Identification And Functional Analysis Of RxLR Effectors Essential For Pathogenicity In Phytophthora Sojae

Phytophthora are a group of oomycete plant pathogens which can cause devastating diseases,such as soybean root rot caused byPhytophthora sojae.As a typical hemibiotroph pathogen,P.sojaerequires living plant tissues to obtain main nutritions at the early stage of infection,while transferring to necrotrophy it may immediately kill its host during late infection,revealing the regulation of pathogen-host interaction might be much more complicated.With the completion of genome sequencing,about four hundred RxLR effectors were found in the genome of P.sojae.Although several RxLR effectors have been shown to suppress plant defense,the biological functions and mechanisms of the vast majority of effectors during infection remain unknown.Compared with fungi in evolutionary,one of the significant differences in pathogenic mechanism is that Phytophthora can secrete a large amount of RxLR effectors.So far,it is still unknown whether effectors have functional redundancy orwhether most of the single effectorsare essential for pathogenicity.In this study,we conducted a transcriptional analysis of the RxLR effectors using previous MicroArraydata andselected 17 highly expressedRxLR effectorgenes for further study.We transformedP.sojae(P6497)with synthetic dsRNA derived from target effector to transiently silence these effector genes to learn their contribution to pathogenicity.We silenced and assessed the virulence contribution of the 17 selectedPsAvh genes.13 of these effectors showed different degrees of reduced virulence,suggestingthe importance of these highly expressed effectors.It revealed that most of the single effectorscould also be determinative for pathogenicity.RNA silencing serves as a major defense mechanism against RNA viruses in plants,fungi and invertebrates during the pathogen-plant co-evolution.Recently,our lab have identified two RxLR effectors(i.e.PSR1 and PSR2)functioning as silencing suppressors from P.sojae.PSR2 specifically controls plant ta-siRNA accumulation to help infection.We found PSR2 was one of the essential effectors by stable silencing.Besides,Kuan et al.identified the host factors that may affect the function of PSR2 in plants.Our study further examined the expression of the selected gene upon pathogen infection.Results indicated the expression of the selected gene was induced during infection by the wild type P.sojae,while kept constant levels of transcription when infected by PSR2 silenced lines.Furthermore,protein structural analysis showed that PSR2 exhibited a highly specific pattern containing 5 whole and 2 half repeat units,and each repeat unit was consisted of the conserved W,Y and L motifs.Our results suggested that one whole repeat closed to Cterminus and two half repeats were essential for RNA silencing suppression activity of PSR2.However,none of these repeats,when deleted,led to PSR2 localization changes in plants.In conclusion,transiently silencing 17 highly expressed RxLR effectors in P.sojae showed most of these effectors are indispensable for pathogenicity.To further study the function of RNA silencing suppressor PSR2,we confirmed PSR2 as one of the essential effectors and identified the important domains for its RNA silencing suppression activity.These results are of great value for understanding the biological significance and relationships between effector function and functional domains,providing important knowledge for further studies of effectors.