mitochondria move independently of EEs. Covisualization studies show that EE-mediated ER motility is not required for PO or LD movement, suggesting that the organelles interact with EEs independently. In the absence of EE motility, POs and LDs cluster at the growing tip, whereas ER is partially retracted to subapical regions. Collectively, our results show that moving EEs interact transiently with other organelles, thereby mediating their directed transport and distribution in the cell.
Abstract.
Author URL.
Schuster M, Kilaru S, Guo M, Sommerauer M, Lin C, Steinberg G (2015). Red fluorescent proteins for imaging Zymoseptoria tritici during invasion of wheat.
Fungal Genet Biol,
79, 132-140.
Abstract:
Red fluorescent proteins for imaging Zymoseptoria tritici during invasion of wheat.
The use of fluorescent proteins (FPs) in plant pathogenic fungi provides valuable insight into their intracellular dynamics, cell organization and invasion mechanisms. Compared with green-fluorescent proteins, their red-fluorescent "cousins" show generally lower fluorescent signal intensity and increased photo-bleaching. However, the combined usage of red and green fluorescent proteins allows powerful insight in co-localization studies. Efficient signal detection requires a bright red-fluorescent protein (RFP), combined with a suitable corresponding filter set. We provide a set of four vectors, suitable for yeast recombination-based cloning that carries mRFP, TagRFP, mCherry and tdTomato. These vectors confer carboxin resistance after targeted single-copy integration into the sdi1 locus of Zymoseptoria tritici. Expression of the RFPs does not affect virulence of this wheat pathogen. We tested all four RFPs in combination with four epi-fluorescence filter sets and in confocal laser scanning microscopy, both in and ex planta. Our data reveal that mCherry is the RFP of choice for investigation in Z. tritici, showing highest signal intensity in epi-fluorescence, when used with a Cy3 filter set, and laser scanning confocal microscopy. However, mCherry bleached significantly faster than mRFP, which favors this red tag in long-term observation experiments. Finally, we used dual-color imaging of eGFP and mCherry expressing wild-type strains in planta and show that pycnidia are formed by single strains. This demonstrates the strength of this method in tracking the course of Z. tritici infection in wheat.
Abstract.
Author URL.
Kilaru S, Steinberg G (2015). Yeast recombination-based cloning as an efficient way of constructing vectors for Zymoseptoria tritici.
Fungal Genet Biol,
79, 76-83.
Abstract:
Yeast recombination-based cloning as an efficient way of constructing vectors for Zymoseptoria tritici.
Many pathogenic fungi are genetically tractable. Analysis of their cellular organization and invasion mechanisms underpinning virulence determinants profits from exploiting such molecular tools as fluorescent fusion proteins or conditional mutant protein alleles. Generation of these tools requires efficient cloning methods, as vector construction is often a rate-limiting step. Here, we introduce an efficient yeast recombination-based cloning (YRBC) method to construct vectors for the fungus Zymoseptoria tritici. This method is of low cost and avoids dependency on the availability of restriction enzyme sites in the DNA sequence, as needed in more conventional restriction/ligation-based cloning procedures. Furthermore, YRBC avoids modification of the DNA of interest, indeed this potential risk limits the use of site-specific recombination systems, such as Gateway cloning. Instead, in YRBC, multiple DNA fragments, with 30bp overlap sequences, are transformed into Saccharomyces cerevisiae, whereupon homologous recombination generates the vector in a single step. Here, we provide a detailed experimental protocol and four vectors, each encoding a different dominant selectable marker cassette, that enable YRBC of constructs to be used in the wheat pathogen Z. tritici.
Abstract.
Author URL.
2014
Higuchi Y, Ashwin P, Roger Y, Steinberg G (2014). Early endosome motility spatially organizes polysome distribution.
J Cell Biol,
204(3), 343-357.
Abstract:
Early endosome motility spatially organizes polysome distribution.
Early endosomes (EEs) mediate protein sorting, and their cytoskeleton-dependent motility supports long-distance signaling in neurons. Here, we report an unexpected role of EE motility in distributing the translation machinery in a fungal model system. We visualize ribosomal subunit proteins and show that the large subunits diffused slowly throughout the cytoplasm (Dc,60S = 0.311 µm(2)/s), whereas entire polysomes underwent long-range motility along microtubules. This movement was mediated by "hitchhiking" on kinesin-3 and dynein-driven EEs, where the polysomes appeared to translate EE-associated mRNA into proteins. Modeling indicates that this motor-driven transport is required for even cellular distribution of newly formed ribosomes. Indeed, impaired EE motility in motor mutants, or their inability to bind EEs in mutants lacking the RNA-binding protein Rrm4, reduced ribosome transport and induced ribosome aggregation near the nucleus. As a consequence, cell growth was severely restricted. Collectively, our results indicate that polysomes associate with moving EEs and that "off- and reloading" distributes the protein translation machinery.
Abstract.
Author URL.
Higuchi Y, Steinberg G (2014). Early endosomes motility in filamentous fungi: How and why they move.
Fungal Biology ReviewsAbstract:
Early endosomes motility in filamentous fungi: How and why they move
Elongate hyphae of filamentous fungi grow predominantly at their tips, whereas organelles are positioned in the subapical parts of the cell. Organelle positioning and long-distance intracellular communication involves active, energy-dependent transport along microtubules (MTs). This is mediated by specialized molecular motors, named kinesins and dynein, which utilize ATP hydrolysis to "walk" along the tubulin polymers. Work in the basidiomycete Ustilago maydis and the ascomycete Aspergillus nidulans has shown that early endosomes (EEs) are one of the major cargos of MT-dependent motors in fungi. EEs are part of the early endocytic pathway, and their motility behavior and the underlying transport machinery is well understood. However, the physiological role of constant bi-directional EE motility remains elusive. Recent reports, conducted in the corn smut fungus U. maydis, have provided novel insights into the cellular function of EE motility. They show that EE motility is crucial for the distribution of the protein synthesis machinery, and also that EEs transmit signals during plant infection that trigger the production of fungal effector proteins, required for successful invasion into host plants.
Abstract.
Steinberg G (2014). Endocytosis and early endosome motility in filamentous fungi.
Curr Opin Microbiol,
20, 10-18.
Abstract:
Endocytosis and early endosome motility in filamentous fungi.
Hyphal growth of filamentous fungi requires microtubule-based long-distance motility of early endosomes. Since the discovery of this process in Ustilago maydis, our understanding of its molecular basis and biological function has greatly advanced. Studies in U. maydis and Aspergillus nidulans reveal a complex interplay of the motor proteins kinesin-3 and dynein, which co-operate to support bi-directional motion of early endosomes. Genetic screening has shed light on the molecular mechanisms underpinning motor regulation, revealing Hook protein as general motor adapters on early endosomes. Recently, fascinating insight into unexpected roles for endosome motility has emerged. This includes septin filament formation and cellular distribution of the machinery for protein translation.
Abstract.
Author URL.
Bielska E, Schuster M, Roger Y, Berepiki A, Soanes DM, Talbot NJ, Steinberg G (2014). Hook is an adapter that coordinates kinesin-3 and dynein cargo attachment on early endosomes.
J Cell Biol,
204(6), 989-1007.
Abstract:
Hook is an adapter that coordinates kinesin-3 and dynein cargo attachment on early endosomes.
Bidirectional membrane trafficking along microtubules is mediated by kinesin-1, kinesin-3, and dynein. Several organelle-bound adapters for kinesin-1 and dynein have been reported that orchestrate their opposing activity. However, the coordination of kinesin-3/dynein-mediated transport is not understood. In this paper, we report that a Hook protein, Hok1, is essential for kinesin-3- and dynein-dependent early endosome (EE) motility in the fungus Ustilago maydis. Hok1 binds to EEs via its C-terminal region, where it forms a complex with homologues of human fused toes (FTS) and its interactor FTS- and Hook-interacting protein. A highly conserved N-terminal region is required to bind dynein and kinesin-3 to EEs. To change the direction of EE transport, kinesin-3 is released from organelles, and dynein binds subsequently. A chimaera of human Hook3 and Hok1 rescues the hok1 mutant phenotype, suggesting functional conservation between humans and fungi. We conclude that Hok1 is part of an evolutionarily conserved protein complex that regulates bidirectional EE trafficking by controlling attachment of both kinesin-3 and dynein.
Abstract.
Author URL.
