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The compound appressoria of Sclerotinia sclerotiorum can produce cell wall‐degrading enzymes, effectors and toxins, which promote penetration and the death of host cells. Subsequently, invasive hyphae (IH) branch rapidly as necrotrophic growth and disease symptoms are observed. S. sclerotiorum can respond to complex stresses and regulate its metabolism to adapt to the external environment. Here we demonstrated that type 2C Ser/Thr phosphatase (PP2C) SsPtc3 responds to nutritional, osmotic, cell wall and oxidative stresses. Loss of function ΔSsptc3 mutants displayed defects in mycelial growth, sclerotia formation and reduced virulence. Phosphoproteomic analyses revealed that SsPtc3 is involved in autophagy and MAPK signalling pathways. We obtained evidence that SsPtc3 negatively modulates the phosphorylation of SsSmk1. SsSmk1 is essential for mycelial growth, compound appressorium formation and pathogenicity, SsPtc3 modulated phosphorylation homeostasis of SsSmk1 to maintain hyphal growth. SsPtc3 interacted with SsAtg1 to influence autophagic flux under starvation. Taken together, these results reveal that SsPtc3 responds to various stresses that modulate autophagy and phosphorylation of SsSmk1‐MAPK, which facilitates the growth and virulence of S. sclerotiorum. [ABSTRACT FROM AUTHOR]

APSES (Asm1p, Phd1p, Sok2p, Efg1p, and StuAp) family transcription factors play crucial roles in various biological processes of fungi, however, their functional characterization in phytopathogenic fungi is limited. In this study, we explored the role of SsStuA, a typical APSES transcription factor, in the regulation of cell wall integrity (CWI), sclerotia formation and pathogenicity of Sclerotinia sclerotiorum, which is a globally important plant pathogenic fungus. A deficiency of SsStuA led to abnormal phosphorylation level of SsSmk3, the key gene SsAGM1 for UDP-GlcNAc synthesis was unable to respond to cell wall stress, and decreased tolerance to tebuconazole. In addition, ΔSsStuA was unable to form sclerotia but produced more compound appressoria. Nevertheless, the virulence of ΔSsStuA was significantly reduced due to the deficiency of the invasive hyphal growth and increased susceptibility to hydrogen peroxide. We also revealed that SsStuA could bind to the promoter of catalase family genes which regulate the expression of catalase genes. Furthermore, the level of reactive oxygen species (ROS) accumulation was found to be increased in ΔSsStuA. In summary, SsStuA, as a core transcription factor involved in the CWI pathway and ROS response, is required for vegetative growth, sclerotia formation, fungicide tolerance and the full virulence of S. sclerotiorum. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

The necrotrophic fungus Sclerotinia sclerotiorum is a devastating pathogen. S. sclerotiorum can cause Sclerotinia stem rot in more than 600 species of plants, which results in serious economic losses every year. Chitin is one of the most important polysaccharides in fungal cell walls. Chitin and β-Glucan form a scaffold that wraps around the cell and determines the vegetative growth and pathogenicity of pathogens. UDP-GlcNAc is a direct precursor of chitin synthesis. During the synthesis of UDP-GlcNAc, the conversion of GlcNAc-6P to GlcNAc-1P that is catalyzed by AGM1 (N -acetylglucosamine-phosphate mutase) is a key step. However, the significance and role of AGM1 in phytopathogenic fungus are unclear. We identified a cytoplasm-localized SsAGM1 in S. sclerotiorum , which is homologous to AGM1 of Saccharomyces cerevisiae. We utilized RNA interference (RNAi) and overexpression to characterize the function of SsAGM1 in S. sclerotiorum. After reducing the expression of SsAGM1 , the contents of chitin and UDP-GlcNAc decreased significantly. Concomitantly, the gene-silenced transformants of SsAGM1 slowed vegetative growth and, importantly, lost the ability to produce sclerotia and infection cushion; it also lost virulence, even on wounded leaves. In addition, SsAGM1 was also involved in the response to osmotic stress and inhibitors of cell wall synthesis. Our results revealed the function of SsAGM1 in the growth, development, stress response, and pathogenicity in S. sclerotiorum. [ABSTRACT FROM AUTHOR]

