Identification of a common secondary mutation in the Neurospora crassa knockout collection conferring a cell fusion-defective phenotype.

Gene-deletion mutants represent a powerful tool to study gene function. The filamentous fungus Neurospora crassa is a well-established model organism, and features a comprehensive gene knockout strain collection. While these mutant strains have been used in numerous studies, resulting in the functional annotation of many Neurospora genes, direct confirmation of gene-phenotype relationships is often lacking, which is particularly relevant given the possibility of background mutations, sample contamination, and/or strain mislabeling. Indeed, spontaneous mutations resulting in phenotypes resembling many cell fusion mutants have long been known to occur at relatively high frequency in N. crassa, and these secondary mutations are common in the Neurospora deletion collection. The identity of these mutations, however, is largely unknown. Here, we report that the Δada-3 strain from the N. crassa knockout collection, which exhibits a cell fusion defect, harbors a secondary mutation responsible for this phenotype. Through whole-genome sequencing and genetic analyses, we found a ~30-Kb deletion in this strain affecting a known cell fusion-related gene, so/ham-1, and show that it is the absence of this gene-and not of ada-3-that underlies its cell fusion defect. We additionally found three other knockout strains harboring the same deletion, suggesting that this mutation may be common in the collection and could have impacted previous studies. Our findings provide a cautionary note and highlight the importance of proper functional validation of strains from mutant collections. We discuss our results in the context of the spread of cell fusion-defective cheater variants in N. crassa cultures. IMPORTANCE This study emphasizes the need for careful and detailed characterization of strains from mutant collections. Specifically, we found a common deletion in various strains from the Neurospora crassa gene knockout collection that results in a cell fusion-defective phenotype. This is noteworthy because this collection is known to contain background mutations-of a largely unclear nature-that produce cell fusion-defective phenotypes. Our results describe an example of such mutations, and highlight how this common genetic defect could have impacted previous studies that have used the affected strains. Furthermore, they provide a cautionary note about the use of Neurospora strains with similar phenotypes. Lastly, these findings offer additional details relevant to our understanding of the origin and spread of cell fusion-defective cheater variants in N. crassa cultures.

Genome-Wide Characterization of Light-Regulated Gene Expression in Botrytis cinerea Reveals Underlying Complex Photobiology.

Botrytis cinerea is a necrotrophic fungus characterized mainly by its wide host range of infected plants. The deletion of the white-collar-1 gene (bcwcl1), which encodes for a blue-light receptor/transcription factor, causes a decrease in virulence, particularly when assays are conducted in the presence of light or photocycles. However, despite ample characterization, the extent of the light-modulated transcriptional responses regulated by BcWCL1 remains unknown. In this study, pathogen and pathogen:host RNA-seq analyses, conducted during non-infective in vitro plate growth and when infecting Arabidopsis thaliana leaves, respectively, informed on the global gene expression patterns after a 60 min light pulse on the wild-type B05.10 or ∆bcwcl1 B. cinerea strains. The results revealed a complex fungal photobiology, where the mutant did not react to the light pulse during its interaction with the plant. Indeed, when infecting Arabidopsis, no photoreceptor-encoding genes were upregulated upon the light pulse in the ∆bcwcl1 mutant. Differentially expressed genes (DEGs) in B. cinerea under non-infecting conditions were predominantly related to decreased energy production in response to the light pulse. In contrast, DEGs during infection significantly differ in the B05.10 strain and the ∆bcwcl1 mutant. Upon illumination at 24 h post-infection in planta, a decrease in the B. cinerea virulence-associated transcripts was observed. Accordingly, after a light pulse, biological functions associated with plant defense appear enriched among light-repressed genes in fungus-infected plants. Taken together, our results show the main transcriptomic differences between wild-type B. cinerea B05.10 and ∆bcwcl1 after a 60 min light pulse when growing saprophytically on a Petri dish and necrotrophically over A. thaliana.

The Botrytis cinerea Gene Expression Browser.

