The Snf5-Hsf1 transcription module synergistically regulates stress responses and pathogenicity by maintaining ROS homeostasis in Sclerotinia sclerotiorum.

The SWI/SNF complex is guided to the promoters of designated genes by its co-operator to activate transcription in a timely and appropriate manner to govern development, pathogenesis, and stress responses in fungi. Nevertheless, knowledge of the complexes and their co-operator in phytopathogenic fungi is still fragmented. We demonstrate that the heat shock transcription factor SsHsf1 guides the SWI/SNF complex to promoters of heat shock protein (hsp) genes and antioxidant enzyme genes using biochemistry and pharmacology. This is accomplished through direct interaction with the complex subunit SsSnf5 under heat shock and oxidative stress. This results in the activation of their transcription and mediates histone displacement to maintain reactive oxygen species (ROS) homeostasis. Genetic results demonstrate that the transcription module formed by SsSnf5 and SsHsf1 is responsible for regulating morphogenesis, stress tolerance, and pathogenicity in Sclerotinia sclerotiorum, especially by directly activating the transcription of hsp genes and antioxidant enzyme genes counteracting plant-derived ROS. Furthermore, we show that stress-induced phosphorylation of SsSnf5 is necessary for the formation of the transcription module. This study establishes that the SWI/SNF complex and its co-operator cooperatively regulate the transcription of hsp genes and antioxidant enzyme genes to respond to host and environmental stress in the devastating phytopathogenic fungi.

Two Putative Pheromone Receptors, but Not Their Cognate Pheromones, Regulate Female Fertility in the Atypical Mating Fungus Colletotrichum fructicola.

Colletotrichum fungi are a group of damaging phytopathogens with atypical mating type loci (harboring only MAT1-2-1 but not MAT1-1-1) and complex sexual behaviors. Sex pheromones and their cognate G-protein-coupled receptors are conserved regulators of fungal mating. These genes, however, lose function frequently among Colletotrichum species, indicating a possibility that pheromone signaling is dispensable for Colletotrichum sexual reproduction. We have identified two putative pheromone-receptor pairs (PPG1:PRE2, PPG2:PRE1) in C. fructicola, a species that exhibits plus-to-minus mating type switching and plus-minus-mediated mating line development. Here, we report the generation and characterization of gene-deletion mutants for all four genes in both plus and minus strain backgrounds. Single-gene deletion of pre1 or pre2 had no effect on sexual development, whereas their double deletion caused self-sterility in both the plus and minus strains. Moreover, double deletion of pre1 and pre2 caused female sterility in plus-minus outcrossing. Double deletion of pre1 and pre2, however, did not inhibit perithecial differentiation or plus-minus-mediated enhancement of perithecial differentiation. Contrary to the results with pre1 and pre2, double deletion of ppg1 and ppg2 had no effect on sexual compatibility, development, or fecundity. We concluded that pre1 and pre2 coordinately regulate C. fructicola mating by recognizing novel signal molecule(s) distinct from canonical Ascomycota pheromones. The contrasting importance between pheromone receptors and their cognate pheromones highlights the complicated nature of sex regulation in Colletotrichum fungi.

Discerning the global phylogeographic distribution of Phyllosticta citricarpa by means of whole genome sequencing.

Phyllosticta citricarpa is a fungal pathogen causing citrus black spot (CBS). As a regulated pest in some countries, the presence of the pathogen limits the export of fruit and is therefore of agricultural and economic importance. In this study, we used high throughput sequencing data to infer the global phylogeographic distribution of this pathogen, including 71 isolates from eight countries, Argentina, Australia, Brazil, China, Cuba, Eswatini, South Africa and the United States of America. We assembled draft genomes and used a pairwise read mapping approach for the detection and enumeration of variants between isolates. We performed SSR marker discovery based on the assembled genome with the best assembly statistics, and generated genotype profiles for all isolates with 1987 SSR markers in silico. Furthermore, we identified 32,560 SNPs relative to a reference sequence followed by population genetic analyses based on the three datasets; pairwise variant counts, SSR genotypes and SNP genotypes. All three analysis approaches gave similar overall results. Possible pathways of dissemination among the populations from China, Australia, southern Africa and the Americas are postulated. The Chinese population is the most diverse, and is genetically the furthest removed from all other populations, and is therefore considered the closest to the origin of the pathogen. Isolates from Australia, Eswatini and the South African province Mpumalanga are closely associated and clustered together with those from Argentina and Brazil. The Eastern Cape, North West, and KwaZulu-Natal populations in South Africa grouped in another cluster, while isolates from Limpopo are distributed between the two aforementioned clusters. Southern African populations showed a close relationship to populations in North America, and could be a possible source of P. citricarpa populations that are now found in North America. This study represents the largest whole genome sequencing survey of P. citricarpa to date and provides a more comprehensive assessment of the population genetic diversity and connectivity of P. citricarpa from different geographic origins. This information could further assist in a better understanding of the epidemiology of the CBS pathogen, its long-distance dispersal and dissemination pathways, and can be used to refine phytosanitary regulations and management programmes for the disease.