The plant pathogenic fungus Sclerotinia sclerotiorum can survive on a wide range of hosts and cause significant losses on crop yields. FKH, a forkhead box (FOX)-containing protein, functions to regulate transcription and signal transduction. As a transcription factor (TF) with multiple biological functions in eukaryotic organisms, little research has been done on the role of FKH protein in pathogenic fungi. Ss Fkh1 encodes a protein which has been predicted to contain FOX domain in S. sclerotiorum. In this study, the deletion mutant of SsFkh1 resulted in severe defects in hyphal development, virulence, and sclerotia formation. Moreover, knockout of SsFkh1 lead to gene functional enrichment in mitogen-activated protein kinase (MAPK) signaling pathway in transcriptome analysis and SsFkh1 was found to be involved in the maintenance of the cell wall integrity (CWI) and the MAPK signaling pathway. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that SsFkh1 interacts with SsMkk1. In addition, we explored the conserved MAPK signaling pathway components, including Bck1, Mkk1, Pkc1, and Smk3 in S. sclerotiorum. Δ Ssmkk1 , Δ Sspkc1 , Δ Ssbck1 , and Δ Sssmk3 knockout mutant strains together with Δ Ssmkk1 com, Δ Sspkc1 com, Δ Ssbck1 com, and Δ Sssmk3 com complementation mutant strains were obtained. The results indicated that Δ Ssmkk1 , Δ Sspkc1 , Δ Ssbck1 , and Δ Sssmk3 displayed similar phenotypes to Δ Ssfkh1 in sclerotia formation, compound appressorium development, and pathogenicity. Taken together, SsFkh1 may be the downstream substrate of SsMkk1 and involved in sclerotia formation, compound appressorium development, and pathogenicity in S. sclerotiorum. [ABSTRACT FROM AUTHOR]

Sclerotinia sclerotiorum, the notorious necrotrophic phytopathogenic fungus with wide distribution, is responsible for sclerotium disease in more than 600 plant species, including many economic crops such as soybean, oilseed rape, and sunflower. The compound appressorium is a crucial multicellular infection structure that is a prerequisite for infecting healthy tissues. Previously, the Forkhead‐box family transcription factors (FOX TFs) SsFoxE2 and SsFKH1 were shown to play a key regulatory role in the hyphae growth, sexual reproduction, and pathogenicity of S. sclerotiorum. However, little is known about the roles of SsFoxE3 regulating growth and development and pathogenicity. Here, we report SsFoxE3 contributes to sclerotium formation and deletion of SsFoxE3 leads to reduced formation of compound appressoria and developmental delays. Transcripts of SsFoxE3 were greatly increased during the initial stage of infection and SsFoxE3 deficiency reduced virulence on the host, while stabbing inoculation could partially restore pathogenicity. The SsFoxE3 mutant showed sensitivity to H2O2, and the expression of reactive oxygen species detoxification and autophagy‐related genes were reduced. Moreover, expression of SsAtg8 was also decreased during the infection process of the SsFoxE3 mutant. Yeast 1‐hybrid tests suggested that SsFoxE3 interacted with the promoter of SsAtg8. Disruption of SsAtg8 resulted in a phenotype similar to that of the SsFoxE3 mutant. Comparative analysis of the level of autophagy in the wild type and SsFoxE3 mutant showed that N starvation‐induced autophagy was reduced in the SsFoxE3 mutant. Taken together, our findings indicate that SsFoxE3 plays an important role in compound appressorium formation and is involved in transcriptional activation of SsAtg8 during infection by S. sclerotiorum. [ABSTRACT FROM AUTHOR]

