Functional Study Of The Transcriptional Activator-Like Effector PsAvh48 Of Phytophthora Sojae

Soybean root rot caused by Phytophthora sojae occurs to varying degrees in soybean production areas around the world and is a devastating disease that threatens soybean production and even global food security.Due to the lack of effective fungicides,current agricultural production relies on breeding resistant varieties to control the disease.Today,all identified soybean resistance genes to resist soybean root rot have been overcome by the pathogen.Development of new genetic resources for resistance is a pressing issue at this stage.Therefore,in-depth research on the pathogenesis and mechanisms of infestation of Phytophthora is important for the development of disease control measures and precise molecular breeding for disease resistance.Recent studies have found that Phytophthora infest the host by secreting many effectors into the host cell or apoplast,thus achieving the purpose of destroying or inhibiting the host’s defense response and promoting its invasion and colonization.A class of effectors with a conserved N-terminal signal peptide and an RxLR structural domain,called RxLR effectors,has been identified in Phytophthora.Previous analysis of the RxLR family of effectors in Phytophthora sojae revealed that PsAvh48 was not found as a homologous protein in other Phytophthora,but two homologs existed in four sequenced strains of Phytophthora sojae.Therefore,speculate that PsAvh48 may be a RxLR effector that evolved directionally in Phytophthora sojae in the face of long-term selection pressure.In the present study,PsAvh48 was found to be up regulated in the pre-infection stage of Phytophthora sojae,which is presumed to play an important virulence role in the pre-infection stage.Structural analysis of PsAvh48 revealed a conserved signal peptide region and nuclear localization signal(NLS)at its N-terminal.Transient expression in tobacco indicates that PsAvh48 can be localized in the plant nucleus.By mutating the nuclear localization signals to nuclear export signal(NES),PsAvh48 loses its original nuclear localization and loses its virulence effect.The transcriptional activation activity of PsAvh48 was confirmed using a nuclear yeast system,and the amino acids 76-95 of PsAvh48 were identified as its transcriptional activation domain by truncating mutations.The study further confirmed the important role of the nuclear localization signal and transcriptional activation structural domain of PsAvh48 in promoting the infestation of Phytophthora.In this study,knockout and complementation transformants of the PsAvh48 gene of Phytophthora sojae were obtained using the CRISPR/Cas9 system.Virulence testing of transformants revealed that knocking out PsAvh48 in Phytophthora sojae reduced the pathogenicity of Phytophthora sojae,while the virulence of the knockout strain was restored to the same level as the wild type after complementation of the PsAvh48 gene.The above results demonstrate that the virulence effect of the RxLR effector PsAvh48 is necessary for the full virulence of Phytophthora sojae.In this study,a total of 43 candidate DNA sequences that might be recognized and specifically bound by PsAvh48 were screened by yeast single-hybrid screening of promoter motif libraries,and it was also preliminarily verified by yeast single hybridization that PsAvh48 has intereaction relationship with the promoter fragment having the GTGGGY motif.In this study,a transcriptional activator-like RxLR effector PsAvh48 was identified for the first time in Phytophthora sojae,which laid the molecular foundation for analyzing the virulence mechanism of host gene transcription manipulation by Phytophthora sojae and provided theoretical support for further analysis of the pathogenesis of Phytophthora sojae and the development of disease prevention and control strategies and molecular breeding for disease resistance.