The C2H2 Transcription Factor SsZFH1 Regulates the Size, Number, and Development of Apothecia in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a notorious phytopathogenic Ascomycota fungus with a host range of >600 plant species worldwide. This homothallic Leotiomycetes species reproduces sexually through a multicellular apothecium that produces and releases ascospores. These ascospores serve as the primary inoculum source for disease initiation in the majority of S. sclerotiorum disease cycles. The regulation of apothecium development for this pathogen and other apothecium-producing fungi remains largely unknown. Here, we report that a C2H2 transcription factor, SsZFH1 (zinc finger homologous protein), is necessary for the proper development and maturation of sclerotia and apothecia in S. sclerotiorum and is required for the normal growth rate of hyphae. Furthermore, ΔSszfh1 strains exhibit decreased H2O2 accumulation in hyphae, increased melanin deposition, and enhanced tolerance to H2O2 in the process of vegetative growth and sclerotia formation. Infection assays on common bean leaves, with thin cuticles, and soybean and tomato leaves, with thick cuticles, suggest that the deletion of Sszfh1 slows the mycelial growth rate, which in turn affects the expansion of leaf lesions. Collectively, our results provide novel insights into a major fungal factor mediating maturation of apothecia with additional effects on hyphae and sclerotia development.

Transcriptional profile of oil palm pathogen, Ganoderma boninense, reveals activation of lignin degradation machinery and possible evasion of host immune response.

BACKGROUND: The white-rot fungi in the genus Ganoderma interact with both living and dead angiosperm tree hosts. Two Ganoderma species, a North American taxon, G. zonatum and an Asian taxon, G. boninense, have primarily been found associated with live palm hosts. During the host plant colonization process, a massive transcriptional reorganization helps the fungus evade the host immune response and utilize plant cell wall polysaccharides. RESULTS: A publicly available transcriptome of G. boninense - oil palm interaction was surveyed to profile transcripts that were differentially expressed in planta. Ten percent of the G. boninense transcript loci had altered expression as it colonized oil palm plants one-month post inoculation. Carbohydrate active enzymes (CAZymes), particularly those with a role in lignin degradation, and auxiliary enzymes that facilitate lignin modification, like cytochrome P450s and haloacid dehalogenases, were up-regulated in planta. Several lineage specific proteins and secreted proteins that lack known functional domains were also up-regulated in planta, but their role in the interaction could not be established. A slowdown in G. boninense respiration during the interaction can be inferred from the down-regulation of proteins involved in electron transport chain and mitochondrial biogenesis. Additionally, pathogenicity related genes and chitin degradation machinery were down-regulated during the interaction indicating G. boninense may be evading detection by the host immune system. CONCLUSIONS: This analysis offers an overview of the dynamic processes at play in G. boninense - oil palm interaction and provides a framework to investigate biology of Ganoderma fungi across plantations and landscape.

The infection cushion of Botrytis cinerea: a fungal 'weapon' of plant-biomass destruction.

The necrotrophic plant-pathogen fungus Botrytis cinerea produces multicellular appressoria dedicated to plant penetration, named infection cushions (IC). A microarray analysis was performed to identify genes upregulated in mature IC. The expression data were validated by RT-qPCR analysis performed in vitro and in planta, proteomic analysis of the IC secretome and biochemical assays. 1231 upregulated genes and 79 up-accumulated proteins were identified. The data support the secretion of effectors by IC: phytotoxins, ROS, proteases, cutinases, plant cell wall-degrading enzymes and plant cell death-inducing proteins. Parallel upregulation of sugar transport and sugar catabolism-encoding genes would indicate a role of IC in nutrition. The data also reveal a substantial remodelling of the IC cell wall and suggest a role for melanin and chitosan in IC function. Lastly, mutagenesis of two upregulated genes in IC identified secreted fasciclin-like proteins as actors in the pathogenesis of B. cinerea. These results support the role of IC in plant penetration and also introduce other unexpected functions for this fungal organ, in colonization, necrotrophy and nutrition of the pathogen.

