The Screening Of HIGS And SIGS Targets In RXLR Effectors From Phytophthora Capsici

Pepper(Capsicum annuum)is one of the most important vegetable and industrial raw material crops.Phytophthora blight,caused by Phytophthora capsici,frequently reaches epidemic levels and causes huge yield losses in pepper production regions worldwide.Phytophthora is one of the pathogenic oomycetes,which is similar to fungi in morphologically but far away different from cell wall components,physiological metabolisms,reproductions,genetic variations and so on,resulting in difficult control by most of the existing fungicides.During the infection,P.capsici could secrete an array of highly divergent RXLR effectors to suppress host immunity to favor successful infection.Efficient and specific targets for disease control have attracted considerable attention for a long time by researchers.The development of RNAi technology provides an effective strategy for pathogen-derived resistance.Host-induced gene silencing(HIGS)and Spray-induced gene silencing(SIGS),which are new technologies based on RNAi technology,have great potential for application in the field of plant-fungus/nematodes interactions and crop improvement against the disease.However,it is rarely reported in the plant-oomycetes interaction system.In this study,we confirmed that P.capsici could uptake external si RNA labeled with fluorescence from the environment.According to the RNA-seq analysis of P.capsici-pepper interaction and q RT-PCR analysis,12 RXLR effector-encoding genes,which were significantly up-regulated during the infection stage,had been identified.Transient overexpression of these RXLR effectors respectively in Capsicum annuum and Nicotiana benthamiana leaves revealed that most of those RXLR effectors could not induce cell death response,excepting RXLR6,RXLR9,RXLR12 and RXLR13.Moreover,transient overexpression analysis and P.capsici inoculation assay indicated that excepting RXLR2 and RXLR3,other 10 of these RXLR effectors could promote the pathogen's infection in varying degree;among them,RXLR1 and RXLR4 were the most obvious.We then found that exogenously applied si RNAs targeting RXLR1 or RXLR4 could significantly restrict growth of P.capsici on the Capsicum annuum and Nicotiana benthamiana detached leaves.When applied RXLR1-si RNA and RXLR4-si RNA simultanenously,the pathogenicity of P.capsici was further weakened.To determine whether this RXLR1/4-targeting RNAi strategy could efficiently control phytophthora blight or not,we introduced the specific RXLR1/4 RNAi fragments into the pepper plants by virus-induced gene silencing(VIGS)system.The results showed that VIGS pepper plants expressing ds RNA/si RNA targeting RXLR1 or RXLR4 respectively can effectively reduce the infection of P.capsici.When VIGS pepper plants expressing RNAi constructs targeting RXLR1 and RXLR4 simultaneously,the pepper plants conferred a more enhanced resistance to phytophthora blight.These findings suggested that exogenous applying si RNAs or in planta expressing the RNAi constructs that target RXLR1 or RXLR4 could restrict growth of P.capsici.It also showed that interfering RXLR1 and RXLR4 simultaneously could confer plants a more enhanced resistance to the disease.In summary,we confirmed that P.capsici could uptake external si RNA from the environment.And then 12 P.capsici RXLR effectors,which were up-regulated during the infection stage,were identified based on our transcriptomic analysis of P.capsici-pepper interactions,and their virulences were screened preliminarily.Furthermore,it also confirmed that exogenous applying si RNAs or in planta expressing RNAi constructs target RXLR1 and RXLR4 could effectively restrict the infection of P.capsici.These results provide a promising foundation to protect plant against the oomycete pathogen Phytophthora capsici base on pathogen-derived resistance,and also provide an important basis for the development of RNAi-based anti-oomycete agents.