For comprehensive gene expression analyses of the phytopathogenic fungus Botrytis cinerea, which infects a number of plant taxa and is a cause of substantial agricultural losses worldwide, we developed BEB, a web-based B. cinerea gene Expression Browser. This computationally inexpensive web-based application and its associated database contain manually curated RNA-Seq data for B. cinerea. BEB enables expression analyses of genes of interest under different culture conditions by providing publication-ready heatmaps depicting transcript levels, without requiring advanced computational skills. BEB also provides details of each experiment and user-defined gene expression clustering and visualization options. If needed, tables of gene expression values can be downloaded for further exploration, including, for instance, the determination of differentially expressed genes. The BEB implementation is based on open-source computational technologies that can be deployed for other organisms. In this case, the new implementation will be limited only by the number of transcriptomic experiments that are incorporated into the platform. To demonstrate the usability and value of BEB, we analyzed gene expression patterns across different conditions, with a focus on secondary metabolite gene clusters, chromosome-wide gene expression, previously described virulence factors, and reference genes, providing the first comprehensive expression overview of these groups of genes in this relevant fungal phytopathogen. We expect this tool to be broadly useful in B. cinerea research, providing a basis for comparative transcriptomics and candidate gene identification for functional assays.

Circadian oscillations in Trichoderma atroviride and the role of core clock components in secondary metabolism, development, and mycoparasitism against the phytopathogen Botrytis cinerea.

Circadian clocks are important for an individual's fitness, and recent studies have underlined their role in the outcome of biological interactions. However, the relevance of circadian clocks in fungal-fungal interactions remains largely unexplored. We sought to characterize a functional clock in the biocontrol agent Trichoderma atroviride to assess its importance in the mycoparasitic interaction against the phytopathogen Botrytis cinerea. Thus, we confirmed the existence of circadian rhythms in T. atroviride, which are temperature-compensated and modulated by environmental cues such as light and temperature. Nevertheless, the presence of such molecular rhythms appears to be highly dependent on the nutritional composition of the media. Complementation of a clock null (Δfrq) Neurospora crassa strain with the T. atroviride-negative clock component (tafrq) restored core clock function, with the same period observed in the latter fungus, confirming the role of tafrq as a bona fide core clock component. Confrontation assays between wild-type and clock mutant strains of T. atroviride and B. cinerea, in constant light or darkness, revealed an inhibitory effect of light on T. atroviride's mycoparasitic capabilities. Interestingly, when confrontation assays were performed under light/dark cycles, T. atroviride's overgrowth capacity was enhanced when inoculations were at dawn compared to dusk. Deleting the core clock-negative element FRQ in B. cinerea, but not in T. atroviride, was vital for the daily differential phenotype, suggesting that the B. cinerea clock has a more significant influence on the result of this interaction. Additionally, we observed that T. atroviride clock components largely modulate development and secondary metabolism in this fungus, including the rhythmic production of distinct volatile organic compounds (VOCs). Thus, this study provides evidence on how clock components impact diverse aspects of T. atroviride lifestyle and how daily changes modulate fungal interactions and dynamics.

Interactions between Core Elements of the Botrytis cinerea Circadian Clock Are Modulated by Light and Different Protein Domains.

Botrytis cinerea possesses a complex light-sensing system composed of eleven photoreceptors. In B. cinerea, bcwcl1 encodes for the BcWCL1 protein, the orthologue of the blue-light photoreceptor WC-1 from Neurospora crassa. The functional partner of BcWCL1 is the BcWCL2 protein, both interacting in the nucleus and forming the B. cinerea white collar complex (BcWCC). This complex is required for photomorphogenesis and circadian regulation. However, no molecular evidence shows a light-dependent interaction between the BcWCC components or light-sensing capabilities in BcWCL1. In this work, by employing a yeast two-hybrid system that allows for the in vivo analysis of protein-protein interactions, we confirm that BcWCL1 and BcWCL2 interact in the absence of light as well as upon blue-light stimulation, primarily through their PAS (Per-Arnt-Sim) domains. Deletion of the PAS domains present in BcWCL1 (BcWCL1PAS∆) or BcWCL2 (BcWCL2PAS∆) severely impairs the interaction between these proteins. Interestingly, the BcWCL1PAS∆ protein shows a blue-light response and interacts with BcWCL2 or BcWCL2PAS∆ upon light stimulation. Finally, we demonstrate that BcWCL1 and BcWCL1PAS∆ respond to blue light by introducing a point mutation in the photoactive cysteine, confirming that both proteins are capable of light sensing. Altogether, the results revealed the complexity of protein-protein interactions occurring between the core elements of the B. cinerea circadian clock.