Bielska E, Higuchi Y, Schuster M, Steinberg N, Kilaru S, Talbot NJ, Steinberg G (2014). Long-distance endosome trafficking drives fungal effector production during plant infection.
Nat Commun,
5Abstract:
Long-distance endosome trafficking drives fungal effector production during plant infection.
To cause plant disease, pathogenic fungi can secrete effector proteins into plant cells to suppress plant immunity and facilitate fungal infection. Most fungal pathogens infect plants using very long strand-like cells, called hyphae, that secrete effectors from their tips into host tissue. How fungi undergo long-distance cell signalling to regulate effector production during infection is not known. Here we show that long-distance retrograde motility of early endosomes (EEs) is necessary to trigger transcription of effector-encoding genes during plant infection by the pathogenic fungus Ustilago maydis. We demonstrate that motor-dependent retrograde EE motility is necessary for regulation of effector production and secretion during host cell invasion. We further show that retrograde signalling involves the mitogen-activated kinase Crk1 that travels on EEs and participates in control of effector production. Fungal pathogens therefore undergo long-range signalling to orchestrate host invasion.
Abstract.
Author URL.
Mirzadi Gohari A, Mehrabi R, Robert O, Ince IA, Boeren S, Schuster M, Steinberg G, de Wit PJGM, Kema GHJ (2014). Molecular characterization and functional analyses of ZtWor1, a transcriptional regulator of the fungal wheat pathogen Zymoseptoria tritici.
Molecular Plant Pathology,
15(4), 394-405.
Abstract:
Molecular characterization and functional analyses of ZtWor1, a transcriptional regulator of the fungal wheat pathogen Zymoseptoria tritici
Zymoseptoria tritici causes the major fungal wheat disease septoria tritici blotch, and is increasingly being used as a model for transmission and population genetics, as well as host-pathogen interactions. Here, we study the biological function of ZtWor1, the orthologue of Wor1 in the fungal human pathogen Candida albicans, as a representative of a superfamily of regulatory proteins involved in dimorphic switching. In Z.tritici, this gene is pivotal for pathogenesis, as ZtWor1 mutants were nonpathogenic and complementation restored the wild-type phenotypes. Inplanta expression analyses showed that ZtWor1 is up-regulated during the initiation of colonization and fructification, and regulates candidate effector genes, including one that was discovered after comparative proteome analysis of the Z.tritici wild-type strain and the ZtWor1 mutant, which was particularly expressed inplanta. Cell fusion and anastomosis occur frequently in ZtWor1 mutants, reminiscent of mutants of MgGpb1, the β-subunit of the heterotrimeric G protein. Comparative expression of ZtWor1 in knock-out strains of MgGpb1 and MgTpk2, the catalytic subunit of protein kinase A, suggests that ZtWor1 is downstream of the cyclic adenosine monophosphate (cAMP) pathway that is crucial for pathogenesis in many fungal plant pathogens. © 2013 BSPP AND JOHN WILEY and SONS LTD.
Abstract.
2013
Schuster M, Kilaru S, Steinberg G (2013). Dynactin is essential for extended retrograde run-length of single dynein motors in Ustilago maydis.
MOLECULAR BIOLOGY OF THE CELL,
24 Author URL.
Mirzadi Gohari A, Mehrabi R, Robert O, Ince IA, Boeren S, Schuster M, Steinberg G, de Wit PJGM, Kema GHJ (2013). Molecular characterization and functional analyses of ZtWor1, a transcriptional regulator of the fungal wheat pathogen Zymoseptoria tritici. Molecular Plant Pathology
Lin C, Ashwin P, Steinberg G (2013). Motor-mediated bidirectional transport along an antipolar microtubule bundle: a mathematical model.
Physical Review E - Statistical, Nonlinear, and Soft Matter Physics,
87(5).
Abstract:
Motor-mediated bidirectional transport along an antipolar microtubule bundle: a mathematical model
Long-distance bidirectional transport of organelles depends on the coordinated motion of various motor proteins on the cytoskeleton. Recent quantitative live cell imaging in the elongated hyphal cells of Ustilago maydis has demonstrated that long-range motility of motors and their endosomal cargo occurs on unipolar microtubules (MTs) near the extremities of the cell. These MTs are bundled into antipolar bundles within the central part of the cell. Dynein and kinesin-3 motors coordinate their activity to move early endosomes (EEs) in a bidirectional fashion where dynein drives motility towards MT minus ends and kinesin towards MT plus ends. Although this means that one can easily assign the drivers of bidirectional motion in the unipolar section, the bipolar orientations in the bundle mean that it is possible for either motor to drive motion in either direction. In this paper we use a multilane asymmetric simple exclusion process modeling approach to simulate and investigate phases of bidirectional motility in a minimal model of an antipolar MT bundle. In our model, EE cargos (particles) change direction on each MT with a turning rate Ω and there is switching between MTs in the bundle at the minus ends. At these ends, particles can hop between MTs with rate q1 on passing from a unipolar to a bipolar section (the obstacle-induced switching rate) or q2 on passing in the other direction (the end-induced switching rate). By a combination of numerical simulations and mean-field approximations, we investigate the distribution of particles along the MTs for different values of these parameters and of Θ, the overall density of particles within this closed system. We find that even if Θ is low, the system can exhibit a variety of phases with shocks in the density profiles near plus and minus ends caused by queuing of particles. We discuss how the parameters influence the type of particle that dominates active transport in the bundle. © 2013 American Physical Society.
Abstract.
2012
Lin C, Ashwin P, Steinberg G (2012). A model for motor-mediated bidirectional transport along an antipolar. microtubule bundle.
Steinberg G (2012). Cytoplasmic fungal lipases release fungicides from ultra-deformable vesicular drug carriers.
PLoS One,
7(5).
Abstract:
Cytoplasmic fungal lipases release fungicides from ultra-deformable vesicular drug carriers.
The Transfersome® is a lipid vesicle that contains membrane softeners, such as Tween 80, to make it ultra-deformable. This feature makes the Transfersome® an efficient carrier for delivery of therapeutic drugs across the skin barrier. It was reported that TDT 067 (a topical formulation of 15 mg/ml terbinafine in Transfersome® vesicles) has a much more potent antifungal activity in vitro compared with conventional terbinafine, which is a water-insoluble fungicide. Here we use ultra-structural studies and live imaging in a model fungus to describe the underlying mode of action. We show that terbinafine causes local collapse of the fungal endoplasmic reticulum, which was more efficient when terbinafine was delivered in Transfersome® vesicles (TFVs). When applied in liquid culture, fluorescently labeled TFVs rapidly entered the fungal cells (T(1/2)~2 min). Entry was F-actin- and ATP-independent, indicating that it is a passive process. Ultra-structural studies showed that passage through the cell wall involves significant deformation of the vesicles, and depends on a high concentration of the surfactant Tween 80 in their membrane. Surprisingly, the TFVs collapsed into lipid droplets after entry into the cell and the terbinafine was released from their interior. With time, the lipid bodies were metabolized in an ATP-dependent fashion, suggesting that cytosolic lipases attack and degrade intruding TFVs. Indeed, the specific monoacylglycerol lipase inhibitor URB602 prevented Transfersome® degradation and neutralized the cytotoxic effect of Transfersome®-delivered terbinafine. These data suggest that (a) Transfersomes deliver the lipophilic fungicide Terbinafine to the fungal cell wall, (b) the membrane softener Tween 80 allows the passage of the Transfersomes into the fungal cell, and (c) fungal lipases digest the invading Transfersome® vesicles thereby releasing their cytotoxic content. As this mode of action of Transfersomes is independent of the drug cargo, these results demonstrate the potential of Transfersomes in the treatment of all fungal diseases.
Abstract.
Author URL.
Steinberg G, Schuster M, Theisen U, Kilaru S, Forge A, Martin-Urdiroz M (2012). Motor-driven motility of fungal nuclear pores organizes chromosomes and fosters nucleocytoplasmic transport.
J Cell Biol,
198(3), 343-355.
Abstract:
Motor-driven motility of fungal nuclear pores organizes chromosomes and fosters nucleocytoplasmic transport.
