Mechanism Of The Effector Pst＿215 Regulating Wheat Immunity In Puccinia Striiformis

Wheat is the staple food and the main food crop for about 1/3 people in the world.However,it is threatened by a variety of diseases,including the stripe rust pathogen Puccinia striiformis f.sp.tritici(Pst)is one of the most serious diseases.However,the loss of resistant varieties in production leads to the continuous epidemic of diseases,which is a major production problem for the prevention and control of Pst.Therefore,it is an urgent problem to cultivate long-lasting disease-resistant varieties.Pst is a biotrophic fugus,which mainly regulates host immunity by secreting a large number of toxic effectors through haustorium,and absorbs nutrients from host,so as to promote infection and cause disease.Therefore,on the basis of the previous identification of some important effectors,this paper deeply analyzed the mechanism of host immunity regulated by Pst effectors and explored how to prevent pathogen effectors from manipulating host immunity.Broad-spectrum disease-resistant materials were created to provide resources for the cultivation of disease-resistant wheat varieties.In the early stage,based on the haustorium transcriptome and functional screening of Pst,the effector Pst＿215 was obtained which was highly abundance-induced in haustorium and significantly inhibited the basic immune response of wheat.On this basis,the regulation mechanism of effector Pst＿215 was deeply studied and the main results are as follows:1.The signal peptide encoded by N-terminal 1-18 amino acid of Pst＿215 was proved to have secretory function by using YSST(yeast secretion signal trap)system.q RT-PCR analysis showed that Pst＿215 was highly induced in 12 and 24 hpi(hour post inoculation)of wheat infected by Pst,which it was speculated that it played a role in the early stage of Pst infection.Transient expression of Pst＿215 in N.benthamiana inhibited callose accumulation and MAPK phosphorylation induced by flg22 and decreased the expression of Nb PR1,Nb PR2 and Nb WRKY12,which further indicated that Pst＿215 had the function of inhibiting plant PTI(PAMP triggered immunity).2.Wheat transgenic materials with stable overexpression and silencing of Pst＿215were created by Agrobacterium-mediated wheat genetic transformation system.After inoculation with CYR23,wild type wheat showed obvious necrotic reaction and Pst＿215-OE plants produced a small amount of urediniospore pustules while producing allergic necrotic reaction;Compared with the wild type,the areas of reactive oxygen species around the infection site of Pst were decreased and the expression of Ta PR1 and Ta PR2 was decreased in Pst＿215-OE plants,the hyphae length and infection areas were increased and the biomass was increased,indicating that the overexpression of Pst＿215 in wheat promoted the infection of Pst.But Pst＿215-RNAi#L1 and #L2 were inoculated with CYR32,compared with the wild type,urediniospore pustules were significantly decreased in Pst＿215-RNAi plants,the hyphae length and infection areas were decreased,the biomass was decreased,the cell necrotic areas were increased,and the expression of Ta PR1 and Ta PR2 was increased,indicating that silencing Pst＿215 decreased the pathogenicity of Pst.The results showed that Pst＿215 was an important effector which significantly affected the pathogenicity of Pst.3.The candidate targets of Pst＿215 were screened by co-immunoprecipitation and mass spectrometry,the interactions between Pst＿215 and Ta MKK2 was further verified by yeast two-hybrid,luciferase complementation and co-immunoprecipitation,indicating that Pst＿215 interacted with Ta MKK2.CRISPR-Cas9 edited Ta MKK2 to create Ta MKK2 knockout mutants,inoculated with CYR23,knockout Ta MKK2 plants produced a small number of urediniospore pustules,and the hyphae length and infection areas were increased.However,when the plants overexpressing Ta MKK2 were inoculated with CYR32,urediniospore pustules of Pst were significantly decreased,the growth and development of Pst was inhibited,the level of MAPK phosphorylation were increased,the areas of H2O2 were increased,and the expression of Ta PR1,Ta PR2 and Ta WRKY53 was increased.Transient expression of Pst＿215 in overexpressed Ta MKK2 plants inhibited the level of MAPK phosphorylation,decreased the expression of defense-related