Efficient genome editing using endogenous U6 snRNA promoter-driven CRISPR/Cas9 sgRNA in Sclerotinia sclerotiorum.

We previously reported on a CRISPR-Cas9 genome editing system for the necrotrophic fungal plant pathogen Sclerotinia sclerotiorum. This system (the TrpC-sgRNA system), based on an RNA polymerase II (RNA Pol II) promoter (TrpC) to drive sgRNA transcription in vivo, was successful in creating gene insertion mutants. However, relatively low efficiency targeted gene editing hampered the application of this method for functional genomic research in S. sclerotiorum. To further optimize the CRISPR-Cas9 system, a plasmid-free Cas9 protein/sgRNA ribonucleoprotein (RNP)-mediated system (the RNP system) and a plasmid-based RNA polymerase III promoter (U6)-driven sgRNA transcription system (the U6-sgRNA system) were established and evaluated. The previously characterized oxaloacetate acetylhydrolase (Ssoah1) locus and a new locus encoding polyketide synthase12 (Sspks12) were targeted in this study to create loss-of-function mutants. The RNP system, similar to the TrpC-sgRNA system we previously reported, creates mutations at the Ssoah1 gene locus with comparable efficiency. However, neither system successfully generated mutations at the Sspks12 gene locus. The U6-sgRNA system exhibited a significantly higher efficiency of genemutation at both loci. This technology provides a simple and efficient strategy for targeted gene mutation and thereby will accelerating the pace of research of pathogenicity and development in this economically important plant pathogen.

Molecular Dissection of Perithecial Mating Line Development in Colletotrichum fructicola, a Species with a Nontypical Mating System Featuring Plus-to-Minus Switch and Plus-Minus-Mediated Sexual Enhancement.

The genetic regulation of Colletotrichum (Glomerella) sexual reproduction does not strictly adhere to the Ascomycota paradigm and remains poorly understood. Morphologically different but sexually compatible strain types, termed plus and minus, have been recognized, but the biological and molecular distinctions between these strain types remain elusive. In this study, we characterized the sexual behaviors of a pair of plus and minus strains of C. fructicola with the aid of live-cell nucleus-localized fluorescent protein labeling, gene expression, and gene mutation analyses. We confirmed a genetically stable plus-to-minus switching phenomenon and demonstrated the presence of both cross-fertilized and self-fertilized perithecia within the mating line (perithecia cluster at the line of colony contact) between plus and minus strains. We demonstrated that pheromone signaling genes (a-factor-like and α-factor-like pheromones and their corresponding GPCR receptors) were differently expressed between vegetative hyphae of the two strains. Moreover, deletion of pmk1 (a FUS/KSS1 mitogen-activate protein kinase) in the minus strain severely limited mating line formation, whereas deletion of a GPCR (FGSG_05239 homolog) and two histone modification factors (hos2, snt2) in the minus strain did not affect mating line development but altered the ratio between cross-fertilization and self-fertilization within the mating line. We propose a model in which mating line formation in C. fructicola involves enhanced protoperithecium differentiation and enhanced perithecium maturation of the minus strain mediated by both cross-fertilization and diffusive effectors. This study provides insights into mechanisms underlying the mysterious phenomenon of plus-minus-mediated sexual enhancement being unique to Colletotrichum fungi. IMPORTANCE Plus-minus regulation of Colletotrichum sexual differentiation was reported in the early 1900s. Both plus and minus strains produce fertile perithecia in a homothallic but inefficient manner. However, when the two strain types encounter each other, efficient differentiation of fertile perithecia is triggered. The plus strain, by itself, can also generate minus ascospore progeny at high frequency. This nontypical mating system facilitates sexual reproduction and is Colletotrichum specific; the underlying molecular mechanisms, however, remain elusive. The current study revisits this longstanding mystery using C. fructicola as an experimental system. The presence of both cross-fertilized and self-fertilized perithecia within the mating line was directly evidenced by live-cell imaging with fluorescent markers. Based on further gene expression and gene mutation analysis, a model explaining mating line development (plus-minus-mediated sexual enhancement) is proposed. Data reported here have the potential to allow us to better understand Colletotrichum mating and filamentous ascomycete sexual regulation.

