The Mechanism Of SsSnf5-SsHsf1 Transcription Module Regulates Stress Responses And Pathogenicity In Sclerotinia Sclerotiorum

The chromatin remodeling complex SWI/SNF regulates eukaryotic development and various stress responses by changing the structure of nucleosomes and acting as a bridge between intracellular and extracellular signals and transcriptional reprogramming.Since the SWI/SNF complex does not have sequence specificity in binding to DNA,it is usually recruited by transcription factors to specific DNA regions to activate transcription of target genes,precisely regulating various life activities in space and time.However,in phytopathogenic fungi,the function of the SWI/SNF complex is limited,and it is still unclear which transcription factors the complex interacts with and how it regulates responses to environmental and host signals.Sclerotinia sclerotiorum(Lib.)de Bary is a worldwide and necrotrophic phytopathogenic fungi with a wide host-range.Sclerotinia stem rot(SSR)caused in soybean and rapeseed by S.sclerotiorum has caused huge economic losses to agricultural production.The life cycle includes the sclerotia of dormant that resist stress and overwinter,and the infection cushion,which penetrate the epidermis of the host.The formation of these morphological structures and the detoxification and antioxidant system ensure the strong stress tolerance and pathogenicity of S.sclerotiorum,and also bring great challenges to the prevention and control of SSR.At present,little is known about the morphogenesis of S.sclerotiorum and the transcriptional regulatory mechanisms in response to environmental and host signals.Therefore,in this study,used genetic,biochemical,molecular biology and pharmacological methods,we elucidated the molecular mechanisms of maintained reactive oxygen species(ROS)homeostasis to regulated morphogenesis,stress response,and pathogenesis in S.sclerotiorum.This is accomplished through the transcription module composed of SWI/SNF complex subunit SsSnf5 and heat shock transcription factor SsHsf1,which mediated the transcription of genes encoding heat shock protein(hsp)and antioxidant enzymes.The main results of the research are as follows:1.SsSnf5 regulates sclerotia development and maintains ROS homeostasis under stress.The SWI/SNF complex promotes chromatin accessibility and manipulates transcriptional regulation,with Snf5 as its core subunit.Bioinformatics and protein-protein interaction analysis confirmed the existence of SsSnf5 as a subunit of the SWI/SNF complex in S.sclerotiorum.To investigate the function of SsSnf5,gene deletion mutants and genetic complementation strains were obtained.Compared with the wild type(WT),ΔSssnf5 mutants showed delayed colony expansion,and the clustered hyphae were mostly white instead of forming black sclerotia.Transcription level analysis of genes encoding seven melanin synthesis-related enzymes and master transcription factors regulating melanin synthesis showed that SsSnf5 positively regulated the expression of melanin synthesis-related genes during sclerotinia stage.High temperature stress experiments showed that ΔSssnf5 was extremely sensitive to high temperature,and high temperature caused a large number of cells of ΔSssnf5 to die and accumulate ROS excessively.Oxidative stress also caused ΔSssnf5 to grow more slowly and accumulate excessive ROS.The expression levels of hsp and antioxidant enzyme genes of WT and ΔSssnf5 under high temperature or oxidative stress were evaluated,and it was found that knockout of SsSnf5 significantly weakened the induced expression of these genes under stress.In conclusion,SsSnf5,as a subunit of the SWI/SNF complex of S.sclerotiorum,positively regulates the transcription of melanin synthesis-related genes during sclerotia formation,and positively regulates the transcription of hsp and antioxidant enzyme genes under stress,thereby regulating sclerotia development and maintaining ROS homeostasis under high temperature and oxidative stress.2.SsSnf5 and SsHsf1 form a module to mediate ROS detoxification under stress.The SWI/SNF complex needs to be guided by transcription factors to the promoter of the target genes,suggesting that there are transcription factors that regulate stress response and can interact with SsSnf5 to regulate stress response.Yeast twohybrid identification of SsSnf5 interaction with two heat-shock transcription factors SsHsf1/2,in vivo co-immunoprecipitation(Co-IP)also confirmed the interaction between SsSnf5 and SsHsf1 in a stress-dependent manner.Hsf is widely involved in stress response,especially by activating hsp gene transcription in response to hightemperature stress,but the function in plant pathogenic fungi is not detailed.Genetic analysis showed that SsHsf1 had a different mechanism from SsSnf5 to regulating sclerotia development.In contrast,similar to ΔSssnf5,ΔSshsf1 was also sensitive to high temperature and oxidative stress,and excessive ROS was accumulated under stress.And knockout of SsHsf1 significantly weakened the expression of stress-induced hsp and antioxidant enzyme genes.In addition,the knockout of SsSnf5 and SsHsf1 also significantly enhanced the