Exchange between the nucleus and the cytoplasm is controlled by nuclear pore complexes (NPCs). In animals, NPCs are anchored by the nuclear lamina, which ensures their even distribution and proper organization of chromosomes. Fungi do not possess a lamina and how they arrange their chromosomes and NPCs is unknown. Here, we show that motor-driven motility of NPCs organizes the fungal nucleus. In Ustilago maydis, Aspergillus nidulans, and Saccharomyces cerevisiae fluorescently labeled NPCs showed ATP-dependent movements at ~1.0 µm/s. In S. cerevisiae and U. maydis, NPC motility prevented NPCs from clustering. In budding yeast, NPC motility required F-actin, whereas in U. maydis, microtubules, kinesin-1, and dynein drove pore movements. In the latter, pore clustering resulted in chromatin organization defects and led to a significant reduction in both import and export of GFP reporter proteins. This suggests that fungi constantly rearrange their NPCs and corresponding chromosomes to ensure efficient nuclear transport and thereby overcome the need for a structural lamina.
Abstract.
Author URL.
Schuster M, Treitschke S, Kilaru S, Molloy J, Harmer NJ, Steinberg G (2012). Myosin-5, kinesin-1 and myosin-17 cooperate in secretion of fungal chitin synthase.
EMBO J,
31(1), 214-227.
Abstract:
Myosin-5, kinesin-1 and myosin-17 cooperate in secretion of fungal chitin synthase.
Plant infection by pathogenic fungi requires polarized secretion of enzymes, but little is known about the delivery pathways. Here, we investigate the secretion of cell wall-forming chitin synthases (CHSs) in the corn pathogen Ustilago maydis. We show that peripheral filamentous actin (F-actin) and central microtubules (MTs) form independent tracks for CHSs delivery and both cooperate in cell morphogenesis. The enzyme Mcs1, a CHS that contains a myosin-17 motor domain, is travelling along both MTs and F-actin. This transport is independent of kinesin-3, but mediated by kinesin-1 and myosin-5. Arriving vesicles pause beneath the plasma membrane, but only ~15% of them get exocytosed and the majority is returned to the cell centre by the motor dynein. Successful exocytosis at the cell tip and, to a lesser extent at the lateral parts of the cell requires the motor domain of Mcs1, which captures and tethers the vesicles prior to secretion. Consistently, Mcs1-bound vesicles transiently bind F-actin but show no motility in vitro. Thus, kinesin-1, myosin-5 and dynein mediate bi-directional motility, whereas myosin-17 introduces a symmetry break that allows polarized secretion.
Abstract.
Author URL.
Dagdas YF, Yoshino K, Dagdas G, Ryder LS, Bielska E, Steinberg G, Talbot NJ (2012). Septin-mediated plant cell invasion by the rice blast fungus, Magnaporthe oryzae.
Science,
336(6088), 1590-1595.
Abstract:
Septin-mediated plant cell invasion by the rice blast fungus, Magnaporthe oryzae.
To cause rice blast disease, the fungus Magnaporthe oryzae develops a pressurized dome-shaped cell called an appressorium, which physically ruptures the leaf cuticle to gain entry to plant tissue. Here, we report that a toroidal F-actin network assembles in the appressorium by means of four septin guanosine triphosphatases, which polymerize into a dynamic, hetero-oligomeric ring. Septins scaffold F-actin, via the ezrin-radixin-moesin protein Tea1, and phosphatidylinositide interactions at the appressorium plasma membrane. The septin ring assembles in a Cdc42- and Chm1-dependent manner and forms a diffusion barrier to localize the inverse-bin-amphiphysin-RVS-domain protein Rvs167 and the Wiskott-Aldrich syndrome protein Las17 at the point of penetration. Septins thereby provide the cortical rigidity and membrane curvature necessary for protrusion of a rigid penetration peg to breach the leaf surface.
Abstract.
Author URL.
Steinberg G (2012). The transport machinery for motility of fungal endosomes. Fungal Genetics and Biology, 49(9), 675-676.
Steinberg G (2012). The transport machinery for motility of fungal endosomes.
Fungal Genet Biol,
49(9), 675-676.
Author URL.
2011
Lin C, Steinberg G, Ashwin P (2011). Bidirectional transport and pulsing states in a multi-lane ASEP model.
Abstract:
Bidirectional transport and pulsing states in a multi-lane ASEP model
In this paper, we introduce an ASEP-like transport model for bidirectional
motion of particles on a multi-lane lattice. The model is motivated by {\em in
vivo} experiments on organelle motility along a microtubule (MT), consisting of
thirteen protofilaments, where particles are propelled by molecular motors
(dynein and kinesin). In the model, organelles (particles) can switch
directions of motion due to "tug-of-war" events between counteracting motors.
Collisions of particles on the same lane can be cleared by switching to
adjacent protofilaments (lane changes).
. We analyze transport properties of the model with no-flux boundary conditions
at one end of a MT ("plus-end" or tip). We show that the ability of lane
changes can affect the transport efficiency and the particle-direction change
rate obtained from experiments is close to optimal in order to achieve
efficient motor and organelle transport in a living cell. In particular, we
find a nonlinear scaling of the mean {\em tip size} (the number of particles
accumulated at the tip) with injection rate and an associated phase transition
leading to {\em pulsing states} characterized by periodic filling and emptying
of the system.
Abstract.
Author URL.
Lin C, Steinberg G, Ashwin P (2011). Bidirectional transport and pulsing states in a multi-lane ASEP model.
Schuster M, Kilaru S, Ashwin P, Lin C, Severs NJ, Steinberg G (2011). Controlled and stochastic retention concentrates dynein at microtubule ends to keep endosomes on track.
EMBO J,
30(4), 652-664.
Abstract:
Controlled and stochastic retention concentrates dynein at microtubule ends to keep endosomes on track.
Bidirectional transport of early endosomes (EEs) involves microtubules (MTs) and associated motors. In fungi, the dynein/dynactin motor complex concentrates in a comet-like accumulation at MT plus-ends to receive kinesin-3-delivered EEs for retrograde transport. Here, we analyse the loading of endosomes onto dynein by combining live imaging of photoactivated endosomes and fluorescent dynein with mathematical modelling. Using nuclear pores as an internal calibration standard, we show that the dynein comet consists of ∼55 dynein motors. About half of the motors are slowly turned over (T(1/2): ∼98 s) and they are kept at the plus-ends by an active retention mechanism involving an interaction between dynactin and EB1. The other half is more dynamic (T(1/2): ∼10 s) and mathematical modelling suggests that they concentrate at MT ends because of stochastic motor behaviour. When the active retention is impaired by inhibitory peptides, dynein numbers in the comet are reduced to half and ∼10% of the EEs fall off the MT plus-ends. Thus, a combination of stochastic accumulation and active retention forms the dynein comet to ensure capturing of arriving organelles by retrograde motors.
Abstract.
Author URL.
Schuster M, Kilaru S, Fink G, Collemare J, Roger Y, Steinberg G (2011). Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array.
Mol Biol Cell,
22(19), 3645-3657.
Abstract:
Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array.
The polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to the growing cell tip (anterograde). Occasionally, EEs run up to 90 μm in one direction. The underlying MT array consists of unipolar MTs at both cell ends and antipolar bundles in the middle region of the cell. Cytoplasmic MT-organizing centers, labeled with a γ-tubulin ring complex protein, are distributed along the antipolar MTs but are absent from the unipolar regions. Dynein colocalizes with EEs for 10-20 μm after they have left the cell tip. Inactivation of temperature-sensitive dynein abolishes EE motility within the unipolar MT array, whereas long-range motility is not impaired. In contrast, kinesin-3 is continuously present, and its inactivation stops long-range EE motility. This indicates that both motors participate in EE motility, with dynein transporting the organelles through the unipolar MT array near the cell ends, and kinesin-3 taking over at the beginning of the medial antipolar MT array. The cooperation of both motors mediates EE movements over the length of the entire cell.
Abstract.
Author URL.
Steinberg G (2011). Motors in fungal morphogenesis: cooperation versus competition.
Curr Opin Microbiol,
14(6), 660-667.
Abstract:
Motors in fungal morphogenesis: cooperation versus competition.
Fungal tip growth underlies substrate invasion and is essential for fungal virulence. It requires the activity of molecular motors that deliver secretory vesicles to the growth region or which mediate bi-directional motility of early endosomes. Visualizing motors and their cargo in living fungal cells revealed unexpected cooperation between motors in membrane trafficking: (1) Class V chitin synthase, which has a class 17 myosin motor domain, moves bi-directionally, with myosin-5 and kinesin-1 cooperating in delivery to the growth region, and dynein taking it back to the cell centre. The myosin-17 motor domain competes with dynein by tethering the chitin synthase to the plasma membrane before exocytosis; (2) Long-range endosome motility is based on a cooperation of kinesin-3 and dynein, but towards the microtubule plus-end dynein competes with kinesin-3 to prevent the organelles from 'falling off the track'. These results reveal a fine-balanced network of cooperative and competitive motor activity, required for fungal morphogenesis.
