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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Genome sequence of Monilinia vaccinii-corymbosi sheds light on mummy berry disease infection of blueberry and mating type.

Mummy berry disease, caused by the fungal pathogen Monilinia vaccinii-corymbosi (Mvc), is one of the most economically important diseases of blueberries in North America. Mvc is capable of inducing two separate blighting stages during its life cycle. Infected fruits are rendered mummified and unmarketable. Genomic data for this pathogen is lacking, but could be useful in understanding the reproductive biology of Mvc and the mechanisms it deploys to facilitate host infection. In this study, PacBio sequencing and Hi-C interaction data were utilized to create a chromosome-scale reference genome for Mvc. The genome comprises nine chromosomes with a total length of 30 Mb, an N50 length of 4.06 Mb, and an average 413X sequence coverage. A total of 9399 gene models were predicted and annotated, and BUSCO analysis revealed that 98% of 1,438 searched conserved eukaryotic genes were present in the predicted gene set. Potential effectors were identified, and the mating-type (MAT) locus was characterized. Biotrophic effectors allow the pathogen to avoid recognition by the host plant and evade or mitigate host defense responses during the early stages of fruit infection. Following locule colonization, necrotizing effectors promote the mummification of host tissues. Potential biotrophic effectors utilized by Mvc include chorismate mutase for reducing host salicylate and necrotrophic effectors include necrosis-inducing proteins and hydrolytic enzymes for macerating host tissue. The MAT locus sequences indicate the potential for homothallism in the reference genome, but a deletion allele of the MAT locus, characterized in a second isolate, indicates heterothallism. Further research is needed to verify the roles of individual effectors in virulence and to determine the role of the MAT locus in outcrossing and population genotypic diversity.

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.

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.

The Formaldehyde Dehydrogenase SsFdh1 Is Regulated by and Functionally Cooperates with the GATA Transcription Factor SsNsd1 in Sclerotinia sclerotiorum.

GATA transcription factors (TFs) are common eukaryotic regulators, and glutathione-dependent formaldehyde dehydrogenases (GD-FDH) are ubiquitous enzymes with formaldehyde detoxification activity. In this study, the formaldehyde dehydrogenase Sclerotinia sclerotiorum Fdh1 (SsFdh1) was first characterized as an interacting partner of a GATA TF, SsNsd1, in S. sclerotiorum Genetic analysis reveals that SsFdh1 functions in formaldehyde detoxification, nitrogen metabolism, sclerotium development, and pathogenicity. Both SsNsd1 and SsFdh1 harbor typical zinc finger motifs with conserved cysteine residues. SsNsd1 regulates SsFdh1 in two distinct manners. SsNsd1 directly binds to GATA-box DNA in the promoter region of Ssfdh1; SsNsd1 associates with SsFdh1 through disulfide bonds formed by conserved Cys residues. The SsNsd1-SsFdh1 interaction and nuclear translocation were found to prevent efficient binding of SsNsd1 to GATA-box DNA. Site-directed point mutation of these Cys residues influences the SsNsd1-SsFdh1 interaction and SsNsd1 DNA binding capacity. SsFdh1 is regulated by and functions jointly with the SsNsd1 factor, providing new insights into the complex transcriptional regulatory mechanisms of GATA factors.IMPORTANCE S. sclerotiorum is a pathogenic fungus with sclerotium and infection cushion development, making S. sclerotiorum one of the most challenging agricultural pathogens with no effective control method. We identified important sclerotium and compound appressorium formation determinants, SsNsd1 and SsFdh1, and investigated their regulatory mechanism at the molecular level. SsNsd1 and SsFdh1 are zinc finger motif-containing proteins and associate with each other in the nucleus. On other hand, SsNsd1, as a GATA transcription factor, directly binds to GATA-box DNA in the promoter region of Ssfdh1 The SsNsd1-SsFdh1 interaction and nuclear translocation were found to prevent efficient binding of SsNsd1 to GATA-box DNA. Our results provide insights into the role of the GATA transcription factor and its regulation of formaldehyde dehydrogenase in stress resistance, fungal sclerotium and compound appressorium development, and pathogenicity.

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.

Introduction of Large Sequence Inserts by CRISPR-Cas9 To Create Pathogenicity Mutants in the Multinucleate Filamentous Pathogen Sclerotinia sclerotiorum.