sensitivity of S.sclerotiorum to cell wall stress,osmotic stress,fungicide stress and nutrient stress,resulting in excessive accumulation of ROS under fungicide stress.Overall,SsSnf5 and SsHsf1 interact in a stress-dependent manner to form a module that mediates the detoxification of ROS under multiple stresses and regulates stress tolerance.3.The SsSnf5-SsHsf1 module activates hsp and antioxidant enzyme gene transcription.Hsf binds to activate transcription at the heat shock element(HSEs)of the hsp genes promoter.HSEs were found in the promoters of hsp and antioxidant enzyme genes in the genome of S.sclerotiorum.Chromatin immunoprecipitationquantitative PCR(Ch IP-q PCR)showed that SsSnf5 could be specifically enriched in HSEs of some hsp and antioxidant enzyme genes,and the enrichment extent was increased under stress.Similarly,Ch IP-q PCR confirmed that SsHsf1 was also enriched in HSEs of these genes and enhanced by stress,and yeast one-hybrid confirmed that SsHsf1 directly binds to HSEs of these genes.In addition,the established a transcribed yeast three-hybrid system found that the co-expression of SsSnf5 and SsHsf1 had stronger binding ability with the HSEs of Sshsp70 or Sscat1 than alone expressing SsHsf1,and heat treatment had a superimposed effect with SsSnf5.The expression of SsSnf5-GFP in ΔSshsf1 confirmed that SsHsf1 was genetically responsible for guiding SsSnf5 to the promoter of hsp and antioxidant enzyme genes to mediating stress response.The SWI/SNF complex promotes transcription by mediating histone displacement.Ch IP-q PCR confirmed that the SsSnf5-SsHsf1 module is required for stress-induced displacement of histones on the promoters of Sshsp70 and Sscat1.Taken together,SsHsf1 guiding SsSnf5 on HSEs of hsp and antioxidant enzyme genes to form a transcription module under stress and to mediate histone displacement and transcriptional activation.4.The SsSnf5-SsHsf1 module regulates pathogenicity.Both the SWI/SNF complex and Hsf have been reported to be involved in pathogenesis.Compared with WT,the infection cushions formed by ΔSssnf5 and ΔSshsf1 were smaller and less aggregated,and the expression of the key enzyme SsPKS13 was also lower in the infection cushions.Inoculation experiments showed that the pathogenicity was significantly reduced during knocking out SsSnf5 or SsHsf1.Early plant immunity relies on ROS production by RBOHD.Comparing the transcription levels of several hsp and antioxidant enzyme genes inoculated on Col-0 and rbohd mutants of Arabidopsis,S.sclerotiorum could induce the expression of hsp and antioxidant enzyme genes in response to ROS produced by plant RBOHD in a SsSnf5-SsHsf1 module-dependent manner.Ch IP-q PCR also confirmed that ROS production of plant RBOHD induced SsSnf5 enrichment in HSEs of these genes.Inoculation experiments showed that ROS produced by plant RBOHD inhibited S.sclerotiorum infection,while SsSnf5-SsHsf1 module weakened the inhibition.These suggest that the SsSnf5-SsHsf1 module contributes the pathogenicity by activating transcription of hsp and antioxidant enzyme genes to oppose host-derived ROS.In addition,host-induced gene silencing(HIGS)confirmed that SsHsp70 also contributed to the pathogenicity of S.sclerotiorum,and the SsSnf5-SsHsf1-SsHsp70 module was used as the HIGS target to effectively prevent S.sclerotiorum infection.5.The SsSnf5-SsHsf1 module is activated by stress-induced phosphorylation.Yeast Hsf1 is hyperphosphorylated upon heat shock to activate downstream transcription.The gel retardation assays of phosphorylation showed that high temperature,menadione,fungicides,and plant-derived stresses all induced hyperphosphorylation of SsHsf1 and phosphorylation of SsSnf5,while the MAPKKsspecific inhibitor U0126 completely inhibited menadione-induced phosphorylation of Erk1/2 and SsSnf5.Co-IP showed that U0126 inhibited menadione-induced SsSnf5 and SsHsf1 interactions,and Ch IP-q PCR showed that U0126 also prevented menadioneinduced SsSnf5 and SsHsf1 enrichment in the Sshsp70 and Sscat1 promoters.In conclusion,stress activates the formation of the SsSnf5-SsHsf1 module by inducing MAPKKs-dependent phosphorylation of SsSnf5.Overall,in this study,it was found that when sensing environmental or hostderived ROS,S.sclerotiorum induces the phosphorylation of the SWI/SNF complex subunit SsSnf5,which then interacts with the heat-shock transcription factor SsHsf1 and forms a transcription module,and was recruited into the promoter of hsp and antioxidant enzyme genes to initiate transcription to maintain ROS homeostasis in response to stress.This study reveals for the first time that the SWI/SNF complex and its cooperative transcription factors synergistically regulate the transcription of hsp and antioxidant enzyme genes in response to host and environmental stresses,providing new insights for understanding the operation of plant pathogenic fungi sensory signals and transcriptional regulatory networks.The molecular mechanism of SsSnf5-Sss Hsf1 module regulating pathogenicity was analyzed,which deepened the understanding of the pathogenic mechanism of S.sclerotiorum and provided new targets for the prevention and control of SSR.