Abstract.
Author URL.
Steinberg G, Schuster M (2011). The dynamic fungal cell.
Fungal Biology Reviews,
25(1), 14-37.
Abstract:
The dynamic fungal cell
Filamentous fungi are fascinating organisms that combine technical amenability with interesting biology and economical and ecological importance. In recent years, intensive research has rapidly extended our knowledge in many of the sub-disciplines in fungal sciences. An upcoming challenge is the integration of this detailed information in a more holistic understanding of the organisation and function of the fungal cell and its interaction with the environment. Here we provide an extensive visual impression of the organisation and motility of cellular structures in hyphae and yeast-like cells of the corn pathogen Ustilago maydis. We show 3D animation of major cytoskeletal elements and organelles in yeast-like and hyphal cells and provide insight into their dynamic behaviour. Using laser-based epi-fluorescence, we also include some more specialised processes, such as the dynamics of microtubules in mitosis, F-actin patches turn-over during endocytic uptake, nuclear import in interphase and in late mitosis and diffusion within the endoplasmic reticulum and the plasma membrane. This collection of 76 previously unpublished movie clips should provide a useful source for teaching fungal cell biology, but also intends to inspire researchers in different areas of fungal science. © 2011 the British Mycological Society.
Abstract.
Schuster M, Lipowsky R, Assmann M-A, Lenz P, Steinberg G (2011). Transient binding of dynein controls bidirectional long-range motility of early endosomes.
Proc Natl Acad Sci U S A,
108(9), 3618-3623.
Abstract:
Transient binding of dynein controls bidirectional long-range motility of early endosomes.
In many cell types, bidirectional long-range endosome transport is mediated by the opposing motor proteins dynein and kinesin-3. Here we use a fungal model system to investigate how both motors cooperate in early endosome (EE) motility. It was previously reported that Kin3, a member of the kinesin-3 family, and cytoplasmic dynein mediate bidirectional motility of EEs in the fungus Ustilago maydis. We fused the green fluorescent protein to the endogenous dynein heavy chain and the kin3 gene and visualized both motors and their cargo in the living cells. Whereas kinesin-3 was found on anterograde and retrograde EEs, dynein motors localize only to retrograde organelles. Live cell imaging shows that binding of retrograde moving dynein to anterograde moving endosomes changes the transport direction of the organelles. When dynein is leaving the EEs, the organelles switch back to anterograde kinesin-3-based motility. Quantitative photobleaching and comparison with nuclear pores as an internal calibration standard show that single dynein motors and four to five kinesin-3 motors bind to the organelles. These data suggest that dynein controls kinesin-3 activity on the EEs and thereby determines the long-range motility behavior of the organelles.
Abstract.
Author URL.
2010
Ashwin PB, Lin C, Steinberg G (2010). Queueing induced by bidirectional motor motion near the end of a microtubule. Phys. Rev. E, 82
Treitschke S, Doehlemann G, Schuster M, Steinberg G (2010). The myosin motor domain of fungal chitin synthase V is dispensable for vesicle motility but required for virulence of the maize pathogen Ustilago maydis.
Plant Cell,
22(7), 2476-2494.
Abstract:
The myosin motor domain of fungal chitin synthase V is dispensable for vesicle motility but required for virulence of the maize pathogen Ustilago maydis.
Class V chitin synthases are fungal virulence factors required for plant infection. They consist of a myosin motor domain fused to a membrane-spanning chitin synthase region that participates in fungal cell wall formation. The function of the motor domain is unknown, but it might deliver the myosin chitin synthase-attached vesicles to the growth region. Here, we analyze the importance of both domains in Mcs1, the chitin synthase V of the maize smut fungus Ustilago maydis. By quantitative analysis of disease symptoms, tissue colonization, and single-cell morphogenic parameters, we demonstrate that both domains are required for fungal virulence. Fungi carrying mutations in the chitin synthase domain are rapidly recognized and killed by the plant, whereas fungi carrying a deletion of the motor domain show alterations in cell wall composition but can invade host tissue and cause a moderate plant response. We also show that Mcs1-bound vesicles exhibit long-range movement for up to 20 microm at a velocity of approximately 1.75 microm/s. Apical Mcs1 localization depends on F-actin and the motor domain, whereas Mcs1 motility requires microtubules and persists when the Mcs1 motor domain is deleted. Our results suggest that the myosin motor domain of ChsV supports exocytosis but not long-range delivery of transport vesicles.
Abstract.
Author URL.
2008
Steinberg G, Perez-Martin J (2008). <em>Ustilago maydis</em>, a new fungal model system for cell biology. Trends in Cell Biology, 18, 61-67.
Theisen U, Straube A, Steinberg G (2008). Dynamic rearrangement of nucleoporins during fungal 'open' mitosis. Molecular Biology of the Cell, 19, 1230-1240.
Read ND, Steinberg G (2008). Editorial. Fungal Biology Reviews, 22(2).
2007
Werner S, Janyce A, Sugui A, Steinberg G & Deising HB (2007). A chitin synthase with a myosin-like motor domain is essential for hyphal growth, appressorium differentiation, and pathogenicity of the maize anthracnose fungus <em>Colletotrichum graminicola</em>. Molecular Plant-Microbe Interactions, 20, 1555-1567.
Steinberg, G. (2007). Hyphal growth: a tale of motors, lipids and the Spitzenkörper. Eukaryotic Cell, 6, 351-360
Steinberg, G. (2007). On the move: Endosomes in fungal growth and pathogenicity. Nature Reviews Microbiology, 5, 309-316
Steinberg, G. (2007). Preparing the way: Fungal motors in microtubule organization. Trends in Microbiology, 15, 14-21
Castillo-Lluval S, Alvarez-Tabares I, Weber I, Steinberg G & Perez-Martin J (2007). Sustained cell polarity and virulence in the phytopathogenic fungus <em>Ustilago maydis</em> depends on an essential cyclin-dependent kinase from the Cdk5/Pho85 family. Journal of Cell Science, 120, 1584-1595.
Münsterkötter, M & Steinberg G (2007). The fungus <em>Ustlilago maydis</em> and humans share disease-related proteins that are not found in <em>Saccharomyces cerevisiae</em>. BMC Genomics, 8, 473-483.
Steinberg, G. (2007). Tracks for traffic: Microtubules in the plant pathogen Ustilago maydis. New Phytologist, 721-733.
2006
Lenz JH, Schuchardt I, Straube A, Steinberg G (2006). A dynein loading zone for retrograde endosome motility at microtubule plus-ends.
EMBO J,
25(11), 2275-2286.
Abstract:
A dynein loading zone for retrograde endosome motility at microtubule plus-ends.
In the fungus Ustilago maydis, early endosomes move bidirectionally along microtubules (MTs) and facilitate growth by local membrane recycling at the tip of the infectious hypha. Here, we set out to elucidate the molecular mechanism of this process. We show that endosomes travel by Kinesin-3 activity into the hyphal apex, where they reverse direction and move backwards in a dynein-dependent manner. Our data demonstrate that dynein, dynactin and Lis1 accumulate at MT plus-ends within the hyphal tip, where they provide a reservoir of inactive motors for retrograde endosome transport. Consistently, endosome traffic is abolished after depletion of the dynein activator Lis1 and in Kinesin-1 null mutants, which was due to a defect in targeting of dynein and dynactin to the apical MT plus-ends. Furthermore, biologically active GFP-dynein travels on endosomes in retrograde and not in anterograde direction. Surprisingly, a CLIP170 homologue was neither needed for dynein localization nor for endosome transport. These results suggest an apical dynein loading zone in the hyphal tip, which ensure that endosomes reach the expanding growth region before they reverse direction.
Abstract.
Author URL.
Straube, A. Hause, G. Fink, G. Steinberg, G. (2006). Conventional kinesin mediates microtubule-microtubule interactions in vivo.
Mol Biol Cell,
17(2), 907-916.
Abstract:
Conventional kinesin mediates microtubule-microtubule interactions in vivo.