A comprehensive transcription factor and DNA-binding motif resource for the construction of gene regulatory networks in Botrytis cinerea and Trichoderma atroviride.

Botrytis cinerea and Trichoderma atroviride are two relevant fungi in agricultural systems. To gain insights into these organisms' transcriptional gene regulatory networks (GRNs), we generated a manually curated transcription factor (TF) dataset for each of them, followed by a GRN inference utilizing available sequence motifs describing DNA-binding specificity and global gene expression data. As a proof of concept of the usefulness of this resource to pinpoint key transcriptional regulators, we employed publicly available transcriptomics data and a newly generated dual RNA-seq dataset to build context-specific Botrytis and Trichoderma GRNs under two different biological paradigms: exposure to continuous light and Botrytis-Trichoderma confrontation assays. Network analysis of fungal responses to constant light revealed striking differences in the transcriptional landscape of both fungi. On the other hand, we found that the confrontation of both microorganisms elicited a distinct set of differentially expressed genes with changes in T. atroviride exceeding those in B. cinerea. Using our regulatory network data, we were able to determine, in both fungi, central TFs involved in this interaction response, including TFs controlling a large set of extracellular peptidases in the biocontrol agent T. atroviride. In summary, our work provides a comprehensive catalog of transcription factors and regulatory interactions for both organisms. This catalog can now serve as a basis for generating novel hypotheses on transcriptional regulatory circuits in different experimental contexts.

Uncovering Divergence in Gene Expression Regulation in the Adaptation of Yeast to Nitrogen Scarcity.

Saccharomyces cerevisiae rewires its transcriptional output to survive stressful environments, such as nitrogen scarcity under fermentative conditions. Although divergence in nitrogen metabolism among natural yeast populations has been reported, the impact of regulatory genetic variants modulating gene expression and nitrogen consumption remains to be investigated. Here, we employed an F1 hybrid from two contrasting S. cerevisiae strains, providing a controlled genetic environment to map cis factors involved in the divergence of gene expression regulation in response to nitrogen scarcity. We used a dual approach to obtain genome-wide allele-specific profiles of chromatin accessibility, transcription factor binding, and gene expression through ATAC-seq (assay for transposase accessible chromatin) and RNA-seq (transcriptome sequencing). We observed large variability in allele-specific expression and accessibility between the two genetic backgrounds, with a third of these differences specific to a deficient nitrogen environment. Furthermore, we discovered events of allelic bias in gene expression correlating with allelic bias in transcription factor binding solely under nitrogen scarcity, where the majority of these transcription factors orchestrates the nitrogen catabolite repression regulatory pathway and demonstrates a cis × environment-specific response. Our approach allowed us to find cis variants modulating gene expression, chromatin accessibility, and allelic differences in transcription factor binding in response to low nitrogen culture conditions. IMPORTANCE Historically, coding variants were prioritized when searching for causal mechanisms driving adaptation of natural populations to stressful environments. However, the recent focus on noncoding variants demonstrated their ubiquitous role in adaptation. Here, we performed genome-wide regulatory variation profiles between two divergent yeast strains when facing nitrogen nutritional stress. The open chromatin availability of several regulatory regions changes in response to nitrogen scarcity. Importantly, we describe regulatory events that deviate between strains. Our results demonstrate a widespread variation in gene expression regulation between naturally occurring populations in response to stressful environments.

Salinity impairs photosynthetic capacity and enhances carotenoid-related gene expression and biosynthesis in tomato (Solanum lycopersicum L. cv. Micro-Tom).