Highly Contiguous Genome Resource of Colletotrichum fructicola Generated Using Long-Read Sequencing.

Colletotrichum fructicola is a plant-pathogenic fungus with a broad host range. It causes significant losses to important crops, including apple, pear, strawberry, and other Rosaceae and non-Rosaceae species. To date, two short read-based C. fructicola genomes are publicly available, but both are fragmented. In this study, we re-sequenced the genome of C. fructicola using nanopore long-read technology and refined the assembly with Hi-C map data. The resulting high-quality assembly is an important resource for further comparative and experimental studies with C. fructicola.

Genomic and transcriptomic insights into Raffaelea lauricola pathogenesis.

BACKGROUND: Laurel wilt caused by Raffaelea lauricola is a lethal vascular disease of North American members of the Lauraceae plant family. This fungus and its primary ambrosia beetle vector Xyleborus glabratus originated from Asia; however, there is no report of laurel wilt causing widespread mortality on native Lauraceae trees in Asia. To gain insight into why R. lauricola is a tree-killing plant pathogen in North America, we generated and compared high quality draft genome assemblies of R. lauricola and its closely related non-pathogenic species R. aguacate. RESULTS: Relative to R. aguacate, the R. lauricola genome uniquely encodes several small-secreted proteins that are associated with virulence in other pathogens and is enriched in secondary metabolite biosynthetic clusters, particularly polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS) and PKS-NRPS anchored gene clusters. The two species also exhibit significant differences in secreted proteins including CAZymes that are associated with polysaccharide binding including the chitin binding CBM50 (LysM) domain. Transcriptomic comparisons of inoculated redbay trees and in vitro-grown fungal cultures further revealed a number of secreted protein genes, secondary metabolite clusters and alternative sulfur uptake and assimilation pathways that are coordinately up-regulated during infection. CONCLUSIONS: Through these comparative analyses we have identified potential adaptations of R. lauricola that may enable it to colonize and cause disease on susceptible hosts. How these adaptations have interacted with co-evolved hosts in Asia, where little to no disease occurs, and non-co-evolved hosts in North America, where lethal wilt occurs, requires additional functional analysis of genes and pathways.

Mechanisms of Broad Host Range Necrotrophic Pathogenesis in Sclerotinia sclerotiorum.

Among necrotrophic fungi, Sclerotinia sclerotiorum is remarkable for its extremely broad host range and for its aggressive host tissue colonization. With full genome sequencing, transcriptomic analyses and the increasing pace of functional gene characterization, the factors underlying the basis of this broad host range necrotrophic pathogenesis are now being elucidated at a greater pace. Among these, genes have been characterized that are required for infection via compound appressoria in addition to genes associated with colonization that regulate oxalic acid (OA) production and OA catabolism. Moreover, virulence-related secretory proteins have been identified, among which are candidates for manipulating host activities apoplastically and cytoplasmically. Coupled with these mechanistic studies, cytological observations of the colonization process have blurred the heretofore clear-cut biotroph versus necrotroph boundary. In this review, we reexamine the cytology of S. sclerotiorum infection and put more recent molecular and genomic data into the context of this cytology. We propose a two-phase infection model in which the pathogen first evades, counteracts and subverts host basal defense reactions prior to killing and degrading host cells. Spatially, the pathogen may achieve this via the production of compatibility factors/effectors in compound appressoria, bulbous subcuticular hyphae, and primary invasive hyphae. By examining the nuances of this interaction, we hope to illuminate new classes of factors as targets to improve our understanding of broad host range necrotrophic pathogens and provide the basis for understanding corresponding host resistance.

Sexual Reproduction in the Citrus Black Spot Pathogen, Phyllosticta citricarpa.