The necrotrophic fungal plant pathogen Sclerotinia sclerotiorum is responsible for substantial global crop losses annually resulting in localized food insecurity and loss of livelihood. Understanding the basis of this broad-host-range and aggressive pathogenicity is hampered by the quantitative nature of both host resistance and pathogen virulence. To improve this understanding, methods for efficient functional gene characterization that build upon the existing complete S. sclerotiorum genome sequence are needed. Here, we report on the development of a clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9)-mediated strategy for creating gene disruption mutants and the application of this technique for exploring roles of known and hypothesized virulence factors. A key finding of this research is that transformation with a circular plasmid encoding Cas9, target single guide RNA (sgRNA), and a selectable marker resulted in a high frequency of targeted, insertional gene mutation. We observed that 100% of the mutants integrated large rearranged segments of the transforming plasmid at the target site facilitated by the nonhomologous end joining (NHEJ) repair pathway. This result was confirmed in multiple target sites within the same gene in three independent wild-type isolates of S. sclerotiorum and in a second independent gene. Targeting the previously characterized Ssoah1 gene allowed us to confirm the loss-of-function nature of the CRISPR-Cas9-mediated mutants and explore new aspects of the mutant phenotype. Applying this technology to create mutations in a second previously uncharacterized gene allowed us to determine the requirement for melanin accumulation in infection structure development and function.IMPORTANCE Fungi that cause plant diseases by rotting or blighting host tissue with limited specificity remain among the most difficult to control. This is largely due to the quantitative nature of host resistance and a limited understanding of fungal pathogenicity. A mechanistic understanding of pathogenicity requires the ability to manipulate candidate virulence genes to test hypotheses regarding their roles in disease development. Sclerotinia sclerotiorum is among the most notorious of these so-called broad-host-range necrotrophic plant pathogens. The work described here provides a new method for rapidly constructing gene disruption vectors to create gene mutations with high efficiency compared with existing methods. Applying this method to characterize gene functions in S. sclerotiorum, we confirm the requirement for oxalic acid production as a virulence factor in multiple isolates of the fungus and demonstrate that melanin accumulation is not required for infection. Using this approach, the pace of functional gene characterization and the understanding of pathogenicity and related disease resistance will increase.

Pathogenic adaptations of Colletotrichum fungi revealed by genome wide gene family evolutionary analyses.

The fungal genus Colletotrichum contains hemibiotrophic phytopathogens being highly variable in host and tissue specificities. We sequenced a C. fructicola genome (1104-7) derived from an isolate of apple in China and compared it with the reference genome (Nara_gc5) derived from an isolate of strawberry in Japan. Mauve alignment and BlastN search identified 0.62 Mb lineage-specific (LS) genomic regions in 1104-7 with a length criterion of 10 kb. Genes located within LS regions evolved more dynamically, and a strongly elevated proportion of genes were closely related to non-Colletotrichum sequences. Two LS regions, containing nine genes in total, showed features of fungus-to-fungus horizontal transfer supported by both gene order collinearity and gene phylogeny patterns. We further compared the gene content variations among 13 Colletotrichum and 11 non-Colletotrichum genomes by gene function annotation, OrthoMCL grouping and CAFE analysis. The results provided a global evolutionary picture of Colletotrichum gene families, and identified a number of strong duplication/loss events at key phylogenetic nodes, such as the contraction of the detoxification-related RTA1 family in the monocot-specializing graminicola complex and the expansions of several ammonia production-related families in the fruit-infecting gloeosporioides complex. We have also identified the acquirement of a RbsD/FucU fucose transporter from bacterium by the Colletotrichum ancestor. In sum, this study summarized the pathogenic evolutionary features of Colletotrichum fungi at multiple taxonomic levels and highlights the concept that the pathogenic successes of Colletotrichum fungi require shared as well as lineage-specific virulence factors.

The GATA-type IVb zinc-finger transcription factor SsNsd1 regulates asexual-sexual development and appressoria formation in Sclerotinia sclerotiorum.

The sclerotium, a multicellular structure composed of the compact aggregation of vegetative hyphae, is critical for the long-term survival and sexual reproduction of the plant-pathogenic fungus Sclerotinia sclerotiorum. The development and carpogenic germination of sclerotia are regulated by integrating signals from both environmental and endogenous processes. Here, we report the regulatory functions of the S. sclerotiorum GATA-type IVb zinc-finger transcription factor SsNsd1 in these processes. SsNsd1 is orthologous to the Aspergillus nidulans NsdD (never in sexual development) and the Neurospora crassa SUB-1 (submerged protoperithecia-1) proteins. Ssnsd1 gene transcript accumulation remains relatively low, but variable, during vegetative mycelial growth and multicellular development. Ssnsd1 deletion mutants (Δnsd1-KOs) produce phialides and phialospores (spermatia) excessively in vegetative hyphae and promiscuously within the interior medulla of sclerotia. In contrast, phialospore development occurs only on the sclerotium surface in the wild-type. Loss of SsNsd1 function affects sclerotium structural integrity and disrupts ascogonia formation during conditioning for carpogenic germination. As a consequence, apothecium development is abolished. The Ssnsd1 deletion mutants are also defective in the transition from hyphae to compound appressorium formation, resulting in a loss of pathogenicity on unwounded hosts. In sum, our results demonstrate that SsNsd1 functions in a regulatory role similar to its ascomycete orthologues in regulating sexual and asexual development. Further, SsNsd1 appears to have evolved as a regulator of pre-penetration infectious development required for the successful infection of its many hosts.