Carotenoids are essential components of the photosynthetic antenna and reaction center complexes, being also responsible for antioxidant defense, coloration, and many other functions in multiple plant tissues. In tomato, salinity negatively affects the development of vegetative organs and productivity, but according to previous studies it might also increase fruit color and taste, improving its quality, which is a current agricultural challenge. The fruit quality parameters that are increased by salinity are cultivar-specific and include carotenoid, sugar, and organic acid contents. However, the relationship between vegetative and reproductive organs and response to salinity is still poorly understood. Considering this, Solanum lycopersicum cv. Micro-Tom plants were grown in the absence of salt supplementation as well as with increasing concentrations of NaCl for 14 weeks, evaluating plant performance from vegetative to reproductive stages. In response to salinity, plants showed a significant reduction in net photosynthesis, stomatal conductance, PSII quantum yield, and electron transport rate, in addition to an increase in non-photochemical quenching. In line with these responses the number of tomato clusters decreased, and smaller fruits with higher soluble solids content were obtained. Mature-green fruits also displayed a salt-dependent higher induction in the expression of PSY1, PDS, ZDS, and LYCB, key genes of the carotenoid biosynthesis pathway, in correlation with increased lycopene, lutein, ß-carotene, and violaxanthin levels. These results suggest a key relationship between photosynthetic plant response and yield, involving impaired photosynthetic capacity, increased carotenoid-related gene expression, and carotenoid biosynthesis.

Defects in the Ferroxidase That Participates in the Reductive Iron Assimilation System Results in Hypervirulence in Botrytis Cinerea.

The plant pathogen Botrytis cinerea is responsible for gray-mold disease, which infects a wide variety of species. The outcome of this host-pathogen interaction, a result of the interplay between plant defense and fungal virulence pathways, can be modulated by various environmental factors. Among these, iron availability and acquisition play a crucial role in diverse biological functions. How B. cinerea obtains iron, an essential micronutrient, during infection is unknown. We set out to determine the role of the reductive iron assimilation (RIA) system during B. cinerea infection. This system comprises the BcFET1 ferroxidase, which belongs to the multicopper oxidase (MCO) family of proteins, and the BcFTR1 membrane-bound iron permease. Gene knockout and complementation studies revealed that, compared to the wild type, the bcfet1 mutant displays delayed conidiation, iron-dependent sclerotium production, and significantly reduced whole-cell iron content. Remarkably, this mutant exhibited a hypervirulence phenotype, whereas the bcftr1 mutant presents normal virulence and unaffected whole-cell iron levels and developmental programs. Interestingly, while in iron-starved plants wild-type B. cinerea produced slightly reduced necrotic lesions, the hypervirulence phenotype of the bcfet1 mutant is no longer observed in iron-deprived plants. This suggests that B. cinerea bcfet1 knockout mutants require plant-derived iron to achieve larger necrotic lesions, whereas in planta analyses of reactive oxygen species (ROS) revealed increased ROS levels only for infections caused by the bcfet1 mutant. These results suggest that increased ROS production, under an iron sufficiency environment, at least partly underlie the observed infection phenotype in this mutant.IMPORTANCE The plant-pathogenic fungus B. cinerea causes enormous economic losses, estimated at anywhere between $10 billion and $100 billion worldwide, under both pre- and postharvest conditions. Here, we present the characterization of a loss-of-function mutant in a component involved in iron acquisition that displays hypervirulence. While in different microbial systems iron uptake mechanisms appear to be critical to achieve full pathogenic potential, we found that the absence of the ferroxidase that is part of the reductive iron assimilation system leads to hypervirulence in this fungus. This is an unusual and rather underrepresented phenotype, which can be modulated by iron levels in the plant and provides an unexpected link between iron acquisition, reactive oxygen species (ROS) production, and pathogenesis in the Botrytis-plant interaction.

The Clock Keeps on Ticking: Emerging Roles for Circadian Regulation in the Control of Fungal Physiology and Pathogenesis.