Conventional kinesin is a ubiquitous organelle transporter that moves cargo toward the plus-ends of microtubules. In addition, several in vitro studies indicated a role of conventional kinesin in cross-bridging and sliding microtubules, but in vivo evidence for such a role is missing. In this study, we show that conventional kinesin mediates microtubule-microtubule interactions in the model fungus Ustilago maydis. Live cell imaging and ultrastructural analysis of various mutants in Kin1 revealed that this kinesin-1 motor is required for efficient microtubule bundling and participates in microtubule bending in vivo. High levels of Kin1 led to increased microtubule bending, whereas a rigor-mutation in the motor head suppressed all microtubule motility and promoted strong microtubule bundling, indicating that kinesin can form cross-bridges between microtubules in living cells. This effect required a conserved region in the C terminus of Kin1, which was shown to bind microtubules in vitro. In addition, a fusion protein of yellow fluorescent protein and the Kin1tail localized to microtubule bundles, further supporting the idea that a conserved microtubule binding activity in the tail of conventional kinesins mediates microtubule-microtubule interactions in vivo.
Abstract.
Fink G, Steinberg G (2006). Dynein-dependent motility of microtubules and nucleation sites supports polarization of the tubulin array in the fungus Ustilago maydis.
Mol Biol Cell,
17(7), 3242-3253.
Abstract:
Dynein-dependent motility of microtubules and nucleation sites supports polarization of the tubulin array in the fungus Ustilago maydis.
Microtubules (MTs) are often organized by a nucleus-associated MT organizing center (MTOC). In addition, in neurons and epithelial cells, motor-based transport of assembled MTs determines the polarity of the MT array. Here, we show that MT motility participates in MT organization in the fungus Ustilago maydis. In budding cells, most MTs are nucleated by three to six small and motile gamma-tubulin-containing MTOCs at the boundary of mother and daughter cell, which results in a polarized MT array. In addition, free MTs and MTOCs move rapidly throughout the cytoplasm. Disruption of MTs with benomyl and subsequent washout led to an equal distribution of the MTOC and random formation of highly motile and randomly oriented MTs throughout the cytoplasm. Within 3 min after washout, MTOCs returned to the neck region and the polarized MT array was reestablished. MT motility and polarity of the MT array was lost in dynein mutants, indicating that dynein-based transport of MTs and MTOCs polarizes the MT cytoskeleton. Observation of green fluorescent protein-tagged dynein indicated that this is achieved by off-loading dynein from the plus-ends of motile MTs. We propose that MT organization in U. maydis involves dynein-mediated motility of MTs and nucleation sites.
Abstract.
Author URL.
Fink, G. Schuchardt, I. Colombelli, J. Stelzer, E. Steinberg, G. (2006). Dynein-mediated pulling forces drive rapid mitotic spindle elongation in <em>Ustilago maydis</em>. EMBO Journal, 25, 4897-4808
Fuchs, U, Hause, G. Schuchardt, I. Steinberg, G. (2006). Endocytosis is essential for pathogenic development in the corn smut fungus <em>Ustilago maydis</em>. The Plant Cell, 18, 2066-2081
Kämper J, Kahmann R, Bölker M, Ma L-J, Brefort T, Saville BJ, Banuett F, Kronstad JW, Gold SE, Müller O, et al (2006). Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis.
Nature,
444(7115), 97-101.
Abstract:
Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis.
Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.
Abstract.
Author URL.
Weber I, Assmann D, Thines E, Steinberg G (2006). Polar localizing class V myosin chitin synthases are essential during early plant infection in the plant pathogenic fungus Ustilago maydis.
Plant Cell,
18(1), 225-242.
Abstract:
Polar localizing class V myosin chitin synthases are essential during early plant infection in the plant pathogenic fungus Ustilago maydis.
Fungal chitin synthases (CHSs) form fibers of the cell wall and are crucial for substrate invasion and pathogenicity. Filamentous fungi contain up to 10 CHSs, which might reflect redundant functions or the complex biology of these fungi. Here, we investigate the complete repertoire of eight CHSs in the dimorphic plant pathogen Ustilago maydis. We demonstrate that all CHSs are expressed in yeast cells and hyphae. Green fluorescent protein (GFP) fusions to all CHSs localize to septa, whereas Chs5-GFP, Chs6-GFP, Chs7-yellow fluorescent protein (YFP), and Myosin chitin synthase1 (Mcs1)-YFP were found at growth regions of yeast-like cells and hyphae, indicating that they participate in tip growth. However, only the class IV CHS genes chs7 and chs5 are crucial for shaping yeast cells and hyphae ex planta. Although most CHS mutants were attenuated in plant pathogenicity, Deltachs6, Deltachs7, and Deltamcs1 mutants were drastically reduced in virulence. Deltamcs1 showed no morphological defects in hyphae, but Mcs1 became essential during invasion of the plant epidermis. Deltamcs1 hyphae entered the plant but immediately lost growth polarity and formed large aggregates of spherical cells. Our data show that the polar class IV CHSs are essential for morphogenesis ex planta, whereas the class V myosin-CHS is essential during plant infection.
Abstract.
Author URL.
2005
Straube A, Weber I, Steinberg, G. (2005). A novel mechanism of nuclear envelope break-down in a fungus: nuclear migration strips off the envelope. The EMBO Journal, 24(9), 2275-2286.
Fuchs, U. & Steinberg, G. (2005). Endocytosis in the plant pathogenic fungus <em>Ustilago maydis</em>. Protoplasma, 226, 75-80.
Schirawski J, Böhnert HU, Steinberg G, Snetselaar K, Adamikowa L, Kahmann R (2005). Endoplasmic reticulum glucosidase II is required for pathogenicity of Ustilago maydis.
Plant Cell,
17(12), 3532-3543.
Abstract:
Endoplasmic reticulum glucosidase II is required for pathogenicity of Ustilago maydis.
We identified a nonpathogenic strain of Ustilago maydis by tagging mutagenesis. The affected gene, glucosidase1 (gas1), displays similarity to catalytic alpha-subunits of endoplasmic reticulum (ER) glucosidase II. We have shown that Gas1 localizes to the ER and complements the temperature-sensitive phenotype of a Saccharomyces cerevisiae mutant lacking ER glucosidase II. gas1 deletion mutants were normal in growth and mating but were more sensitive to calcofluor and tunicamycin. Mutant infection hyphae displayed significant alterations in the distribution of cell wall material and were able to form appressoria and penetrate the plant surface but arrested growth in the epidermal cell layer. Electron microscopy analysis revealed that the plant-fungal interface between mutant hyphae and the plant plasma membrane was altered compared with the interface of penetrating wild-type hyphae. This may indicate that gas1 mutants provoke a plant response.
Abstract.
Author URL.
Fuchs, U. Manns, I. & Steinberg, G. (2005). Microtubules are dispensable for the initial pathogenic development but required for long-distance hyphal growth in the corn smut fungus <em>Ustilago maydis</em>. Molecular Biology of the Cell, 16, 2746-2758.
Schuchardt I, Assmann D, Thines E, Schuberth C, Steinberg G (2005). Myosin-V, Kinesin-1, and Kinesin-3 cooperate in hyphal growth of the fungus Ustilago maydis.
Mol Biol Cell,
16(11), 5191-5201.
Abstract:
Myosin-V, Kinesin-1, and Kinesin-3 cooperate in hyphal growth of the fungus Ustilago maydis.
Long-distance transport is crucial for polar-growing cells, such as neurons and fungal hyphae. Kinesins and myosins participate in this process, but their functional interplay is poorly understood. Here, we investigate the role of kinesin motors in hyphal growth of the plant pathogen Ustilago maydis. Although the microtubule plus-ends are directed to the hyphal tip, of all 10 kinesins analyzed, only conventional kinesin (Kinesin-1) and Unc104/Kif1A-like kinesin (Kinesin-3) were up-regulated in hyphae and they are essential for extended hyphal growth. deltakin1 and deltakin3 mutant hyphae grew irregular and remained short, but they were still able to grow polarized. No additional phenotype was detected in deltakin1rkin3 double mutants, but polarity was lost in deltamyo5rkin1 and deltamyo5rkin3 mutant cells, suggesting that kinesins and class V myosin cooperate in hyphal growth. Consistent with such a role in secretion, fusion proteins of green fluorescent protein and Kinesin-1, Myosin-V, and Kinesin-3 accumulate in the apex of hyphae, a region where secretory vesicles cluster to form the fungal Spitzenkörper. Quantitative assays revealed a role of Kin3 in secretion of acid phosphatase, whereas Kin1 was not involved. Our data demonstrate that just two kinesins and at least one myosin support hyphal growth.
