Funtional Analysis Of Survival Factor 1 Gene SsSvf1 In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum?Lib.?de Bary is an important necrotrophic plants pathogenic fungus with a world-wide distribution.The fungus can infect more than 400 plants and cause enormous losses to the quality and yield of agricultural products.Pathogens need to resist or tolerate oxidative stress due to oxidative burst of reactive oxygen species?ROS?during infection.However,the molecular mechanism involved in ROS modulation in S.sclerotiorum is still in its infancy.In previous study,the expression level of a S.sclerotiorum gene named SsSvf1?SS1G₀1919?was significantly increased during the initial stage of infection.The gene encodes a protein showed high similarity to Saccharomyes cerevisiae survival factor 1?Svf1?which can help to cope with oxidative stress and maintain cell viability.In this research,the function of SsSvf1 gene was deeply analyzed to reveal the mechanism involved in coping with oxidative stress and pathogenicity of S.sclerotiorumAnalysis of SsSvf1 gene using bioinformatics technology,the coding region of SsSvf1 gene is 1146 bp in length and encodes 381 amino acids.Conserved domain analysis indicated that the C-terminal contains the survival factor 1 domain.By constructing phylogenetic tree,SsSvf1 and XP₀01548941 of Botrytis cinerea,ESZ99098 of Sclerotinia borealis is clustered into a small branch,indicating that the relationship between the three proteins is closer.The expression pattern of S.sclerotiorum SsSvf1 gene was analyzed by real-time RT-PCR.The results showed that the expression of this gene was significantly up-regulated under oxidative stress and during early stage of infection,indicating that the expression of this gene was induced by oxidative stress and the gene may play an important role during the pathogenic process.In this study,a gene-silencing vector of SsSvf1 gene was constructed and the SsSvf1gene-silenced transformants were obtained by PEG-mediated protoplast transformation.The hyphae and colony morphology of the SsSvf1 gene-silenced transformants were observed,and the number and dry weight of sclerotia were analyzed.The results showed that the morphology of hyphae and colony,sclerotia formation were no significantly differences from those of the wild-type strain.The hyphal growth of SsSvf1gene-silenced transformants were significantly inhibited in PDA medium supplemented with sucrose,sorbitol and sodium chloride,indicating that SsSvf1 is involved in coping with hyperosmotic stress.The growth of SsSvf1 gene-silenced transformants was also more significantly inhibited in the medium containing sodium dialkyl sulfate?SDS?and congo red?CR?,indicating that the gene is associated with cell wall integrity.SsSvf1gene-silenced transformants were more sensitive to oxidative stress,indicating that SsSvf1 is closely related to oxidative stress.Reactive oxygen species?ROS?in gene-silenced transformants were detected by nitroblue tetrazolium?NBT?staining.The results showed that silenced of this gene led to excessive accumulation of ROS.The quantitative analysis of the secreted oxalic acid by HPLC showed that the inhibition of SsSvf1 expression did not cause a change in the accumulation of oxalic acid.However,the virulence of SsSvf1 gene-silenced transformants in leaves of Brassica napus and Arabidopsis leaves was significantly reduced,while the artificially wounds could partially restore the pathogenicity of gene-silenced transformants.Trypan blue staining further showed the compound appressorial formation of the gene-silenced transformants were abnormal,indicating that the gene is closely related to the formation of compound appressoria.This study suggests that SsSvf1 plays an important role involved in coping with oxidative stress and in the pathogenicity of S.sclerotiorum.In this study,the fusion expression vector of SsSvf1 with FLAG-tag was constructed,and the transformant was obtained by PEG-mediated protoplast transformation.The transformant expressed the FLAG-tagged SsSvf1 protein was vertified by RT-PCR and Western blot.The proteins which may interact with S.sclerotiorum SsSvf1 was identified throught CO-IP and LC-MS/MS analysis.The interactions between the proteins and SsSvf1 were vertified by yeast two-hybrid.The results showed that the catalase SsCat2?SS1G₀0547?interacts with SsSvf1 directly.Itis speculated that S.sclerotiorum SsSvf1 regulate the production of ROS during the infection throught its interaction with catalase 2,and play a key role in the pathogenicity of S.sclerotiorum.This study analyzed the function of S.sclerotiorum SsSvf1 gene in oxidative stress and pathogenesis,which not only provides a theoretical basis for revealing the pathogenic mechanism of S.sclerotiorum,but also offers important clues to control Sclerotnia disease.This study also provides an important reference for exploring the function of SsSvf1 homologous proteins in other fungi.