Tic-tac, tic-tac, the sound of time is familiar to us, yet, it also silently shapes daily biological processes conferring 24-hour rhythms in, among others, cellular and systemic signaling, gene expression, and metabolism. Indeed, circadian clocks are molecular machines that permit temporal control of a variety of processes in individuals, with a close to 24-hour period, optimizing cellular dynamics in synchrony with daily environmental cycles. For over three decades, the molecular bases of these clocks have been extensively described in the filamentous fungus Neurospora crassa, yet, there have been few molecular studies in fungi other than Neurospora, despite evidence of rhythmic phenomena in many fungal species, including pathogenic ones. This chapter will revise the mechanisms underlying clock regulation in the model fungus N. crassa, as well as recent findings obtained in several fungi. In particular, this chapter will review the effect of circadian regulation of virulence and organismal interactions, focusing on the phytopathogen Botrytis cinerea, as well as several entomopathogenic fungi, including the behavior-manipulating species Ophiocordyceps kimflemingiae and Entomophthora muscae. Finally, this review will comment current efforts in the study of mammalian pathogenic fungi, while highlighting recent circadian lessons from parasites such as Trypanosoma and Plasmodium. The clock keeps on ticking, whether we can hear it or not.

WRKY7, -11 and -17 transcription factors are modulators of the bZIP28 branch of the unfolded protein response during PAMP-triggered immunity in Arabidopsis thaliana.

Plants must defend themselves against pathogens. The defense response requires greater protein synthesis, which generates endoplasmic reticulum (ER) stress, yet failure to attenuate this stress has detrimental effects. WRKY7/11/17 transcription factors (TFs) are negative regulators of immunity since mutants are more resistant to Pseudomonas syringae pv tomato (Pst) infection. Here, we reveal a connection between ER-stress and the molecular mechanisms underlying the wrky mutant phenotype. The bZIP28 TF upregulates ER-chaperone expression (BiP1/2, ERdj3B, and SDF2) upon exposure of Arabidopsis to a bacterial defense elicitor, flagellin 22 (Flg22). Also, the activation of ER-chaperones is more sustained in double and triple wrky mutants treated with Flg22, suggesting that WRKY7/11/17 TFs downregulate these genes. Moreover, wrky mutants accumulate more bZIP28 transcripts in response to Flg22, indicating that WRKY7/11/17 transcriptionally repress this TF. Using Arabidopsis protoplasts, we also demonstrate that WRKYs bind to the bZIP28 promoter via W-box elements. Additionally, triple wrky mutants are more resistant, whilst bzip28 mutants are more susceptible, to Pst infection. Finally, we postulate a model of PAMP-Triggered Immunity regulation, where Flg22 activates bZIP28-signaling inducing the expression of ER-stress genes, as well as WRKY7/11/17 expression, which in turn inhibits PTI by downregulating bZIP28, controlling physiological responses in the Arabidopsis-Pst interaction.

Spontaneous circadian rhythms in a cold-adapted natural isolate of Aureobasidium pullulans.

Circadian systems enable organisms to synchronize their physiology to daily and seasonal environmental changes relying on endogenous pacemakers that oscillate with a period close to 24 h even in the absence of external timing cues. The oscillations are achieved by intracellular transcriptional/translational feedback loops thoroughly characterized for many organisms, but still little is known about the presence and characteristics of circadian clocks in fungi other than Neurospora crassa. We sought to characterize the circadian system of a natural isolate of Aureobasidium pullulans, a cold-adapted yeast bearing great biotechnological potential. A. pullulans formed daily concentric rings that were synchronized by light/dark cycles and were also formed in constant darkness with a period of 24.5 h. Moreover, these rhythms were temperature compensated, as evidenced by experiments conducted at temperatures as low as 10 °C. Finally, the expression of clock-essential genes, frequency, white collar-1, white collar-2 and vivid was confirmed. In summary, our results indicate the existence of a functional circadian clock in A. pullulans, capable of sustaining rhythms at very low temperatures and, based on the presence of conserved clock-gene homologues, suggest a molecular and functional relationship to well-described circadian systems.

Recent Advances in the Study of the Plant Pathogenic Fungus Botrytis cinerea and its Interaction with the Environment.

