ABSTRACT
Filamentous fungi of the genus Aspergillus are of particular interest for biotechnological applications due to their natural capacity to secrete carbohydrate-active enzymes (CAZy) that target plant biomass. The presence of easily metabolizable sugars such as glucose, whose concentrations increase during plant biomass hydrolysis, results in the repression of CAZy-encoding genes in a process known as carbon catabolite repression (CCR), which is undesired for the purpose of large-scale enzyme production. To date, the C2H2 transcription factor CreA has been described as the major CC repressor in Aspergillus spp. although little is known about the role of posttranslational modifications in this process. In this work, phosphorylation sites were identified by mass spectrometry on Aspergillus nidulans CreA, and subsequently, the previously identified but uncharacterized site S262, the characterized site S319, and the newly identified sites S268 and T308 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was investigated. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 was not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. All sites were shown to be important for glycogen and trehalose metabolism. This study highlights the importance of CreA phosphorylation sites for the regulation of CCR. These sites are interesting targets for biotechnological strain engineering without the need to delete essential genes, which could result in undesired side effects.
IMPORTANCE in filamentous fungi, the transcription factor CreA controls carbohydrate metabolism through the regulation of genes encoding enzymes required for the use of alternative carbon sources. In this work, phosphorylation sites were identified on Aspergillus nidulans CreA, and subsequently, the two newly identified sites S268 and T308, the previously identified but uncharacterized site S262, and the previously characterized site S319 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was characterized. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 is not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. This work characterized novel CreA phosphorylation sites under carbon catabolite-repressing conditions and showed that they are crucial for CreA protein turnover, control of carbohydrate utilization, and biotechnologically relevant enzyme production.

© 2020 the Authors in certain niches, microbes encounter environmental challenges that are temporally linked. In such cases, microbial fitness is enhanced by the evolution of anticipatory responses where the initial challenge simultaneously activates pre-emptive protection against the second impending challenge. The accumulation of anticipatory responses in domesticated yeasts, which have been termed 'adaptive prediction', has led to the emergence of 'core stress responses' that provide stress cross-protection. Protective anticipatory responses also seem to be common in fungal pathogens of humans. These responses reflect the selective pressures that these fungi have faced relatively recently in their evolutionary history. Consequently, some pathogens have evolved 'core environmental responses' which exploit host signals to trigger immune evasion strategies that protect them against imminent immune attack.

ABSTRACT
The high-osmolarity glycerol (HOG) response pathway is a multifunctional signal transduction pathway that specifically transmits ambient osmotic signals. Saccharomyces cerevisiae Hog1p has two upstream signaling branches, the sensor histidine kinase Sln1p and the receptor Sho1p. The Sho1p branch includes two other proteins, the Msb2p mucin and Opy2p. Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Here, we investigated the roles played by A. fumigatus SlnASln1p, ShoASho1p, MsbAMsb2p, and OpyAOpy2p putative homologues during the activation of the mitogen-activated protein kinase (MAPK) HOG pathway. The shoA, msbA, and opyA singly and doubly null mutants are important for the cell wall integrity (CWI) pathway, oxidative stress, and virulence as assessed by a Galleria mellonella model. Genetic interactions of ShoA, MsbA, and OpyA are also important for proper activation of the SakAHog1p and MpkASlt2 cascade and the response to osmotic and cell wall stresses. Comparative label-free quantitative proteomics analysis of the singly null mutants with the wild-type strain upon caspofungin exposure indicates that the absence of ShoA, MsbA, and OpyA affects the osmotic stress response, carbohydrate metabolism, and protein degradation. The putative receptor mutants showed altered trehalose and glycogen accumulation, suggesting a role for ShoA, MsbA, and OpyA in sugar storage. Protein kinase a activity was also decreased in these mutants. We also observed genetic interactions between SlnA, ShoA, MsbA, and OpyA, suggesting that both branches are important for activation of the HOG/CWI pathways. Our results help in the understanding of the activation and modulation of the HOG and CWI pathways in this important fungal pathogen.
IMPORTANCE Aspergillus fumigatus is an important human-pathogenic fungal species that is responsible for a high incidence of infections in immunocompromised individuals. A. fumigatus high-osmolarity glycerol (HOG) and cell wall integrity pathways are important for the adaptation to different forms of environmental adversity such as osmotic and oxidative stresses, nutrient limitations, high temperatures, and other chemical and mechanical stresses that may be produced by the host immune system and antifungal drugs. Little is known about how these pathways are activated in this fungal pathogen. Here, we characterize four A. fumigatus putative homologues that are important for the activation of the yeast HOG pathway. A. fumigatus SlnASln1p, ShoASho1p, MsbAMsb2p, and OpyAOpy2p are genetically interacting and are essential for the activation of the HOG and cell wall integrity pathways. Our results contribute to the understanding of A. fumigatus adaptation to the host environment.

