Functional Analysis And Host-interacting Proteins Identification Of A Secreted Protein SsCTP1 From Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum(Lib.)de Bary is a plant pathogenic fungus with a wide range of host.It can infect more than 600 plant species.Crop Sclerotinia stem rot caused by S.sclerotiorum is one of the widespread soil-borne diseases around the world,which seriously affects the yield and quality of crops such as soybean,canola oil and sunflower.A large number of effectors can be secreted to participate the molecular interaction and co-evolution of soybean during infection.Several virulent secretory proteins promoted the infection of S.sclerotiorum after transient expression of N.benthamiana.SsCTP1(Chloroplast-targeted Protein 1)is highly up-regulated in the stage of infection and localized in chloroplasts.The study used SsCTP1 as the target gene.And the functions of SsCTP1 in the growth and pathogenicity of S.sclerotiorum were studied by gene knockout,q RT-PCR,and protein interaction verification.The main results are as follows:1.Functional analysis of SsCTP1 in S.sclerotiorumRNA-seq and q RT-PCR results showed that the expression of SsCTP1 was significantly up-regulated in the early stage of host infection.SsCTP1 is localized in chloroplasts and does not trigger Hypersensitive reaction(HR).SsCTP1 can not inhibit the PCD triggered by BAX/INF1.After induction with flg22 and chitin,SsCTP1 can inhibit callose deposition and up-regulated expression of defense genes.The study successfully obtained the knock-out mutant △Ssctp1.There is no significant difference between the wild type and △Ssctp1 in the growth and development,but the pathogenicity of △Ssctp1 to different hosts was significantly decreased.Transgenic Arabidopsis thaliana overexpressing SsCTP1 showed a higher susceptibility to S.sclerotiorum.These results indicated that SsCTP1,as an effector protein of S.sclerotiorum,was involved in the pathogenesis of S.sclerotiorum.It was a virulence related factor.2.Identification the interaction proteins of SsCTP1Two interacting proteins oxygen-dependent coproporphyrinogen-III oxidase(Gm CPX)and shikimic acid kinase 2(Gm SKL2)were identified by screening soybean yeast two-hybrid library,and their interactions in vivo were confirmed by split-luciferase complementation(Split-LUC)and Co-immunoprecipitation(Co-IP).Confocal microscopy showed that SsCTP1,Gm CPX and Gm SKL2 were localized in chloroplasts.The virus-induced gene silencing mediated by Tobacco rattle virus was used to silence Nb CPX in tobacco.The results showed that Nb CPX silenced plants were more resistant to S.sclerotiorum.These results indicate that SsCTP1 is an effector protein and participate the pathogenicity of S.sclerotiorum.Gm CPX and Gm SKL2 were located in chloroplasts.The Nb CPX silenced plants were more resistant to S.sclerotiorum.This study provided a theoretical basis for the analysis of the pathogenic mechanism and host interaction.At the same time,it provides theoretical support for cultivating crop varieties resistant to Sclerotinia stem rot.