Abstract.
Author URL.
Feldbrügge, M, Bölker, M. Steinberg, G. Kämper, J. & Kahmann, R. (2005). Regulatory and structural networks orchestrating mating, dimorphism, cell shape, and pathogenesis in <em>Ustilago maydis</em>. In Fischer K (Ed) The Mycota I, Growth, Differentiation and Sexuality.
2004
Adamíková L, Straube A, Schulz I, Steinberg G (2004). Calcium signaling is involved in dynein-dependent microtubule organization.
Mol Biol Cell,
15(4), 1969-1980.
Abstract:
Calcium signaling is involved in dynein-dependent microtubule organization.
The microtubule cytoskeleton supports cellular morphogenesis and polar growth, but the underlying mechanisms are not understood. In a screen for morphology mutants defective in microtubule organization in the fungus Ustilago maydis, we identified eca1 that encodes a sarcoplasmic/endoplasmic calcium ATPase. Eca1 resides in the endoplasmic reticulum and restores growth of a yeast mutant defective in calcium homeostasis. Deletion of eca1 resulted in elevated cytosolic calcium levels and a severe growth and morphology defect. While F-actin and myosin V distribution is unaffected, Deltaeca1 mutants contain longer and disorganized microtubules that show increased rescue and reduced catastrophe frequencies. Morphology can be restored by inhibition of Ca(2+)/calmodulin-dependent kinases or destabilizing microtubules, indicating that calcium-dependent alterations in dynamic instability are a major cause of the growth defect. Interestingly, dynein mutants show virtually identical changes in microtubule dynamics and dynein-dependent ER motility was drastically decreased in Deltaeca1. This indicates a connection between calcium signaling, dynein, and microtubule organization in morphogenesis of U. maydis.
Abstract.
Author URL.
Garcia-Muse T, Steinberg G, Perez-Martin J (2004). Characterization of B-type cyclins in the smut fungus Ustilago maydis: roles in morphogenesis and pathogenicity.
JOURNAL OF CELL SCIENCE,
117(3), 487-506.
Author URL.
Feldbrügge M, Kämper J, Steinberg G, Kahmann R (2004). Regulation of mating and pathogenic development in Ustilago maydis.
Curr Opin Microbiol,
7(6), 666-672.
Abstract:
Regulation of mating and pathogenic development in Ustilago maydis.
The plant pathogenic fungus Ustilago maydis induces disease only in its dikaryotic stage that is generated after mating. This process involves coordinated cAMP and mitogen-activated protein kinase signalling to regulate transcriptional as well as morphological responses. Among the induced products is the key regulator for pathogenic development. Recent advances identified crucial nodes that interconnect these pathways. The key regulator orchestrates a complex transcriptional cascade, the components of which have been uncovered by genomic strategies. This is complemented by insights into organization, dynamics and function of the cytoskeleton, which begin to establish the links between signalling, intracellular transport processes and morphology.
Abstract.
Author URL.
Steinberg G, Fuchs U (2004). The role of microtubules in cellular organization and endocytosis in the plant pathogen Ustilago maydis.
J Microsc,
214(Pt 2), 114-123.
Abstract:
The role of microtubules in cellular organization and endocytosis in the plant pathogen Ustilago maydis.
Microtubules are an important part of the eukaryotic cytoskeleton, which participates in numerous essential cellular processes. In fungi interphase microtubules mediate cell polarity and participate in polar growth. However, our understanding of their detailed role in fungal growth is just at the beginning. In growing cells of the plant pathogenic fungus Ustilago maydis microtubules are organized by polar microtubule organizing centres that focus the microtubule minus ends at the small bud. Two opposing motor complexes utilize this microtubule polarity. Cytoplasmic dynein and a kinesin of the Unc104/Kif1A family of kinesins mediate rapid bi-directional transport of early endosomes. A balance of their activity is required for cell cycle-dependent accumulation of early endosomes at the growth site, the rear cell pole and the region of cell cleavage. Mutant phenotypes suggest that these endosomes participate in polar growth, bud site selection and cell separation. Therefore, our data suggest that endocytotic membrane recycling participates in local exocytosis, and that the microtubule cytoskeleton has a crucial role in this process.
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Author URL.
Basse CW, Steinberg G (2004). Ustilago maydis, model system for analysis of the molecular basis of fungal pathogenicity.
Mol Plant Pathol,
5(2), 83-92.
Abstract:
Ustilago maydis, model system for analysis of the molecular basis of fungal pathogenicity.
UNLABELLED: SUMMARY Ustilago maydis, a facultative biotrophic basidiomycete fungus, causes smut disease in maize. A hallmark of this disease is the induction of large plant tumours that are filled with masses of black-pigmented teliospores. During the last 15 years U. maydis has become an important model system to unravel molecular mechanisms of fungal phytopathogenicity. This review highlights recent insights into molecular mechanisms of complex signalling pathways that are involved in the transition from budding to filamentous growth and operate during the pathogenic growth phase. In addition, we describe recent progress in understanding the structural basis of morphogenesis and polar growth in different stages of U. maydis development. Finally, we present an overview of recently identified genes related to pathogenic development and summarize novel molecular and genomic approaches that are powerful tools to explore the genetic base of pathogenicity. TAXONOMY: Ustilago maydis (DC) Corda (synonymous with Ustilago zeae Ung.)-Kingdom Eukaryota, Phylum Fungi, Order Basidiomycota, Family Ustilaginomycetes, Genus Ustilago. HOST RANGE: Infects aerial parts of corn plants (Zea mays) and its progenitor teosinte (Zea mays ssp. parviglumis). Maize smut is distributed throughout the world. Disease symptoms: U. maydis causes chlorotic lesions in infected areas, the formation of anthocyanin pigments, necrosis, hyperplasia and hypertrophy of infected organs. Infection by U. maydis can inhibit development and lead to stunting of infected plants. A few days after infection plant tumours develop in which massive fungal proliferation and the formation of the black-pigmented, diploid teliospores occurs. Under natural conditions tumours predominantly develop on sexual organs (tassels and ears), stems and nodal shoots. Tumours may vary in size from minute pustules to several centimetres in diameter and contain up to 200 billion spores. Useful web site: http://www-genome.wi.mit.edu/annotation/fungi/ustilago_maydis/
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Author URL.
2003
Weber I, Gruber C, Steinberg G (2003). A class-V myosin required for mating, hyphal growth, and pathogenicity in the dimorphic plant pathogen Ustilago maydis.
Plant Cell,
15(12), 2826-2842.
Abstract:
A class-V myosin required for mating, hyphal growth, and pathogenicity in the dimorphic plant pathogen Ustilago maydis.
In the early stages of plant infection, yeast-like haploid sporidia of Ustilago maydis respond to pheromone secreted by compatible partners by forming conjugation tubes. These then fuse to generate a dikaryotic hypha that forms appressoria to penetrate the host plant. As a first step toward understanding the structural requirements for these transitions, we have identified myo5, which encodes a class-V myosin. Analysis of conditional and null mutants revealed that Myo5 plays nonessential roles in cytokinesis and morphogenesis in sporidia and is required for hyphal morphology. Consistent with a role in morphogenesis, a functional green fluorescent protein-Myo5 fusion protein localized to the bud tip and the hyphal apex as well as to the septa and the spore wall during later stages of infection. However, the loss of Myo5 did not affect the tip growth of hyphae and sporidia. By contrast, Myo5 was indispensable for conjugation tube formation. Furthermore, myo5 mutants were impaired in the perception of pheromones, which indicates a particular importance of Myo5 in the mating process. Consequently, few mutant hyphae were formed that penetrated the plant epidermis but did not continue invasive growth. These results indicate a crucial role of Myo5 in the morphogenesis, dimorphic switch, and pathogenicity of U. maydis.
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Author URL.
Straube A, Brill M, Oakley BR, Horio T, Steinberg G (2003). Microtubule organization requires cell cycle-dependent nucleation at dispersed cytoplasmic sites: polar and perinuclear microtubule organizing centers in the plant pathogen Ustilago maydis.
Mol Biol Cell,
14(2), 642-657.
Abstract:
Microtubule organization requires cell cycle-dependent nucleation at dispersed cytoplasmic sites: polar and perinuclear microtubule organizing centers in the plant pathogen Ustilago maydis.
