Pathogenic Mechanism Of Chorismate Mutase SsCm1 In The Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum(Lib.)de Bary is a plant pathogenic fungus with a wide range of hosts.Sclerotinia stem rot caused by S.sclerotiorum greatly limited the yield and quality of soybean(Glycine max L.Merr.).Chorismate mutase catalyzes the formation of prephenylic acid into phenylalanine and tyrosine.It is worth noting that chorismate is also an important precursor for the synthesis of salicylic acid(SA).The chorismate mutase of biotrophic pathogen Ustilago maydis(Um Cmu1)reduces the level of salicylic acid by forming dimer with the host's chorismate mutase,thereby inhibiting the immune responses.Bioinformatics analysis shows that S.sclerotiorum is the only necrotrophic that has homologous protein with Um Cmu1.And the functions of SsCm1 in the development and pathogenicity of S.sclerotiorum were studied.The main research results are as follows:1.Functional analysis of the chorismate mutase SsCm1 in S.sclerotiorumq RT-PCR results show that the expression of SsCm1 is significantly up-regulated during infection.SsCm1 is localized in the cytoplasm and does not trigger hypersensitive reaction(HR).SsCm1 which localized in chloroplast but not cytoplasm inhibits the PCD triggered by BAX/INF.There is no significant difference between the wild type and SsCm1 knock-out mutant(?SsCm1)in the growth and development,but the pathogenicity of ?SsCm1 to different hosts was significantly decreased.Transgenic Arabidopsis thaliana overexpressing SsCm1 is more susceptible to S.sclerotiorum.These results suggested that SsCm1 might be an effector and participate in the pathogenicity of S.sclerotiorum.Both SsCm1 and Um Cmu1 belong to the Aro Q family of chorismate mutase and can form homodimer,It was proved by Y2 H.Y2H results also show that SsCm1 could form heterodimer with chorismate mutases in soybean which localized in the chloroplast and cytoplasm,respectively.It is suggested that SsCm1 might have similar function to Um Cmu1.2.Identification the interaction proteins of SsCm1In order to further explore the pathogenesis between S.sclerotiorum and soybean,UF-1 is used to infect susceptible soybean(Jidadou 1),and collected the leaves inoculated for 0 h,24 h,36 h and 48 h and mixed to construct a yeast two-hybrid library,and 11 interacting proteins were obtained by Y2 H library screening.Among them,the two proteins with the highest frequency were selected as candidate proteins,namely multi-organelle RNA editing factor(Gm MORF6)and plastocyanin(Gm PETE).The interactions were verified by Y2 H,Bi FC and Co-IP assays.Confocal microscopy showed that SsCm1 was localized in cytoplasm,while Gm MORF6 and Gm PETE were localized in chloroplasts.The co-localization of SsCm1 with Gm MORF6 and Gm PETE was all in the chloroplast,suggesting that the interaction between SsCm1 and candidate proteins causes SsCm1 to enter the host chloroplast.3.Interaction mechanism between SsCm1 and MORF6In order to investigate the function of the candidate proteins,the interaction between SsCm1 and the homologous protein of Gm MORF6 in tobacco(Nb MORF2b)was verified by Co-IP.The virus-induced gene silencing mediated by TRV(Tobacco rattle virus)was used to silence Nb MORF2 b in tobacco.The results showed that Nb MORF2 b silenced plants were more resistant to S.sclerotiorum.The expression of Gm MORF6 was significantly inhibited during the infection of S.sclerotiorum.Transient expression of Nb MORF2 b and Gm MORF6 in tobacco inhibited the PCD triggered by BAX and INF.Taken together,these results suggest that MORF6 is a negative regulator of plant immunity.Our results suggest that SsCm1,as an effector,may affect the immune response of the host by affecting the accumulation of salicylic acid and targeting to the host chloroplast.