Citrus black spot (Phyllosticta citricarpa) causes fruit blemishes and premature fruit drop, resulting in significant economic losses in citrus growing areas with summer rainfall across the globe. The mating type locus of P. citricarpa has recently been characterized, revealing the heterothallic nature of this pathogen. However, insight into the occurrence of mating and the impact of completing the sexual cycle of P. citricarpa was lacking. To investigate the occurrence and impact of sexual reproduction, we developed a method to reliably, and for the first time, produce ascospores of P. citricarpa on culture media. To demonstrate meiosis during the mating process, we identified recombinant genotypes through multilocus genotyping of single ascospores. Because the process of fertilization was not well understood, we experimentally determined that fertilization of P. citricarpa occurs via spermatization. Our results demonstrate that P. citricarpa is heterothallic and requires isolates of different MAT idiomorphs to be in direct physical contact, or for spermatia to fulfill their role as male elements to fertilize the receptive organs, in order to initiate the mating process. The impact of mating on the epidemiology of citrus black spot in the field is discussed.

The Arabidopsis Mediator Complex Subunit16 Is a Key Component of Basal Resistance against the Necrotrophic Fungal Pathogen Sclerotinia sclerotiorum.

Although Sclerotinia sclerotiorum is a devastating necrotrophic fungal plant pathogen in agriculture, the virulence mechanisms utilized by S. sclerotiorum and the host defense mechanisms against this pathogen have not been fully understood. Here, we report that the Arabidopsis (Arabidopsis thaliana) Mediator complex subunit MED16 is a key component of basal resistance against S. sclerotiorum. Mutants of MED16 are markedly more susceptible to S. sclerotiorum than mutants of 13 other Mediator subunits, and med16 has a much stronger effect on S. sclerotiorum-induced transcriptome changes compared with med8, a mutation not altering susceptibility to S. sclerotiorum. Interestingly, med16 is also more susceptible to S. sclerotiorum than coronatine-insensitive1-1 (coi1-1), which is the most susceptible mutant reported so far. Although the jasmonic acid (JA)/ethylene (ET) defense pathway marker gene PLANT DEFENSIN1.2 (PDF1.2) cannot be induced in either med16 or coi1-1, basal transcript levels of PDF1.2 in med16 are significantly lower than in coi1-1. Furthermore, ET-induced suppression of JA-activated wound responses is compromised in med16, suggesting a role for MED16 in JA-ET cross talk. Additionally, MED16 is required for the recruitment of RNA polymerase II to PDF1.2 and OCTADECANOID-RESPONSIVE ARABIDOPSIS ETHYLENE/ETHYLENE-RESPONSIVE FACTOR59 (ORA59), two target genes of both JA/ET-mediated and the transcription factor WRKY33-activated defense pathways. Finally, MED16 is physically associated with WRKY33 in yeast and in planta, and WRKY33-activated transcription of PDF1.2 and ORA59 as well as resistance to S. sclerotiorum depends on MED16. Taken together, these results indicate that MED16 regulates resistance to S. sclerotiorum by governing both JA/ET-mediated and WRKY33-activated defense signaling in Arabidopsis.

Mating Type and Simple Sequence Repeat Markers Indicate a Clonal Population of Phyllosticta citricarpa in Florida.

Phyllosticta citricarpa, the citrus black spot pathogen, was first identified in Florida in March 2010. Subsequently, this pathogen has become established in Florida but can be easily confused with the endemic nonpathogenic citrus endophyte P. capitalensis. In this study, the mating-type (MAT) loci of P. citricarpa and P. capitalensis were identified via draft genome sequencing and were characterized at the structural and sequence levels. P. citricarpa was determined to have an idiomorphic, heterothallic MAT locus structure, whereas P. capitalensis was found to have a single MAT locus consistent with a homothallic mating system. A survey of P. citricarpa isolates from Florida revealed that only the MAT1-2 idiomorph existed in the Floridian population. In contrast, isolates collected from Australia exhibited a 1:1 ratio of MAT1-1 and MAT1-2 isolates. Development and analysis of simple sequence repeat markers revealed a single multilocus genotype (MLG) in the Floridian population (n = 70) and 11 MLG within the Australian population (n = 24). These results indicate that isolates of P. citricarpa from Florida are likely descendent from a single clonal lineage and are reproducing asexually. The disease management focus in Florida will need to be concentrated on the production and dispersal of pycnidiospores.

Characterizing the Palm Pathogenic Thielaviopsis Species from Florida.