The primary contact between the fungal phytopathogen Botrytis cinerea and its host takes place at the cell surface of both organisms. The fungal cell wall is generally composed of an inner skeletal layer consisting of various polysaccharides surrounded by a layer of glycoproteins. Some of these glycoproteins have structural or enzymatic functions, or are involved in conidial adhesion. After landing on the host surface and sensing appropriate signals, B. cinerea conidia produce a germ tube and secrete phytotoxic fungal metabolites and cell wall-degrading enzymes (CWDEs), facilitating host penetration. In fact, 118 genes encoding putative Carbohydrate-Active Enzymes (CAZymes) have been identified in the B. cinerea genome. This large enzymatic repertoire could explain, at least in part, the ability of B. cinerea to infect a vast number of plant species. In recent years, several genes and signaling factors have been identified as playing key roles in pathogenesis, particularly in appressorium formation and penetration. These include the NOX Complex, MAPK cascades, heterotrimeric G proteins, histidine kinases and cAMP signaling pathways. Some of these pathways could also be responsible for controlling the expression and secretion of CWDEs and/or secondary metabolites during infection. Herein, putative virulence factors that are linked to the cell wall, as well as recentlydescribed genes and components that allow the sensing of environmental cues, are highlighted.

Circadian clocks and the regulation of virulence in fungi: Getting up to speed.

You cannot escape time. Therefore, it seems wise to learn how to keep track of it and use it to your advantage. Circadian clocks are molecular circuits that allow organisms to temporally coordinate a plethora of processes, including gene expression, with a close to 24h rhythm, optimizing cellular function in synchrony with daily environmental cycles. The molecular bases of these clocks have been extensively studied in the fungus Neurospora crassa, providing a detailed molecular description. Surprisingly, there is scarce molecular information of clocks in fungi other than Neurospora, despite the existence of rhythmic phenomena in many fungal species, including pathogenic ones. This review will comment on the overall importance of clocks, what is known in Neurospora and what has been described in other fungi including new insights on the evolution of fungal clock components. The molecular description of the circadian system of the phytopathogenic fungus Botrytis cinerea will be revisited, as well as time-of-the-day variation in host-pathogen interaction dynamics, utilizing an Arabidopsis-Botrytis system, including also what is known regarding circadian regulation of defense mechanisms in the Arabidopsis thaliana plant model. Finally, this review will mention how little is known about circadian regulation of human pathogenic fungi, commenting on potential future directions and the overall perspective of fungal circadian studies.

Around the Fungal Clock: Recent Advances in the Molecular Study of Circadian Clocks in Neurospora and Other Fungi.

Night follows day and as a consequence, organisms have evolved molecular machineries that allow them to anticipate and respond to the many changes that accompany these transitions. Circadian clocks are precise yet plastic pacemakers that allow the temporal organization of a plethora of biological process. Circadian clocks are widespread across the tree of life and while their exact molecular components differ among phyla, they tend to share common design principles. In this review, we discuss the circadian system of the filamentous fungus Neurospora crassa. Historically, this fungus has served a key role in the genetic and molecular dissection of circadian clocks, aiding in their detailed mechanistic understanding. Recent studies have provided new insights into the daily molecular dynamics that constitute the Neurospora circadian oscillator, some of which have questioned traditional paradigms describing timekeeping mechanisms in eukaryotes. In addition, recent reports support the idea of a dynamic network of transcription factors underlying the rhythmicity of thousands of genes in Neurospora, many of which oscillate only under specific conditions. Besides Neurospora, which harbors the best characterized circadian system among filamentous fungi, the recent characterization of the circadian system of the plant-pathogenic fungus Botrytis cinerea has provided additional insights into the physiological impact of the clock and potential additional functions of clock proteins in fungi. Finally, we speculate on the presence of FRQ or FRQ-like proteins in diverse fungal lineages.

Transcriptome analysis reveals regulatory networks underlying differential susceptibility to Botrytis cinerea in response to nitrogen availability in Solanum lycopersicum.

