Study On Molecular Mechanism Of A Puccinia Striiformis Effector Ps3439 Promoting Pathogenicity By Regmlating A Wheat ATPase

Wheat stripe rust is one of the most serious diseases on wheat.Puccinia striiformis f.sp.tritici（Pst）,the causal agent of wheat stripe rust,is a kind of obligate biotrophic fungus and virulence variation frequently happens.A large number of studies have shown that Pst inhibits the host defense response by secreting effectors to promote its infection and pathogenicty.In recent years,the whole genome sequencing of Pst has been completed successively,and numerous secreted proteins have been found.However,little is known about the roles of secreted protein in the infection process of Pst and its regulation mechanism.This study was based on the preliminary study of haustoria transcriptome sequencing of Pst,a candidate effector gene Ps3439 was then screened,and the functions of Ps3439 in the process of infection and pathogenesis was identified.The obtained results may lay a foundation for understanding the interactions between wheat and Pst and provide theoretical and technical support for the persistent prevention strategy of wheat stripe rust.The key results are as below:1.Analysis of secretion and expression profile of Ps3439.The effector gene Ps3439,which was 303bp,encoded 118 amino acids.The first 25amino acids at the N-terminal were signal peptide,and the isoelectric point（p I）is 9.70.The molecular weight was 8.6 k Da,which doesn’t contain cysteine（Cys）.Transmembrane regions and conserved domain was not found.Yeast signal sequence trap assay revealed that the N-terminal signal peptide of Ps3439 could be secreted from yeast cells;Subcellular localization of Ps3439 mediated by Agrobacterium showed that the Ps3439 was located in the cytoplasm and nucleus.The transcript expression levels of Ps3439 during Pst infection of wheat were analyzed by qRT-PCR.The results showed that the transcription level of Ps3439was increased by more than 40 times at 6,12,18 and 24 hours after inoculation,sμggesting that Ps3439 may play a key function at the early stage of Pst infection.2.Ps3439 interacts with a wheat ATPase named TaXB24.To analyze the regulation mechanism of Ps3439,we screened and verified a candidate effector named TaXB24 from wheat,based on the yeast two-hybrid system of the wheat-Pst interaction.The two proteins were expressed in vitro with the prokaryotic cells,and then pull down assay verified their mutual effect.The BiFC assay and the Co-IP experiments also confirmed the interactions between Ps3439 and TaXB24 in vivo.Those evidences indicate that there is mutual effect between Ps3439 and TaXB24.3.Ps3439 significantly increases TaXB24 enzyme activityTaXB24 had a closer evolutionary relationship with other plant proteins（Triticum urartu,barley,rice）containing the ATPase motif by phylogenetic analyses.Based on this,prokaryotic expression of TaXB24 was performed and enzyme properties were determined.TaXB24 was found to have ATPase activity in vitro,and Mg²⁺had a higher positive effect on its activity.To determine whether Ps3439 had an effect upon the ATPase activity of TaXB24,Ps3439both were expressed throμgh prokaryotes and purified.The results showed that enzyme activity of TaXB24 was significantly enhanced with the increasing amount of Ps3439.4.TaXB24 plays a negative role in the resistance process of wheat.To obtain insight into a reasonable function of TaXB24 during Pst infection,we analyzed the transcript abundance of TaXB24 in compatible and incompatible systems by qRT-PCR.The results showed that compared with the control,TaXB24 was down-regulated in wheat resistance to stripe rust infection.Subcellular localization of TaXB24 mediated by Agrobacterium showed that the TaXB24 was located in the cytoplasm.Knocking down TaXB24 by virus induced gene silencing showed that compared with the control,the silencing plants had significantly reduced sporulation,and histological observations revealed that the colony area decreased significantly at 48 hpi and 120 hpi,and the development of the mycelia was limited,indicating that TaXB24 plays a negative role in the resistance process of wheat.