Thielaviopsis paradoxa sensu lato is a soilborne fungal pathogen that causes Thielaviopsis trunk rot and heart rot in palms. The loss of structural integrity resulting from trunk rot can cause the palm trunk to collapse suddenly and poses a serious threat to life and property. Even though rudimentary knowledge about the Thielaviopsis infection process in palms is available, nothing is known about the T. paradoxa species complex in the US. The aim of this study was to characterize T. paradoxa s. lat. isolates collected from diseased palms grown in Florida. Multi-locus phylogeny using three genes, ITS, ß-tubulin, and tef1-α, revealed that the isolates separate into two distinct clades with high bootstrap support. The majority of the isolates clustered with the species T. ethacetica, while two isolates formed a separate clade, distinct from T. musarum, and might represent an undescribed Thielaviopsis species. One representative isolate from each clade, when grown on three distinct media and at four different temperatures, showed differences in gross colony morphology, as well as growth rates. The T. ethacetica isolate TP5448 and the Thielaviopsis sp. isolate PLM300 grew better at opposite ends of the temperature spectrum tested in this study, i.e., 35 °C and 10 °C, respectively. In pathogenicity assays on whole plants, the T. ethacetica isolate proved to be more aggressive than Thielaviopsis sp. isolate PLM300, as it produced larger lesions when inoculated on wounded leaflets. An unequal distribution was observed for the mating-type locus of T. ethacetica, as 12 isolates carried the MAT1-1-1 allele, while the status for four isolates remained undefined. Variation in mycelial growth in response to different fungicides was also observed between the two clades. These results demonstrate the existence of two Thielaviopsis clades that can infect palms in Florida and underscore the need for targeted sampling to help uncover the diversity of Thielaviopsis species across palm-growing regions in the US.

Genome comparisons reveal accessory genes crucial for the evolution of apple Glomerella leaf spot pathogenicity in Colletotrichum fungi.

Apple Glomerella leaf spot (GLS) is an emerging fungal disease caused by Colletotrichum fructicola and other Colletotrichum species. These species are polyphyletic and it is currently unknown how these pathogens convergently evolved to infect apple. We generated chromosome-level genome assemblies of a GLS-adapted isolate and a non-adapted isolate in C. fructicola using long-read sequencing. Additionally, we resequenced 17 C. fructicola and C. aenigma isolates varying in GLS pathogenicity using short-read sequencing. Genome comparisons revealed a conserved bipartite genome architecture involving minichromosomes (accessory chromosomes) shared by C. fructicola and other closely related species within the C. gloeosporioides species complex. Moreover, two repeat-rich genomic regions (1.61 Mb in total) were specifically conserved among GLS-pathogenic isolates in C. fructicola and C. aenigma. Single-gene deletion of 10 accessory genes within the GLS-specific regions of C. fructicola identified three that were essential for GLS pathogenicity. These genes encoded a putative non-ribosomal peptide synthetase, a flavin-binding monooxygenase and a small protein with unknown function. These results highlight the crucial role accessory genes play in the evolution of Colletotrichum pathogenicity and imply the significance of an unidentified secondary metabolite in GLS pathogenesis.

Cross-Talk and Multiple Control of Target of Rapamycin (TOR) in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a necrotrophic phytopathogenic fungus that cross-talks with its hosts for control of cell-death pathways for colonization. Target of rapamycin (TOR) is a central regulator that controls cell growth, intracellular metabolism, and stress responses in a variety of eukaryotes, but little is known about TOR signaling in S. sclerotiorum. In this study, we identified a conserved TOR signaling pathway and characterized SsTOR as a critical component of this pathway. Hyphal growth of S. sclerotiorum was retarded by silencing SsTOR, moreover, sclerotia and compound appressoria formation were severely disrupted. Notably, pathogenicity assays of strains shows that the virulence of the SsTOR-silenced strains were dramatically decreased. SsTOR was determined to participate in cell wall integrity (CWI) by regulating the phosphorylation level of SsSmk3, a core MAP kinase in the CWI pathway. Importantly, the inactivation of SsTOR induced autophagy in S. sclerotiorum potentially through SsAtg1 and SsAtg13. Taken together, our results suggest that SsTOR is a global regulator controlling cell growth, stress responses, cell wall integrity, autophagy, and virulence of S. sclerotiorum. IMPORTANCE TOR is a conserved protein kinase that regulates cell growth and metabolism in response to growth factors and nutrient abundance. Here, we used gene silencing to characterize SsTOR, which is a critical component of TOR signaling pathway. SsTOR-silenced strains have limited mycelium growth, and the virulence of the SsTOR-silenced strains was decreased. Phosphorylation analysis indicated that SsTOR influenced CWI by regulating the phosphorylation level of SsSmk3. Autophagy is essential to preserve cellular homeostasis in response to cellular and environmental stresses. Inactivation of SsTOR induced autophagy in S. sclerotiorum potentially through SsAtg1 and SsAtg13. These findings further indicated that SsTOR is a global regulator of the growth, development, and pathogenicity of S. sclerotiorum in multiple ways.