Growth of most eukaryotic cells requires directed transport along microtubules (MTs) that are nucleated at nuclear-associated microtubule organizing centers (MTOCs), such as the centrosome and the fungal spindle pole body (SPB). Herein, we show that the pathogenic fungus Ustilago maydis uses different MT nucleation sites to rearrange MTs during the cell cycle. In vivo observation of green fluorescent protein-MTs and MT plus-ends, tagged by a fluorescent EB1 homologue, provided evidence for antipolar MT orientation and dispersed cytoplasmic MT nucleating centers in unbudded cells. On budding gamma-tubulin containing MTOCs formed at the bud neck, and MTs reorganized with >85% of all minus-ends being focused toward the growth region. Experimentally induced lateral budding resulted in MTs that curved out of the bud, again supporting the notion that polar growth requires polar MT nucleation. Depletion or overexpression of Tub2, the gamma-tubulin from U. maydis, affected MT number in interphase cells. The SPB was inactive in G2 phase but continuously recruited gamma-tubulin until it started to nucleate mitotic MTs. Taken together, our data suggest that MT reorganization in U. maydis depends on cell cycle-specific nucleation at dispersed cytoplasmic sites, at a polar MTOC and the SPB.
Abstract.
Author URL.
García-Muse T, Steinberg G, Pérez-Martín J (2003). Pheromone-induced G<inf>2</inf> arrest in the phytopathogenic fungus Ustilago maydis.
Eukaryotic Cell,
2(3), 494-500.
Abstract:
Pheromone-induced G2 arrest in the phytopathogenic fungus Ustilago maydis
In the corn smut fungus Ustilago maydis, pathogenic development is initiated when two compatible haploid cells fuse and form the infectious dikaryon. Mating is dependent on pheromone recognition by compatible cells. In this report, we set out to evaluate the relationship between the cell cycle and the pheromone response in U. maydis. To achieve this, we designed a haploid pheromone-responsive strain that is able to faithfully reproduce the native mating response in nutrient-rich medium. Addition of synthetic pheromone to the responsive strain induces the formation of mating structures, and this response is abolished by mutations in genes encoding components of the pheromone signal transduction cascade. After recognition of pheromone, U. maydis cells arrest the cell cycle in a postreplicative stage. Visualization of the nucleus and microtubule organization indicates that the arrest takes place at the G2 phase. Chemical-induced cell cycle arrest and release in the presence of pheromone further support this conclusion.
Abstract.
2002
GSteinb, Friedrich MW, Straube A, Wedlich-Soldner R (2002). Balance of KIF1A-like kinesin and dynein organizes early endosomes in the fungus Ustilago maydis. The EMBO Journal, 21(12), 2946-2957.
Wedlich-Söldner R, Schulz I, Straube A, Steinberg G (2002). Dynein supports motility of endoplasmic reticulum in the fungus Ustilago maydis.
Mol Biol Cell,
13(3), 965-977.
Abstract:
Dynein supports motility of endoplasmic reticulum in the fungus Ustilago maydis.
The endoplasmic reticulum (ER) of most vertebrate cells is spread out by kinesin-dependent transport along microtubules, whereas studies in Saccharomyces cerevisiae indicated that motility of fungal ER is an actin-based process. However, microtubules are of minor importance for organelle transport in yeast, but they are crucial for intracellular transport within numerous other fungi. Herein, we set out to elucidate the role of the tubulin cytoskeleton in ER organization and dynamics in the fungal pathogen Ustilago maydis. An ER-resident green fluorescent protein (GFP)-fusion protein localized to a peripheral network and the nuclear envelope. Tubules and patches within the network exhibited rapid dynein-driven motion along microtubules, whereas conventional kinesin did not participate in ER motility. Cortical ER organization was independent of microtubules or F-actin, but reformation of the network after experimental disruption was mediated by microtubules and dynein. In addition, a polar gradient of motile ER-GFP stained dots was detected that accumulated around the apical Golgi apparatus. Both the gradient and the Golgi apparatus were sensitive to brefeldin a or benomyl treatment, suggesting that the gradient represents microtubule-dependent vesicle trafficking between ER and Golgi. Our results demonstrate a role of cytoplasmic dynein and microtubules in motility, but not peripheral localization of the ER in U. maydis.
Abstract.
Author URL.
2001
Straube, A. Berner A, Enard W, Kahmann R, Steinberg, G. (2001). A split motor domain in a cytoplasmic dynein. The EMBO Journal, 20(18), 5091-5100.
Steinberg G, Wedlich-Söldner R, Brill M, Schulz I (2001). Microtubules in the fungal pathogen Ustilago maydis are highly dynamic and determine cell polarity.
J Cell Sci,
114(Pt 3), 609-622.
Abstract:
Microtubules in the fungal pathogen Ustilago maydis are highly dynamic and determine cell polarity.
Many fungal pathogens undergo a yeast-hyphal transition during their pathogenic development that requires rearrangement of the cytoskeleton, followed by directed membrane traffic towards the growth region. The role of microtubules and their dynamic behavior during this process is not well understood. Here we set out to elucidate the organization, cellular role and in vivo dynamics of microtubules in the dimorphic phytopathogen Ustilago maydis. Hyphae and unbudded yeast-like cells of U. maydis contain bundles of spindle pole body-independent microtubules. At the onset of bud formation two spherical tubulin structures focus microtubules towards the growth region, suggesting that they support polar growth in G(2), while spindle pole body-nucleated astral microtubules participate in nuclear migration in M and early G(1). Conditional mutants of an essential alpha-tubulin gene from U. maydis, tub1, confirmed a role for interphase microtubules in determination of cell polarity and growth. Observation of GFP-Tub1 fusion protein revealed that spindle pole body-independent and astral microtubules are dynamic, with elongation and shrinkage rates comparable to those found in vertebrate systems. In addition, very fast depolymerization was measured within microtubule bundles. Unexpectedly, interphase microtubules underwent bending and rapid translocations within the cell, suggesting that unknown motor activities participate in microtubule organization in U. maydis. Movies available on-line: http://www.biologists.com/JCS/movies/jcs1792.html
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Author URL.
2000
Wedlich-Söldner, R. Bölker, M. Kahmann, R. & Steinberg, G. (2000). A putative endosomal t-SNARE links exo- and endocytosis in the phytopathogenic fungus <em>Ustilago maydis</em>. EMBO Journal, 19, 1974-1986.
Steinberg G (2000). The cellular roles of molecular motors in fungi.
TRENDS IN MICROBIOLOGY,
8(4), 162-168.
Author URL.
Kahmann, R, Steinberg, G. Basse, C. Feldbrügge, M. Kämper, J. (2000). Ustilago maydis, the causive agent of corn smut disease. In Kronstadt J (Ed) Fungal Pathology, Amsterdam: Kluwer Academic Publisher, 347-371.
1999
Heath IB, Steinberg G (1999). Mechanisms of hyphal tip growth: Tube dwelling amebae revisited.
FUNGAL GENETICS AND BIOLOGY,
28(2), 79-93.
Author URL.
1998
Steinberg G, McIntosh JR (1998). Effects of the myosin inhibitor 2,3-butanedione monoxime on the physiology of fission yeast.
EUROPEAN JOURNAL OF CELL BIOLOGY,
77(4), 284-293.
Author URL.
Steinberg G, Schliwa M, Lehmler C, Bolker M, Kahmann R, McIntosh JR (1998). Kinesin from the plant pathogenic fungus Ustilago maydis is involved in vacuole formation and cytoplasmic migration.
JOURNAL OF CELL SCIENCE,
111, 2235-2246.
Author URL.
Steinberg G (1998). Organelle transport and molecular motors in fungi.
Fungal Genet Biol,
24(1-2), 161-177.
Abstract:
Organelle transport and molecular motors in fungi.
Polarized growth, secretion of exoenzymes, organelle inheritance, and organelle positioning require vectorial transport along cytoskeletal elements. The discovery of molecular motors and intensive studies on their biological function during the past 3 years confirmed a central role of these mechanoenzymes in morphogenesis and development of yeasts and filamentous fungi. Saccharomyces cerevisiae proved to be an excellent model system, in which the complete set of molecular motors is presumed to be known. Genetic studies combined with cell biological methods revealed unexpected functional relationships between these motors and has greatly improved our understanding of nuclear migration, exocytosis, and endocytosis in yeasts. Tip growth of elongated hyphae, compared to budding, however, does require vectorial transport over long distances. The identification of ubiquitous motors that are not present in yeast indicates that studies on filamentous fungi might be helpful to elucidate the role of motors in long-distance organelle transport within higher eukaryotic cells. Copyright 1998 Academic Press.