ABSTRACT
Filamentous fungi are remarkable producers of enzymes dedicated to the degradation of sugar polymers found in the plant cell wall. Here, we integrated transcriptomic data to identify novel transcription factors (TFs) related to the control of gene expression of lignocellulosic hydrolases in Trichoderma reesei and Aspergillus nidulans. Using various sets of differentially expressed genes, we identified some putative cis-regulatory elements that were related to known binding sites for Saccharomyces cerevisiae TFs. Comparative genomics allowed the identification of six transcriptional factors in filamentous fungi that have corresponding S. cerevisiae homologs. Additionally, a knockout strain of T. reesei lacking one of these TFs (S. cerevisiae AZF1 homolog) displayed strong reductions in the levels of expression of several cellulase-encoding genes in response to both Avicel and sugarcane bagasse, revealing a new player in the complex regulatory network operating in filamentous fungi during plant biomass degradation. Finally, RNA sequencing (RNA-seq) analysis showed the scope of the AZF1 homologue in regulating a number of processes in T. reesei, and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) provided evidence for the direct interaction of this TF in the promoter regions of cel7a, cel45a, and swo. Therefore, we identified here a novel TF which plays a positive effect in the expression of cellulase-encoding genes in T. reesei.
IMPORTANCE in this work, we used a systems biology approach to map new regulatory interactions in Trichoderma reesei controlling the expression of genes encoding cellulase and hemicellulase. By integrating transcriptomics related to complex biomass degradation, we were able to identify a novel transcriptional regulator which is able to activate the expression of these genes in response to two different cellulose sources. In vivo experimental validation confirmed the role of this new regulator in several other processes related to carbon source utilization and nutrient transport. Therefore, this work revealed novel forms of regulatory interaction in this model system for plant biomass deconstruction and also represented a new approach that could be easy applied to other organisms.

ABSTRACT
In filamentous fungi, an important kinase responsible for adaptation to changes in available nutrients is cyclic AMP (cAMP)-dependent protein kinase (protein kinase a [PKA]). This kinase has been well characterized at a molecular level, but its systemic action and direct/indirect targets are generally not well understood in filamentous fungi. In this work, we used a pkaA deletion strain (ΔpkaA) to identify Aspergillus nidulans proteins for which phosphorylation is dependent (either directly or indirectly) on PKA. A combination of phosphoproteomic and transcriptomic analyses revealed both direct and indirect targets of PKA and provided a global perspective on its function. One of these targets was the transcription factor CreA, the main repressor responsible for carbon catabolite repression (CCR). In the ΔpkaA strain, we identified a previously unreported phosphosite in CreA, S319, which (based on motif analysis) appears to be a direct target of Stk22 kinase (AN5728). Upon replacement of CreA S319 with an alanine (i.e. phosphonull mutant), the dynamics of CreA import to the nucleus are affected. Collectively, this work provides a global overview of PKA function while also providing novel insight regarding significance of a specific PKA-mediated phosphorylation event.
IMPORTANCE the cyclic AMP (cAMP)-dependent protein kinase a (PKA) signaling pathway is well conserved across eukaryotes, and previous work has shown that it plays an important role in regulating development, growth, and virulence in a number of fungi. PKA is activated in response to extracellular nutrients and acts to regulate metabolism and growth. While a number of components in the PKA pathway have been defined in filamentous fungi, current understanding does not provide a global perspective on PKA function. Thus, this work is significant in that it comprehensively identifies proteins and functional pathways regulated by PKA in a model filamentous fungus. This information enhances our understanding of PKA action and may provide information on how to manipulate it for specific purposes.

Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. β-glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have β-glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasitism of Trichoderma spp. only a few functional analysis studies have been conducted on glucanases. In the present study, we used a functional genomics approach to investigate the functional role of the gluc31 gene, which encodes an endo-β-1,3-glucanase belonging to the GH16 family in Trichoderma harzianum ALL42. We demonstrated that the absence of the gluc31 gene did not affect the in vivo mycoparasitism ability of mutant T. harzianum ALL42; however, gluc31 evidently influenced cell wall organization. Polymer measurements and fluorescence microscopy analyses indicated that the lack of the gluc31 gene induced a compensatory response by increasing the production of chitin and glucan polymers on the cell walls of the mutant hyphae. The mutant strain became more resistant to the fungicide benomyl compared to the parental strain. Furthermore, qRT-PCR analysis showed that the absence of gluc31 in T. harzianum resulted in the differential expression of other glycosyl hydrolases belonging to the GH16 family, because of functional redundancy among the glucanases.

ABSTRACT
The pathogenic fungus Aspergillus fumigatus is able to adapt to extremely variable environmental conditions. The A. fumigatus genome contains four genes coding for mitogen-activated protein kinases (MAPKs), which are important regulatory knots involved in diverse cellular responses. From a clinical perspective, MAPK activity has been connected to salvage pathways, which can determine the failure of effective treatment of invasive mycoses using antifungal drugs. Here, we report the characterization of the Saccharomyces cerevisiae Fus3 ortholog in A. fumigatus, designated MpkB. We demonstrate that MpkB is important for conidiation and that its deletion induces a copious increase of dihydroxynaphthalene (DHN)-melanin production. Simultaneous deletion of mpkB and mpkA, the latter related to maintenance of the cell wall integrity, normalized DHN-melanin production. Localization studies revealed that MpkB translocates into the nuclei when A. fumigatus germlings are exposed to caspofungin stress, and this is dependent on the cross-talk interaction with MpkA. Additionally, DHN-melanin formation was also increased after deletion of genes coding for the Gα protein GpaA and for the G protein-coupled receptor GprM. Yeast two-hybrid and coimmunoprecipitation assays confirmed that GpaA and GprM interact, suggesting their role in the MpkB signaling cascade.
IMPORTANCE Aspergillus fumigatus is the most important airborne human pathogenic fungus, causing thousands of deaths per year. Its lethality is due to late and often inaccurate diagnosis and the lack of efficient therapeutics. The failure of efficient prophylaxis and therapy is based on the ability of this pathogen to activate numerous salvage pathways that are capable of overcoming the different drug-derived stresses. A major role in the protection of A. fumigatus is played by melanins. Melanins are cell wall-associated macromolecules classified as virulence determinants. The understanding of the various signaling pathways acting in this organism can be used to elucidate the mechanism beyond melanin production and help to identify ideal drug targets.