Nitrogen (N) is one of the main limiting nutrients for plant growth and crop yield. It is well documented that changes in nitrate availability, the main N source found in agricultural soils, influences a myriad of developmental programs and processes including the plant defense response. Indeed, many agronomical reports indicate that the plant N nutritional status influences their ability to respond effectively when challenged by different pathogens. However, the molecular mechanisms involved in N-modulation of plant susceptibility to pathogens are poorly characterized. In this work, we show that Solanum lycopersicum defense response to the necrotrophic fungus Botrytis cinerea is affected by plant N availability, with higher susceptibility in nitrate-limiting conditions. Global gene expression responses of tomato against B. cinerea under contrasting nitrate conditions reveals that plant primary metabolism is affected by the fungal infection regardless of N regimes. This result suggests that differential susceptibility to pathogen attack under contrasting N conditions is not only explained by a metabolic alteration. We used a systems biology approach to identify the transcriptional regulatory network implicated in plant response to the fungus infection under contrasting nitrate conditions. Interestingly, hub genes in this network are known key transcription factors involved in ethylene and jasmonic acid signaling. This result positions these hormones as key integrators of nitrate and defense against B. cinerea in tomato plants. Our results provide insights into potential crosstalk mechanisms between necrotrophic defense response and N status in plants.

A circadian oscillator in the fungus Botrytis cinerea regulates virulence when infecting Arabidopsis thaliana.

The circadian clock of the plant model Arabidopsis thaliana modulates defense mechanisms impacting plant-pathogen interactions. Nevertheless, the effect of clock regulation on pathogenic traits has not been explored in detail. Moreover, molecular description of clocks in pathogenic fungi--or fungi in general other than the model ascomycete Neurospora crassa--has been neglected, leaving this type of question largely unaddressed. We sought to characterize, therefore, the circadian system of the plant pathogen Botrytis cinerea to assess if such oscillatory machinery can modulate its virulence potential. Herein, we show the existence of a functional clock in B. cinerea, which shares similar components and circuitry with the Neurospora circadian system, although we found that its core negative clock element FREQUENCY (BcFRQ1) serves additional roles, suggesting extracircadian functions for this protein. We observe that the lesions produced by this necrotrophic fungus on Arabidopsis leaves are smaller when the interaction between these two organisms occurs at dawn. Remarkably, this effect does not depend solely on the plant clock, but instead largely relies on the pathogen circadian system. Genetic disruption of the B. cinerea oscillator by mutation, overexpression of BcFRQ1, or by suppression of its rhythmicity by constant light, abrogates circadian regulation of fungal virulence. By conducting experiments with out-of-phase light:dark cycles, we confirm that indeed, it is the fungal clock that plays the main role in defining the outcome of the Arabidopsis-Botrytis interaction, providing to our knowledge the first evidence of a microbial clock modulating pathogenic traits at specific times of the day.

bcpmr1 encodes a P-type Ca(2+)/Mn(2+)-ATPase mediating cell-wall integrity and virulence in the phytopathogen Botrytis cinerea.

The cell wall of fungi is generally composed of an inner skeletal layer consisting of various polysaccharides surrounded by a layer of glycoproteins. These usually contain both N- and O-linked oligosaccharides, coupled to the proteins by stepwise addition of mannose residues by mannosyltransferases in the endoplasmic reticulum and the Golgi apparatus. In yeast, an essential luminal cofactor for these mannosyltransferases is Mn(2+) provided by the Ca(2+)/Mn(2+)-ATPase known as Pmr1. In this study, we have identified and characterized the Botrytis cinerea pmr1 gene, the closest homolog of yeast PMR1. We hypothesized that bcpmr1 also encodes a Ca(2+)/Mn(2+)-ATPase that plays an important role in the protein glycosylation pathway. Phenotypic analysis showed that bcpmr1 null mutants displayed a significant reduction in conidial production, radial growth and diameter of sclerotia. Significant alterations in hyphal cell wall composition were observed including a 83% decrease of mannan levels and an increase in the amount of chitin and glucan. These changes were accompanied by a hypersensitivity to cell wall-perturbing agents such as Calcofluor white, Congo red and zymolyase. Importantly, the Δbcpmr1 mutant showed reduced virulence in tomato (leafs and fruits) and apple (fruits) and reduced biofilm formation. Together, our results highlight the importance of bcpmr1 for protein glycosylation, cell wall structure and virulence of B. cinerea.

Analysis of the Phlebiopsis gigantea genome, transcriptome and secretome provides insight into its pioneer colonization strategies of wood.

Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh-cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.