Functional Study Of Candidate Effector Proteins In Ustilaginoidea Virens

Rice false smut caused by Ustilaginoidea virens is a kind of rice fungal disease on rice panicle.In recent years,it has become increasingly serious in China.Rice false smut disease not only affects the yield and quality of rice,but it also produces toxins during the infection and poses a serious threat to human and animal health.Therefore,the prevention and control of rice false smut is one of the important topics of rice production in China.In this study,the proteins secreted by the U.virens during the infection stage were analyzed.Selected proteins were investigated by the agrobacterium-mediated plant transient expression system to identify effectors that could inhibit Bax-induced programmed cell death（PCD）or hypersensitive response（HR）in N.benthamiana.A total of 12 effectors were found to be able to inhibit Bax-induced PCD in N.benthamiana.Three of them showed full-length inhibition activity while signal peptide deleted mutants did not.Eight of them showed that the full length could not,while the signal peptide deleted mutant could inhibit the PCD.Another effector,GME3212,showed that both full-length and signal peptide deleted mutant could inhibit the PCD.The validation of the secretory function of signal peptides for these effectors showed that except for GME₃186,the predicted signal peptide regions of the 11 candidate effectors all had secretory function.And many effectors（wild type or mutant）that inhibit Bax-induced PCD in N.benthamiana can promote the infection of Phytophthora capsici on tobacco leaves.In addition,GME₅236 can cause cell death in N.benthamiana.Subcellular localization analysis showed that GME₅236 was likely located on the chloroplast of tobacco leaf cells.Surprisingly,GME₅236 can also promote the infection of P.capsici on tobacco leaves.Analysis of the GME₅236 protein sequence revealed that GME₅236 contained 6 cysteines and contained a conserved defensin domain.The transient expression results of the point mutants showed that 4 cysteines were indispensable for the function of GME₅236.These results lay the foundation for further exploration of the function of these effectors in the infection of U.virens.This study also provided a reference for the research on the infection mechanism of U.virens.