| Rice false smut,caused by Ustilaginoidea virens,is one of the three major rice diseases in China,which seriously affecting rice yield and quality and endangering human and animal health.Studying the pathogenic mechanism of U.virens has important theoretical significance and application value for reducing diseases and cultivating disease resistant varieties.In this study,the gene functions of 8 genes from subtilisin-like proteases(SBT)family of U.virens were preliminarily explored,and Uv8b＿1567 gene was selected to further explore its pathogenic mechanism,and the research results are as follows:(1)The protein domains encoded by 8 genes from SBT family of U.virens: Uv8b＿44,Uv8b＿1567,Uv8b＿4545,Uv8b＿6002,Uv8b＿6469,Uv8b＿7084,Uv8b＿7700 and Uv8b＿8155 were predicted and analyzed.It was found that all proteins have the same conserved Peptidase S8 domain.The proteins encoded by Uv8b＿44,Uv8b＿1567 and Uv8b＿8155 contain Inhibitor I9 domain,and the proteins encoded by Uv8b＿6469 and Uv8b＿7700 contain Fn-III domain.Except Uv8b＿4545 and Uv8b＿7084 gene,all the other proteins contain a signal peptide domain.All genes were knocked out and complemented,we found that deletion of the Uv8b＿44,Uv8b＿1567 and Uv8b＿8155 genes,the mycelial growth rate was significantly decreased and the sensitivity to pressure stress was also significantly changed.The conidiation of ΔUv8b＿1567 and ΔUv8b＿8155 mutants were significantly increased.The pathogenicity of ΔUv8b＿44 and ΔUv8b＿1567 mutants were significantly decreased.Furthermore,the pathogenicity of ΔUv8b＿8155 mutants were completely loss.(2)The pathogenic mechanism of Uv8b＿1567(UvPr1a)gene was further explored.UvPr1 a contained a signal peptide(SP),the yeast secretory activity test proved that the SP of UvPr1 a had secretory activity.Subcellular localization of UvPr1 a in the cytoplasm of tobacco cells.Abundant expression of UvPr1 a to inhibit Bax-induced programmed cell death,heterologous overexpression of UvPr1 a transgenic rice plants significantly reduced the resistance to rice false smut,rice blast and bacterial blight,indicating that the effector UvPr1 a could suppress the immune response of plants.The interacting R protein OsSGT1 was screened from the rice library using yeast two-hybrid,and further used yeast twohybrid,Co-IP,BiFC and GST pull-down to prove that UvPr1 a and OsSGT1 interact in the cytoplasm.Knock out of OsSGT1 reduces the resistance to rice false smut,rice blast and bacterial blight,while overexpression of OsSGT1 enhances the resistance to rice false smut,rice blast and bacterial blight,indicating that OsSGT1 positively regulates broad-spectrum resistance against rice pathogens.When UvPr1 a and OsSGT1 were co-expressed in Nicotiana benthamiana,we found that UvPr1 a can inhibit the HR response induced by OsSGT1.After prokaryotic purification of UvPr1 a and OsSGT1,in vitro co-incubation experiments showed that UvPr1 a could directly degrade OsSGT1 protein.These results of this study indicate that the effector UvPr1 a interacts with the rice R protein OsSGT1,and UvPr1 a can directly degrade the OsSGT1,thereby affecting the rice immune response regulated by OsSGT1.Taken together,we identified the functions of the subtilisin family genes of U.virens,analyzed the molecular pathogenicity mechanism of the effector UvPr1 a,and provided genetic resources for the creation of new rice varieties with resistance to rice false smut. |
