Study On Immune Inducing Mechanism Of Phytophthora Capsici Effector RxLR129113

Plant immune attractant is a new concept biological preparation,which can use protein inductors from plant pathogens to activate plant immune system and induce plants to resist diseases.Phytophthora capsici is a kind of extremely harmful plant pathogen oomycetes in nature.During the process of infecting the host plants,P.capsici can secrete a range of effectors which can manipulate host innate immunity.These effectors may be used to develop plant immune attractants.Previous studies have demonstrated that the effector RxLR129113 cloned and isolated from the strain SD33 of P.capsici is located in the nucleus and cytoplasm,can induce hypersensitive reaction of the Nicotiana benthamiana.In this study,the effector RxLR129113 was used as the object of study,and N.benthamiana was used as the plant material.Using agrobacterium-mediated transient expression technology,we mainly studied and analyzed immune-inducing mechanism of RxLR129113 on plants during the infection of P.capsici.The main research contents and results are as follows:(1)After transient expression of RxLR129113 in N.benthamiana,infected the leaves with zoospores of P.capsici.The results prove that RxLR129113 can inhibit the infection of P.capsici compared with the empty vector.(2)DAB staining was performed on the leaves of N.benthamiana infected by zoospores of P.capsici after transient expression of RxLR129113.The results show that active oxygen can accumulate during the infection of P.capsici.Eight resistance-related genes in N.benthamiana such as PR1 were veryfied by q RT-PCR to express differentially during the infection of P.capsici.Therefore,it is speculated that RxLR129113 can stimulate plant immunity mediated by the salicylic acid pathway.(3)RxLR129113 was fused with nuclear localization signal and nuclear export signal to express instantaneously in N.benthamiana for infection of P.capsici.The results prove that RxLR129113 can inhibit the infection of P.capsici when it is located in the cytoplasm.When it is located in the nucleus,it has no significant effect on the infection of P.capsici.(4)According to the truncations of the three-dimensional structure of RxLR129113 protein crystals in the previous experiments,it was verified that W1-3 was a key functional domain that induced cell hypersensitive reaction.Based on the previous results,two truncated bodies of W1-3 and W4-5 were constructed for infection of P.capsici.The results show that W1-3 is also a key functional domain of RxLR129113 to inhibit the infection of P.capsici.(5)In order to further clarify the key functional sites of RxLR129113 to inhibit the infection of P.capsici,the loop3 of each WY domain of W1-3 was mutated,and nine mutants were constructed to verify the function.The results show that W1-3 could no longer inhibit the infection of P.capsici after the mutation from the 167 th to 171 th amino acid.(6)Used the virus-induced gene silencing technology to transiently silence the protein PR1 interacting with RxLR129113 in N.benthamiana,RxLR129113 was instantaneously expressed in these leaves and the infection of P.capsici still is inhibited.This indicate that PR1 has no direct effect on the function of RxLR129113.The above results confirmed that RxLR129113 can stimulate plant immunity and preliminarily explained its mechanism.This study provides an important theoretical basis for the prevention and control of P.capsici.disease.At the same time,it also provides a new candidate protein for the research and development of plant immune attractant.