ABSTRACTAspergillus fumigatusmitogen-activated protein kinases (MAPKs) are involved in maintaining the normal morphology of the cell wall and providing resistance against cell wall-damaging agents. Upon cell wall stress, cell wall-related sugars need to be synthesized from carbohydrate storage compounds. Here we show that this process is dependent on cAMP-dependent protein kinase a (PKA) activity and regulated by the high-osmolarity glycerol response (HOG) MAPKs SakA and MpkC. These protein kinases are necessary for normal accumulation/degradation of trehalose and glycogen, and the lack of these genes reduces glucose uptake and glycogen synthesis. Alterations in glycogen synthesis were observed for thesakAandmpkCdeletion mutants, which also displayed alterations in carbohydrate exposure on the cell wall. Carbohydrate mobilization is controlled by SakA interaction with PkaC1 and PkaR, suggesting a putative mechanism where the PkaR regulatory subunit leaves the complex and releases the SakA-PkaC1 complex for activation of enzymes involved in carbohydrate mobilization. This work reveals the communication between the HOG and PKA pathways for carbohydrate mobilization for cell wall construction.IMPORTANCEAspergillus fumigatusis an opportunistic human pathogen causing allergic reactions or systemic infections such as invasive pulmonary aspergillosis, especially in immunocompromised patients. The fungal cell wall is the main component responsible for recognition by the immune system, due to the specific composition of polysaccharide carbohydrates exposed on the surface of the fungal cell wall called pathogen-associated molecular patterns (PAMPs). Key enzymes in the fungal cell wall biosynthesis are a good target for fungal drug development. This report elucidates the cooperation between the HOG and PKA pathways in the mobilization of carbohydrates for fungal cell wall biosynthesis. We suggest that the reduced mobilization of simple sugars causes defects in the structure of the fungal cell wall. In summary, we propose that SakA is important for PKA activity, therefore regulating the availability and mobilization of monosaccharides for fungal cell wall biosynthesis during cell wall damage and the osmotic stress response.

ABSTRACTThe attachment of one or more ubiquitin molecules by SCF (Skp–Cullin–F-box) complexes to protein substrates targets them for subsequent degradation by the 26S proteasome, allowing the control of numerous cellular processes. Glucose-mediated signaling and subsequent carbon catabolite repression (CCR) are processes relying on the functional regulation of target proteins, ultimately controlling the utilization of this carbon source. In the filamentous fungusAspergillus nidulans, CCR is mediated by the transcription factor CreA, which modulates the expression of genes encoding biotechnologically relevant enzymes. Although CreA-mediated repression of target genes has been extensively studied, less is known about the regulatory pathways governing CCR and this work aimed at further unravelling these events. The Fbx23 F-box protein was identified as being involved in CCR and the Δfbx23mutant presented impaired xylanase production under repressing (glucose) and derepressing (xylan) conditions. Mass spectrometry showed that Fbx23 is part of an SCF ubiquitin ligase complex that is bridged via the GskA protein kinase to the CreA-SsnF-RcoA repressor complex, resulting in the degradation of the latter under derepressing conditions. Upon the addition of glucose, CreA dissociates from the ubiquitin ligase complex and is transported into the nucleus. Furthermore, casein kinase is important for CreA function during glucose signaling, although the exact role of phosphorylation in CCR remains to be determined. In summary, this study unraveled novel mechanistic details underlying CreA-mediated CCR and provided a solid basis for studying additional factors involved in carbon source utilization which could prove useful for biotechnological applications.IMPORTANCEThe production of biofuels from plant biomass has gained interest in recent years as an environmentally friendly alternative to production from petroleum-based energy sources. Filamentous fungi, which naturally thrive on decaying plant matter, are of particular interest for this process due to their ability to secrete enzymes required for the deconstruction of lignocellulosic material. A major drawback in fungal hydrolytic enzyme production is the repression of the corresponding genes in the presence of glucose, a process known as carbon catabolite repression (CCR). This report provides previously unknown mechanistic insights into CCR through elucidating part of the protein-protein interaction regulatory system that governs the CreA transcriptional regulator in the reference organismAspergillus nidulansin the presence of glucose and the biotechnologically relevant plant polysaccharide xylan.

