Functional Research On Sshp1 In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum（Lib.）de Bary is an important worldwide plant pathogenic fungus.it can infect hundreds of plant species,including oilseed rape,causing huge losses to the production of oilseed rape.Because of its soil-and air-borne characteristics and complex pathogenesis,It is difficult to completely control in production.In this study,the up-regulated gene Sshp1（SS1G＿01463）was obtained from the transcriptome data of Sclerotinia sclerotiorum infected oilseed rape,it was speculated that it might be involved in regulating the pathogenic process of Sclerotinia sclerotiorum.It can express a functionally unknown protein with DUF3759 domain,named Ss HP1（Sclerotinia sclerotiorum hypothetical protein 1）.The full-length sequence of Sshp1 is 956 bp,including92 bp of 5’UTR,232 bp of 3’UTR and 632 bp of the coding sequence（CDS）,the Sshp1gene was separated by 3 introns.The knock-out mutant strain and the complement strain were obtained by PEG-mediated protoplast transformation and agrobacterium tumefaciens mediated transformation respectively.The homozygous mutant strains were obtained by isolation of ascosporesand verified by PCR and RT-PCR.Biological assays and pathogenicity assays were performed on wild type strains（WT）and knock-out mutants.knock-out mutants had no significant difference in the number of sclerotia formation compared with WT,but the total dry weight of sclerotia was lower,suggesting that Sshp1 might be involved in the development and maturation of sclerotia;The inhibition effect of the mutant was significantly greater than that of the WT under hypersaline hypertonic conditions,suggesting that Sshp1 may affect the hypersaline hypertonic sensitivity of S.sclerotiorum.The results of the cell membrane integrity assay indicated that Ss HP1 may be involved in regulating cell membrane integrity;Sshp1 mutant was significantly more inhibited than WT under high H₂O₂ concentration,suggesting that Sshp1 may be involved in regulating genes related to reactive oxygen species（ROS）metabolism;the pathogenicity of theΔSshp1 strain was significantly reduced in Nicotiana benthamiana,Brassica napus and Arabidopsis thaliana compared with WT,The number and extent of infection cushions formed by theΔSshp1 strain were less than those of WT,suggesting that Ss HP1 might participate in the pathogenesis of S.sclerotiorum by regulating the formation of infection cushions.In conclusion,this study preliminarily suggests that the Sshp1 gene of Sclerotinia sclerotiorum may play a role in regulating the development of sclerotia,formation of infection cushions,and pathogenicity,providing a theoretical basis for screening target fungicides and creating SSR resistant germplasm resources.