Functionalanalysis Of Eight RxLR Effectors From Phytophthora Capsici

Phytophthora belonging to the oomycete,is known as the "destroyer of plants"because of the serious plant diseases it causes.Phytophthora capsici is one of the important species found in Mexico in 1922,since which it has been a huge threaten to agricultural production.According to statistics,economic losses caused by phytophthora capsici every year amounts to billions of dollars.Because of the wide host range,infection of mode plant Arabidopsis and Nicotiana benthamiana,convenient operation,Phytophthora capsici has become an important model of oomycetes.RxLR effector is a class of intracellular effector widely studied in recent years.Almost all the oomycete pathogens secrete a large amount of RxLR effectors to the host cells to interfer with the normal cell physiology and function during the pathogen-host-interaction process.More than 200 RxLR effectors have been identified from Phytophthora capsici.Study on the function and virulence mechanisms of RxLR effectors is of great significance on the prevention and control of pepper Phytophthora blight disease.Identification and functional analysis of PiAvr3a homologous gene:Phytophthora infestans RxLR effector PiAvr3a can inhibit the immune response of plants through regulating the stability of the E3 ubiquitin ligase CMPG1 proteins.Here,we identified 9 PiAvr3a-like genes in P.capsici by BLAST searching and demonstrated that this gene group was widely distributed in both P.capsici and P.sojae,suggesting their potential importance in virulence.We successfully cloned 5 PiAvr3a-like genes from P.capsici for further functional characterization.Expressional profiling showed that several members of PiAvr3a-likes were induced at early infection stage,suggesting that they play important roles in early infection.Cell-death-suppression assay demonstrated that none of 5 tested PiAvr3a-likes can suppress cell death triggered by INF1.Inoculation assay showed that PcAvh128 could promote P.capsici infection when it was transiently expressed in Nicotiana benthamiana,indicating that PcAvh128 suppresses plant immunity by other mechanism.However,4 other PiAvr3a-likes failed to promote pathogen infection when they were expressed in N.benthamiana,suggestive of divergent functions of PiAvr3a-likes in P.capsici.Functional study of three RxLR effectors:In our previous study,a high-throughout screening was carried out to identify the functions of the RxLR effectors of P.capsici.We found that some effectors including RxLR22、RxLR25 can induce plants cell death and several effectors including RxLR172 can interact with plant resistance-related proteins.Here,we examined the expression patterns of these three effector genes during the infection of P.capsici on Nicotiana benthamiana and found that the expression level of RxLR172 is 16 times up-regulated during the early stage of infection,while the RxLR22 and RxLR25 are down-regulated for 80%and 70%in the mid-late stage.In order to determine the specific function of these three effectors in the infection stage,we silenced them in P.capsici through PEG mediated transformation of Phytophthora.Colony diameters of the RxLR172-silenced transformants were smaller than those of the wild type,respectively 76%and 89%while the control transformants is 79%,which can not fully show the effect of RxLR172 on the growth of Phytophthora capsici.Inoculation assay using mycelialplugs showed that the virulence of two silenced transformants was reduced.Phytophthora biomass assay further demonstrated that the P.capsici/N.benthamiana biomass ratio was significantly lower(59.9%or 34.8%)in leaves inoculated with the RxLR172-silcnced transformants than that infected in the wild type(99.2%).These results suggested that RxLR172 contributes positively to pathogen virulence.No significant differences were observed between the RxLR22-silenced transformants and the wild-type LT263 in colony morphology and growth speed of hypha;However,significantly smaller lesion areas were observed on N.benthamiana leaves inoculated with the silenced transformants compared to the wild type strain 24 h after the inoculation;By quantitative real time PCR analyse,we found the hypha colonization rate of the three silenced transformants of RxLR22 were respectively 42.3%,9%and 44.9%compared with the wild type,which is significantly lower.These results indicate that RxLR22 effector may enhances pathogenicity of P.capsici.The colony diameters of RxLR25-silenced transformants were 89%and 85%of wild type and in contrast the control transformants was 95%,suggestive of a light effect on P.capsici growth.