ABSTRACT
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. Aspergillus fumigatus is an opportunistic fungal pathogen that causes invasive aspergillosis (IA), a life-threatening disease in immunocompromised humans. The echinocandin caspofungin, adopted as a second-line therapy in combating IA, is a β-1,3-glucan synthase inhibitor, which, when used in high concentrations, reverts the anticipated A. fumigatus growth inhibition, a phenomenon called the “caspofungin paradoxical effect” (CPE). The CPE has been widely associated with increased chitin content in the cell wall due to a compensatory upregulation of chitin synthase-encoding genes. Here, we demonstrate that the CPE is dependent on the cell wall integrity (CWI) mitogen-activated protein kinase MpkAMPK1 and its associated transcription factor (TF) RlmARLM1, which regulate chitin synthase gene expression in response to different concentrations of caspofungin. Furthermore, the calcium- and calcineurin-dependent TF CrzA binds to and regulates the expression of specific chitin synthase genes during the CPE. These results suggest that the regulation of cell wall biosynthetic genes occurs by several cellular signaling pathways. In addition, CrzA is also involved in cell wall organization in the absence of caspofungin. Differences in the CPE were also observed between two A. fumigatus clinical isolates, which led to the identification of a novel basic leucine zipper TF, termed ZipD. This TF functions in the calcium-calcineurin pathway and is involved in the regulation of cell wall biosynthesis genes. This study therefore unraveled additional mechanisms and novel factors governing the CPE response, which ultimately could aid in developing more effective antifungal therapies. IMPORTANCE Systemic Aspergillus fumigatus infections are often accompanied by high mortality rates. The fungal cell wall is important for infection as it has immunomodulatory and immunoevasive properties. Paradoxical growth of A. fumigatus in the presence of high concentrations of the cell wall-disturbing agent caspofungin has been observed for more than a decade, although the mechanistic nature of this phenomenon remains largely uncharacterized. Here, we show that the CWI pathway components MpkA and RlmA as well as the calcium/calcineurin-responsive transcription factor CrzA regulate the expression of cell wall biosynthetic genes during the caspofungin paradoxical effect (CPE). Furthermore, an additional, novel calcium/calcineurin-responsive transcription factor was identified to play a role in cell wall biosynthesis gene expression during the CPE. This work paints a crucial role for calcium metabolism in the CPE and provides further insight into the complex regulation of cell wall biosynthesis, which could ultimately lead to the development of more efficient antifungal therapies. IMPORTANCE Systemic Aspergillus fumigatus infections are often accompanied by high mortality rates. The fungal cell wall is important for infection as it has immunomodulatory and immunoevasive properties. Paradoxical growth of A. fumigatus in the presence of high concentrations of the cell wall-disturbing agent caspofungin has been observed for more than a decade, although the mechanistic nature of this phenomenon remains largely uncharacterized. Here, we show that the CWI pathway components MpkA and RlmA as well as the calcium/calcineurin-responsive transcription factor CrzA regulate the expression of cell wall biosynthetic genes during the caspofungin paradoxical effect (CPE). Furthermore, an additional, novel calcium/calcineurin-responsive transcription factor was identified to play a role in cell wall biosynthesis gene expression during the CPE. This work paints a crucial role for calcium metabolism in the CPE and provides further insight into the complex regulation of cell wall biosynthesis, which could ultimately lead to the development of more efficient antifungal therapies.

Agaricus blazei (Murrill) ss. Heinem. é um cogumelo nativo do Brasil que vem despertando a atenção de vários pesquisadores em todo o mundo, devido às suas propriedades farmacológicas e nutricionais. Este trabalho teve por objetivo, analisar a variabilidade genética de alguns isolados utilizados comercialmente, por meio de marcadores RAPD. Foram analisados nove isolados de A. blazei, provenientes de diferentes regiões do Brasil e, como controle, dois isolados de A. bisporus. Todos eles fazem parte da coleção de fungos do Laboratório de Cogumelos Comestíveis e Medicinais do DBI/UFLA. Diferentes primers aleatórios foram utilizados, gerando um total de 139 bandas polimórficas. Procedeu-se a avaliação de similaridade genética entre os isolados pelo coeficiente de Dice e análise de agrupamento pelo método UPGMA. Os resultados revelaram que dos 9 isolados de A. blazei, 6 (CS1, CS3, CS4, CS6, CS8 e CS9) apresentaram uma alta similaridade genética, sendo considerados isolados de uma mesma origem ou clones. O isolado CS2 foi o que apresentou a maior divergência genética em relação aos demais, seguido dos isolados CS5 e CS7, os quais formaram cada um, um grupo a parte, com médias de 60,6%, 88,7% e 91,3% de similaridade genética, respectivamente.