genes,and weakened the disease resistance of Ta MKK2.The results showed that Ta MKK2 was positively regulating the resistance of wheat to Pst and the effector Pst＿215 inhibited the resistance of Ta MKK2 to Pst.4.The interaction between Ta MKK2 and Ta MAPK4/6 was confirmed by yeast two-hybrid,luciferase complementarity and Co-IP experiments.Virus-induced gene silencing technique was used to silence Ta MAPK6.The urediniospore pustules and biomass of Pst were increased and the reactive oxygen species areas were decreased on silenced Ta MAPK6 plants,which indicating that silencing of Ta MAPK6 reduced the resistance of wheat to Pst.Ta MAPK4/6 was phosphorylated by Ta MKK2 and phosphorylation was inhibited in the presence of GST-Pst＿215 by in vitro phosphorylation assays and the phosphorylation band of Ta MAPK4/6 was decreased with the increase of GST-Pst＿215concentration.Moreover,the binding affinity of Pst＿215 to Ta MKK2 was higher than that of Ta MAPK4/6 and Pst＿215 reduced the interaction between Ta MKK2 and Ta MAPK4/6.5.Yeast two-hybrid,luciferase complementation and immunoprecipitation experiments showed that Ta MAPK6 interacted with Ta SGT1,while Ta MKK2 and Ta MAPK4 did not interact with Ta SGT1.In vitro phosphorylation assay showed that Ta MAPK6 phosphorylated with Ta SGT1.S290 and S365 were the key phosphorylation sites of Ta SGT1 and the mutation eliminated Ta MAPK6 phosphorylated Ta SGT1.Overexpression and gene knockout mutants of Ta SGT1 were identified for disease resistance.Ta SGT1-OE transgenic lines showed significant reduction in the number of urediniospore pustules after inoculated with CYR32,the areas of reactive oxygen species and cell death were significantly increased,and the resistance to Pst was enhanced.After inoculated with CYR23,the Ta SGT1 knockout mutant plants produced fewer urediniospore,the biomass of Pst and the infection areas of Pst were significantly increased.Transient expression of Ta SGT1S290DS365D(phosphorylation activation)in N.benthamiana showed increase in the proportion of Ta SGT1 protein accumulation in the nucleus.Ta SGT1S290AS365A(phosphorylation inactivation)and Ta SGT1S290DS365 D were transiently expressed in Ta SGT1 knockout mutants.It was found that the Ta SGT1 knockout mutants expressed by Ta SGT1S290AS365 A produced small number of urediniospore pustules when inoculated with CYR23,while the transient expression of Ta SGT1S290DS365 D produced obvious hypersensitive necrosis reaction,increased expression of defense-related genes and high resistance to Pst,indicating that Ta SGT1 simulated phosphorylation enhanced the resistance of wheat to Pst.The amount of nuclear Ta SGT1 in overexpressed Ta SGT1 plants inoculated with CYR23 was higher than that of non-inoculated plants,indicating that the amount of Ta SGT1 in the nucleus increased in the response to disease resistance.Further study showed that CYR23 inoculation enhanced the Ta SGT1 protein level in the nuclei rather than non-inoculated plants of overexpressed Ta SGT1,indicating that Ta SGT1 protein level in the nucleus increased in the response to disease resistance.Transient expression of Pst＿215 decreased the level of Ta SGT1 protein in nucleus and weakened the resistance of wheat.Transient expression of Ta SGT1 with nuclear localization signal in Ta SGT1-KO plants resulted in hypersensitive necrosis reaction.Transient expression of Ta SGT1 with nuclear output signal in Ta SGT1-KO plants resulted in enhanced disease resistance,but a small number of urediniospore pustules were still produced,indicating that nuclear localization of Ta SGT1 enhanced wheat resistance to Pst.In summary,the function and mechanism of Pst＿215 were explored,Pst＿215 was revealed as an important virulence factor of Pst.Pst＿215 regulates the distribution of Ta SGT1 in the nucleus and cytoplasm through the Ta MKK2-Ta MAPK6-Ta SGT1 phosphorylation cascade pathway,thereby affecting wheat resistance to Pst.It enriches the understanding of the regulation of plant immunity by Pst.In the study,transgenic materials of effector Pst＿215 and Ta MKK2-Ta MAPK6-Ta SGT1 cascade pathway members were created by RNAi,CRISPPR-Cas9,overexpression and other techniques,which improved wheat disease resistance and provided genetic resources and materials for the creation of long-lasting broad-spectrum disease-resistant wheat materials.