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Author URL.
1997
Steinberg G (1997). A kinesin-like mechanoenzyme from the zygomycete Syncephalastrum racemosum shares biochemical similarities with conventional kinesin from Neurospora crassa.
EUROPEAN JOURNAL OF CELL BIOLOGY,
73(2), 124-131.
Author URL.
Lehmler C, Steinberg G, Snetselaar K, Schliwa M, Kahmann R, Bolker M (1997). Identification of a motor protein required for filamentous growth in Ustilago maydis.
EMBO JOURNAL,
16(12), 3464-3473.
Author URL.
Seiler S, Nargang FE, Steinberg G, Schliwa M (1997). Kinesin is essential for cell morphogenesis and polarized secretion in Neurospora crassa.
EMBO J,
16(11), 3025-3034.
Abstract:
Kinesin is essential for cell morphogenesis and polarized secretion in Neurospora crassa.
Kinesin is a force-generating molecule that is thought to translocate organelles along microtubules, but its precise cellular function is still unclear. To determine the role of kinesin in vivo, we have generated a kinesin-deficient strain in the simple cell system Neurospora crassa. Null cells exhibit severe alterations in cell morphogenesis, notably hyphal extension, morphology and branching. Surprisingly, the movement of organelles visualized by video microscopy is hardly affected, but apical hyphae fail to establish a Spitzenkörper, an assemblage of secretory vesicles intimately linked to cell elongation and morphogenesis in Neurospora and other filamentous fungi. As cell morphogenesis depends on polarized secretion, our findings demonstrate that a step in the secretory pathway leading to cell shape determination and cell elongation cannot tolerate a loss of kinesin function. The defect is suggested to affect the transport of small, secretory vesicles to the site involved in protrusive activity, resulting in the uncoordinated insertion of new cell wall material over much of the cell surface. These observations have implications for the presumptive function of kinesin in more complex cell systems.
Abstract.
Author URL.
1996
Steinberg G, Schliwa M (1996). Characterization of the biophysical and motility properties of kinesin from the fungus Neurospora crassa.
J Biol Chem,
271(13), 7516-7521.
Abstract:
Characterization of the biophysical and motility properties of kinesin from the fungus Neurospora crassa.
Neurospora kinesin (Nkin) is a distant relative of the family of conventional kinesins, members of which have been identified in various animal species. As in its animal counterparts, Nkin most likely is an organelle motor. Because it is a functional homologue of the kinesin heavy chain of higher eukaryotes, its biophysical and motility properties were compared with those of other conventional kinesins. Purified Nkin behaves as a homodimeric complex composed of two subunits of a 105-kDa polypeptide. Based on its hydrodynamic properties (Stokes radius and sedimentation coefficient), Nkin is an elongated molecule, although it is more compact than its animal counterparts. A detailed comparison of the motility properties of Nkin with those of animal conventional kinesins reveals similarities and some intriguing differences. Nkin is less effective than other kinesins in the use of natural nucleoside triphosphates but responds to a selection of ATP analogues in a similar fashion as mammalian kinesin. Even in the presence of saturating concentrations of ATP, Nkin is significantly more sensitive to ADP or tripolyphosphate than other kinesins. Both the ATP-driven microtubule gliding activity and the microtubule-stimulated ATPase activity of Nkin obey Michaelis-Menten kinetics. Surprisingly, however, the Km values for both these activities are approximately an order of magnitude higher than those of other kinesins. Whether the low affinity for ATP suggested by these high Km values is related to the high rate of motility remains to be determined.
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1995
Steinberg G, Schliwa M (1995). The Neurospora organelle motor: a distant relative of conventional kinesin with unconventional properties.
Mol Biol Cell,
6(11), 1605-1618.
Abstract:
The Neurospora organelle motor: a distant relative of conventional kinesin with unconventional properties.
The "conventional" kinesins comprise a conserved family of molecular motors for organelle transport that have been identified in various animal species. Organelle motors from other phyla have not yet been analyzed at the molecular level. Here we report the identification, biochemical and immunological characterization, and molecular cloning of a cytoplasmic motor in a "lower" eukaryote, the Ascomycete fungus Neurospora crassa. This motor, termed Nkin (for Neurospora kinesin), exhibits several unique structural and functional features, including a high rate of microtubule transport, a lack of copurifying light chains, a second P-loop motif, and an overall sequence organization reminiscent of a kinesin-like protein. However, a greater than average sequence homology in the motor domain and the presence of a highly conserved region in the C-terminus identify Nkin as a distant relative of the family of conventional kinesins. A molecular phylogenetic analysis suggests Nkin to have diverged early in the evolution of this family of motors. The discovery of Nkin may help identify domains important for specific biological functions in conventional kinesins.
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1993
Steinberg G, Kollmann R (1993). A quantitative analysis of the interspecific plasmodesmata in the non-division walls of the plant chimera Laburnocytisus adamii (Poit.) Schneid.
Planta,
192(1), 75-83.
Abstract:
A quantitative analysis of the interspecific plasmodesmata in the non-division walls of the plant chimera Laburnocytisus adamii (Poit.) Schneid.
Non-division walls in petals of the chimera Laburnocytisus adamii (Poit.) Schneid, were screened for the occurrence and distribution of symplasmic connections. The secondary plasmodesmata (PD) between epidermal cells of Cytisus purpureus Scop, and subepidermal cells of Laburnum anagyroides Medik. were compared with the PD of corresponding cell walls in petals of the two parental species. The non-division walls in the petals of L. adamii were traversed mainly by continuous PD and a few half-PD, both being grouped in pit fields. The secondary PD were characterized by a high percentage of branching (82%), with more than 40% consisting of a single strand at the Cytisus cell side interconnected by a median cavity with two strands of the Laburnum subepidermal cell. In addition, more than 30% of all PD showed secondary branching in the subepidermal wall portion. As a consequence, the cross-sectional areas of plasmodesmatal strands on each side of the central cavity differed remarkably in size, representing a "bottleneck" in the epidermal wall portion. In contrast, PD in the petals of the parental species were symmetrically branched. The comparison of cross-sectional areas of PD in the cell wall between the epidermis and subepidermis of petals of L. anagyroides showed a well-tuned system. The occurrence of half-PD in the intraspecific wall indicates a secondary origin. We conclude that, in the chimera, both genotypically different cells take part in the formation of the interspecific PD. © 1994 Springer-Verlag.
Abstract.
PATTERSON DJ, NYGAARD K, STEINBERG G, TURLEY CM (1993). HETEROTROPHIC FLAGELLATES AND OTHER PROTISTS ASSOCIATED WITH OCEANIC DETRITUS THROUGHOUT THE WATER COLUMN IN THE MID NORTH-ATLANTIC.
JOURNAL OF THE MARINE BIOLOGICAL ASSOCIATION OF THE UNITED KINGDOM,
73(1), 67-95.
Author URL.
Steinberg G, Schliwa M (1993). Organelle movements in the wild type and wall-less fz;sg;os-1 mutants of Neurospora crassa are mediated by cytoplasmic microtubules.
J Cell Sci,
106 ( Pt 2), 555-564.
Abstract:
Organelle movements in the wild type and wall-less fz;sg;os-1 mutants of Neurospora crassa are mediated by cytoplasmic microtubules.
The cellular basis of organelle transport in filamentous fungi is still unresolved. Here we have studied the intracellular movement of mitochondria and other organelles in the fungus Neurospora crassa. Four different model systems were employed: hyphae, protoplasts, a cell wallless mutant, and experimentally generated small, flattened cell fragments of the mutant cells. Organelle movements were visualized by DIC optics and computer-enhanced video microscopy. In all cell models the transport of organelles was vectorial and saltatory in nature. The mean velocities for mitochondria, particles and nuclei were 1.4, 2.0, and 0.9 microns/s, respectively. Treatment with 10 microM nocodazole for 30 minutes caused a complete disappearance of microtubules and reversibly blocked directed transport of virtually all organelles, whereas cytochalasin D up to 20 microM was without effect. Correlative video and immunofluorescence microscopy of small fragments of wall-less mutant cells revealed a clear match between microtubule distribution and the tracks of moving organelles. We conclude that organelle movement in the filamentous fungus Neurospora crassa is a microtubule-dependent process.
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