Cloning And Functional Analysis Of RxLR Effectors From Phytophthora Capsici

Phytophthora capsici,a ubiquitous facultative parasited,can attack solanaceous and cucurbit hosts,which causing pepper blight disease in the world wide.Nowadays,the prevention of P.capsici still remains in the primary stage of chemical control,supplemented by agricultural control,therefore these methods are inefficient,costly and unsafe.It may cause wide broke of P.capsici when challenged with climate.Pesticide residues and other problems still threaten the pepper production,human health and environmental safety.With the continuous advancement of research on oomycetes,oomycete RxLR effectors were found to play a critical role in the interaction between Phytophthora and plants.Pathogens secrete a large number of RxLR effectors during infection.The P.capsici encodes nearly 400 RxLR effectors,most of which are still unknown.Phytophthora sojae and Phytophthora infestans have been studied a lot,but we still know a little about P.capsici.P.capsici are also of great significance in agricultural production,therefore,it is very necessary to study the effectors of P.capsici.First of all,two RxLR effectors were cloned and constructed on the pBIN-GFP plant expression vector using P.capsici strain SD33 as the material.Agrobacterium tumefaciens-mediated transient expression was performed to study the function,and Western blot was used to analysis protein expression level,both of which were detected in Nicotiana benthamiana.The results suggested that RxLR13887 and RxLR115890 were all normally expressed in N.benthamiana,and RxLR115890 could induce slight necrosis of N.benthamiana cells.What's more,they couldn't inhibit Bax-induced PCD and PTI induced by INF1,as well as cell death induced by CRN4,nor RxLR13887 could they inhibit pathogenicity of P.capsici.Because RxLR13887 did not have a research function,RxLR115890 could only cause cell death and had no other inhibitory function,which is not conducive to the development of follow-up experiments.Therefore,in order to ensure the continuity of the experiment,the follow-up study of this experiment selected RxLR101 as the research object,and RxLR13887,RxLR115890 no longer develop a series of subsequent studies.RxLR101 was expressed at high levels at the early stage of infection by expression pattern analysis.By predicting the secondary structure,RxLR101 was truncated into five domains.Through functional verification of the truncations,RxLR101nudix(240aa-263aa)was found to inhibit Bax-mediated cell death as a key candidate functional domains,and suppress the pathogenicity.In order to confirm the conservation of RxLRnudix,the homologous gene RxLR101-P.i from P.infestans and RxLR101-P.p from P.parasitica were obtained by sequence alignment,all of which contained conserved nudix motifs.After function analysis,RxLR101-P.i,RxLR101-P.p were found to inhibit Bax-mediated PCD as well as the infection of zoospores.The subcellular localization of RxLR101 was located in both the cytoplasm and the nucleus,nuclear localization signals(NLS)and nuclear export signals(NES)were fused to the C-terminal of RxLR101 to ensure the specific functional subcellular localization.The results suggested that cytoplasm localization of RxLR101 was more efficient to suppress Bax,and inhibit pathogenicity of P.capsici when localized to nuclear,respectively.Simultaneously,NLS and NES were also fused to RxLRnudix to detect the function.Transient expression on N.benthamiana indicated that RxLRnudix could inhibit pathogenicity efficiently when located to nucleus.Furthermore,Bax-meditated PCD was significantly suppressed by cytoplasm localization of RxLRnudix.Supplementally,Agrobacterium co-expression technology was exploited to observe the co-subcellular localization of RxLR101 and VPS,which was a candidate interaction protein of RxLR101.The results showed that the location of VPS was not affected by RxLR101.Through this study,RxLR101 was speculated to play a crucial role in the infection period of P.capsici,and remains active during the interaction with the host.These results will provide a foundation for the further exploration of P.capsici pathogenesis,and offer a reference to reveal the interaction mechanism between effectors and interacting proteins.