Regulatory Mechanism Of Autophagy-related Protein Kinase SsAtg1 In Sclerotia Development And Pathogenicity In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum(Lib.)de Bary,a necrotrophic type phytopathogenic fungus,has an extensive host rang.S.sclerotiorum caused Sclerotinia stem rot which can infect more than 600 species of plants including soybean,peanut,oilseed rape as well as medicinal plants,vegetables and flowers.Sclerotinia stem rot is widely distributed,seriously harmful and caused great economic losses.Polymorphism is a significant characteristic of S.sclerotiorum,which contain sclerotia,compound appressoria and apothecia.Sclerotia is not only an indispensable part of asexual and sexual reproduction,but also a central stage of the life and infection cycle of S.sclerotiorum.However,the formation of sclerotia is subject to complex and exquisite regulation,exploring its developmental mechanism is conducive to a complete grasp of the developmental biological information of S.sclerotiorum,so as to effectively prevent and control Sclerotinia stem rot.Autophagy is an evolutionarily conserved cellular reaction mechanism for degradation and turnover of intracellular materials in eukaryotes.Recently researches demonstrated that autophagy is correlated with the growth and development,morphogenesis and cell metabolism of phytopathogen fungus,however,its biological function has not been elucidated in S.sclerotiorum.Therefore,this study used genetics,molecular biology,biochemistry and other means to explore the mechanism of autophagy pathway related genes on mycelium growth,sclerotia development,compound appressorium formation and pathogenicity of S.sclerotiorum,clarified the correlation between TOR(Target of rapamycin)signaling pathway and PP2 C protein phosphatases(Type 2C protein phosphatases)SsPtc3 and autophagy,the results of the research are as following:1.Autophagy related gene SsATG1 induced autophagy and affected the sclerotia formation and pathogenicity of S.sclerotiorum.Autophagy is regulated by hierarchical Au Topha Gy-related genes(Atgs),and Atg1 complex is the most upstream factor involved in the initiation of autophagy.Through transcriptome analysis of mycelium and sclerotia,it was found that the expression level of SsATG1 was increased significantly in sclerotia stage.After SsATG1 was targeted knockout by Split-marker method,MDC staining and Western blot showed that autophagy pathway of S.sclerotiorum was blocked.Biological function analysis showed that SsATG1 is involved in the stress response of S.sclerotiorum to affected mycelium growth.Moreover,ΔSsatg1 could not form sclerotia and the expression levels of sclerotia development related genes SsPAC1,SsSCD1 and SsTHR1 were significantly lower than those of the wild type.Pathogenicity analysis showed that the deficiency of development and penetration of ΔSsatg1 compound appressorium resulted in decreased pathogenicity on different hosts.In conclusion,SsATG1 is the key gene that regulated autophagy in S.sclerotiorum,which played an important role in stress response,sclerotia formation,compound appressorium development and pathogenicity.These results provided a theoretical basis for further analysis of the biological functions and regulatory mechanisms of autophagy in S.sclerotiorum.2.SsPtc3 interacted with SsAtg1,mediating stress response and pathogenicity of S.sclerotiorum.SsAtg1 is the only protein kinase in Atg proteins and has critical functions in stress response.In order to reveal the functions of SsAtg1 in regulating sclerotia formation and pathogenicity,then the candidate interactors were obtained by screening the c DNA library of the S.sclerotiorum,which including SsPtc3,SsAtg13 and SsPho85.SsPtc3 belongs to PP2 C phosphatase,which is an important protein regulating cell function in response to external stress.The interaction between SsAtg1 and SsPtc3 was verified by yeast two-hybrid and pull down.After SsPTC3 gene deletion,autophagy level was decreased.The mycelium growth of ΔSsptc3 was slowed,the stress response and sclerotia development were abnormal,moreover,the pathogenicity of ΔSsptc3 was significantly reduced.Further analysis showed that the compound appressorium of ΔSsptc3 developed normally but the invasive mycelia spreaded slowly in the onion epidermis.In conclusion,PP2 C phosphatase SsPtc3 and SsAtg1 were involved in response to stress signals and affected sclerotia formation and pathogenicity of S.sclerotiorum.3.The SsAtg1 interacting protein SsAtg13 did not affected the pathogenicity of S.sclerotiorum.Atg13 is an important component of the Atg1 complex,regulating the activity of Atg1 kinase and plays an important role in maintaining cell activity in yeast under starvation conditions.The interaction between SsAtg1 and SsAtg13 was verified by yeast two-hybrid,in order to elucidate the role of Atg1 complex in regulating the growth,development and pathogenicity of S.sclerotiorum,SsATG13 gene knockout was performed.The autophagy level of ΔSsatg13 was slightly lower than that of the wild type and the growth rate of mycelia in nutrient deficiency medium was lower.However,unlike ΔSsatg1,ΔSsatg13 can form sclerotia.In addition,there was no significant difference between the compound appressorium formation and the virulence of ΔSsatg13 and the wild type.Studies have shown that SsAtg13 interacted with SsAtg1,but did not play a major role in sclerotia development and pathogenicity,further demonstrated that the functional diversity of Atg13 and that SsAtg1 is an important protein mediated sclerotia formation and pathogenicity of S.sclerotiorum.4.SsTOR regulated autophagy and cell wall integrity of S.sclerotiorum.TOR is a highly conserved Ser/Thr protein kinase,which is an important regulatory factor for autophagy.There was only one homologous protein(Sscle＿02g011660)similar to Saccharomyces cerevisiae TOR kinases Tor1(47.25%)and Tor2(48.7%)in the S.sclerotiorum genome,therefore,named Sscle＿02g011660 as SsTOR.In order to explore the relationship between TOR signaling pathway and the mechanism of SsAtg1 response to nutrient stress,RNAi was performed to silenced SsTOR.Hyphal growth of S.sclerotiorum was retarded by silencing SsTOR,moreover,sclerotia and compound appressoria formation were severely disrupted.Notably,pathogenicity assays of strains showed that the virulence of the SsTOR-silenced strains were dramatically decreased.Importantly,the inactivation of SsTOR induced autophagy in S.sclerotiorum.Phosphorylation analysis showed that SsTOR involved in the cell wall integrity pathway(CWI)by regulating the phosphorylation level of SsSmk3.In summary,results suggested that SsTOR was a global regulator controlling cell growth,stress responses,cell wall integrity,autophagy and virulence of S.sclerotiorum.Overall,in this study,it was confirmed that autophagy related genes SsATG1 and SsATG13 affected autophagy of S.sclerotiorum,and play an important role in regulating stress response,mycelium growth,sclerotia formation and pathogenicity of S.sclerotiorum.The mechanism of interaction between SsAtg1 and SsPtc3 involved in stress response and pathogenicity was analyzed.Also clarified that SsTOR is a global regulator of the growth and development of S.sclerotiorum,and the molecular mechanism of SsTOR on autophagy was explored.These results provide new insights into the cross talk of autophagy protein kinase SsAtg1 with other metabolic pathways,and deepened the understanding of autophagy on mycelium growth,sclerotia development and pathogenic mechanism of S.sclerotiorum,and provided an important molecular basis for the effective prevention and control of Sclerotinia stem rot.