Sclerotinia sclerotiorum is a challenging agricultural pathogen for management, causing large global economic losses annually. The sclerotia and infection cushions are critical for its long-term survival and successful penetration on a wide spectrum of hosts. The mitogen-activated protein kinase (MAPK) cascades serve as central signaling complexes that are involved in various aspects of sclerotia development and infection. In this study, the putative downstream transcription factor of MAPK pathway, SsSte12, was analyzed in S. sclerotiorum. Silencing SsSte12 in S. sclerotiorum resulted in phenotypes of delayed vegetative growth, reduced size of sclerotia, and fewer appressoria formation. Consequently, the SsSte12 RNAi mutants showed attenuated pathogenicity on the host plants due to the defect compound appressorium. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation assays demonstrated that the SsSte12 interacts with SsMcm1. However, the SsMcm1 expression is independent of the regulation of SsSte12 as revealed by qRT-PCR analysis in SsSte12 RNAi mutants. Together with high accumulation of SsSte12 transcripts in the early development of S. sclerotiorum , our results demonstrated that SsSte12 function was essential in the vegetative mycelial growth, sclerotia development, appressoria formation and penetration-dependent pathogenicity. Moreover, the SsSte12-SsMcm1 interaction might play a critical role in the regulation of the genes encoding these traits in S. sclerotiorum. [ABSTRACT FROM AUTHOR]

SFH1 (for S n f 5 h omolog) protein, comprised in the RSC (R emodels S tructure of C hromatin) chromatin remodeling complex, functions as a transcription factor (TF) to specifically regulate gene transcription and chromatin remodeling. As one of the well-conserved TFs in eukaryotic organisms, little is known about the roles of SFH1 protein in the filamentous fungi. In Sclerotinia sclerotiorum , one of the notorious plant fungal pathogens, there are nine proteins predicted to contain GATA-box domain according to GATA family TF classification, among which Sssfh1 (SS1G_01151) encodes a protein including a GATA-box domain and a SNF5 domain. Here, we characterized the roles of Sssfh1 in the developmental process and fungal pathogenicity by using RNA interference (RNAi)-based gene silencing in S. sclerotiorum. RNA-silenced strains with significantly reduced Sssfh1 RNA levels exhibited slower hyphal growth and decreased reactive oxygen species (ROS) accumulation in hyphae compared to the wild-type (WT) strain. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays demonstrated that SsSFH1 interacts with SsMSG5, a MAPK phosphatase in S. sclerotiorum. Furthermore, Sssfh1 -silenced strains exhibited enhanced tolerance to NaCl and H2O2. Results of infection assays on soybean and common bean (Phaseolus vulgaris) leaves indicated that Sssfh1 is required for full virulence of S. sclerotiorum during infection in the susceptible host plants. Collectively, our results suggest that the TF SsSFH1 is involved in growth, ROS accumulation and virulence in S. sclerotiorum. [ABSTRACT FROM AUTHOR]

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic plant pathogen with a worldwide distribution. The sclerotia of S. sclerotiorum are pigmented multicellular structures formed from the aggregation of vegetative hyphae. These survival structures play a central role in the life and infection cycles of this pathogen. Here, we characterized an atypical forkhead (FKH)-box-containing protein, SsFKH1, involved in sclerotial development and virulence. To investigate the role of SsFkh1 in S. sclerotiorum, the partial sequence of SsFkh1 was cloned and RNA interference (RNAi)-based gene silencing was employed to alter the expression of SsFkh1. RNA-silenced mutants with significantly reduced SsFkh1 RNA levels exhibited slow hyphal growth and sclerotial developmental defects. In addition, the expression levels of a set of putative melanin biosynthesis-related laccase genes and a polyketide synthase-encoding gene were significantly down-regulated in silenced strains. Disease assays demonstrated that pathogenicity in RNAi-silenced strains was significantly compromised with the development of a smaller infection lesion on tomato leaves. Collectively, the results suggest that SsFkh1 is involved in hyphal growth, virulence and sclerotial formation in S. sclerotiorum. [ABSTRACT FROM AUTHOR]