Sclerotinia sclerotiorum γ-glutamyl transpeptidase (Ss-Ggt1) is required for regulating glutathione accumulation and development of sclerotia and compound appressoria.

Transcripts encoding Sclerotinia sclerotiorum γ-glutamyl transpeptidase (Ss-Ggt1) were found to accumulate specifically during sclerotium, apothecium, and compound appressorium development in S. sclerotiorum. To determine the requirement of this protein in these developmental processes, gene deletion mutants of Ss-ggt1 were generated and five independent homokaryotic ΔSs-ggt1 mutants were characterized. All deletion mutants overproduced sclerotial initials that were arrested in further development or eventually produced sclerotia with aberrant rind layers. During incubation for carpogenic germination, these sclerotia decayed and failed to produce apothecia. Total glutathione accumulation was approximately 10-fold higher and H(2)O(2) hyperaccumulated in ΔSs-ggt1 sclerotia compared with the wild type. Production of compound appressoria was also negatively affected. On host plants, these mutants exhibited a defect in infection efficiency and a delay in initial symptom development unless the host tissue was wounded prior to inoculation. These results suggest that Ss-Ggt1 is the primary enzyme involved in glutathione recycling during these key developmental stages of the S. sclerotiorum life cycle but Ss-Ggt1 is not required for host colonization and symptom development. The accumulation of oxidized glutathione is hypothesized to negatively impact these developmental processes by disrupting the dynamic redox environment associated with multicellular development.

Evidence for morphological, vegetative, genetic, and mating-type diversity in Sclerotinia homoeocarpa.

Morphology, vegetative compatibility groups, and molecular characteristics were compared among 47 isolates of the dollar spot pathogen Sclerotinia homoeocarpa. Isolates were collected from cool- and warm-season turfgrasses in Florida and the northern United States. Mycelial pigment accumulation, substratal stromata formation, and symptom development were used to separate the collection into two distinct morphological types: a common-type (C-type) and a Floridian-type (F-type). Phylogenetic relationships estimated from ITS sequences supported the morphological typing. Identification and characterization of the S. homoeocarpa mating-type locus revealed an idiomorphic organization for both C- and F-types with nearly equal frequencies of each mating types present in both groups. These findings suggest heterothallic control of mating and indicate potential for outcrossing in both groups. Dollar spot disease of turfgrass in Florida is caused by two distinct morphological types of S. homoeocarpa which may be cryptic species. These findings could have implications for disease management.

The cAMP-dependent protein kinase A pathway perturbs autophagy and plays important roles in development and virulence of Sclerotinia sclerotiorum.

Previous research has demonstrated that sclerotia production is suppressed by exogenous cyclic AMP (cAMP) in Sclerotinia sclerotiorum and enhanced upon deletion of the adenylate cyclase gene. This study focuses on further functionally characterizing the cAMP-dependent protein kinase A (PKA) signaling pathway in S. sclerotiorum. Here, we demonstrate functions for two components of cAMP signaling: the catalytic, SsPKA, and the regulatory, SsPKAR, subunits of cAMP-dependent PKA. Growth and virulence were greatly reduced by disruption of either Sspka2 or SspkaR in addition to deficiencies in appressorium development. Surprisingly, disruption of both Sspka2 (dSspka2) and SspkaR (dSspkaR) display an up-regulation of autophagy without nutrient starvation suggesting that properly regulated PKA activity is required for control of autophagy. SsPKAR is demonstrated to be required for carbohydrate metabolism and mobilization, which are required for appressorium development and sclerotium initiation. A closer examination of dSspkaR during Nicotiana benthamiana infection revealed that an oxalic acid (OA)-independent necrosis protein(s) or metabolite(s) may be involved in the lesion development in dSspkaR-N. benthamiana interaction. In summary, these data demonstrate that the cAMP-dependent PKA signaling is essential for multiple forms of S. sclerotiorum development as well as virulence which rely on optimal regulation of autophagy.