Functional Study Of Phytophthora Capsici Effector RxLR553394

Phytophthora capsici is a widespread and destructive soil-borne pathogen,which can infect a variety of host crops and cause significant economic losses worldwide.With the deepening of the research on oomycetes,it has been found that RxLR effector can be recognized by R protein of host plants to induce HR response,and also inhibit the defense response of host plants.Therefore,RxLR effector plays a key role in the interaction between oomycete pathogens and host plants.Hundreds of highly diverse RxLR effector genes have been found in the oomycete genome now.Understanding the function and structure of these effectors is of great significance to the study of new strategies for the prevention of P.capsici control.In order to explore the function and pathogenic mechanism of RxLR effector in Phytophthora capsici,LT1534,a standard strain of Phytophthora capsici preserved in our laboratory,was selected as experimental material in this study.Based on the existing theoretical basis and experimental conditions,the effect factor RxLR553394 was selected to study.Firstly,the gene sequence and amino acid sequence of RxLR553394 were screened out by JGI database,and then RxLR553394 was cloned from Phytophthora capsici LT1534.The sequence was aligned with Fungi DB according to the sequencing results.The results were identical,and then bioinformatics analysis of RxLR553394 was carried out.The RxLR553394 was constructed into pBIN-GFP2 expression vector.The function of RxLR553394 was explored by Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana.Western blot was used to analysis protein expression level.The results showed that RxLR553394 could express normally in Nicotiana benthamiana,and couldn’t cause leaf necrosis or inhibit INF1-induced PTI,but could inhibit Bax-induced PCD.After instantaneous expression of RxLR553394 in Nicotiana benthamiana,the subcellular localization of RxLR553394 was observed under confocal microscopy.The results showed that RxLR553394 was localized in the nucleus and cytoplasm.Subsequently,in order to further explore the function of the effector RxLR553394,two silent transformants were obtained by RNA interference technique in Phytophthora capsici.LT1534,no-load transformants and two silent transformants were inoculated into capsici and Nicotiana benthamiana to analyze the biological characteristics and pathogenicity of silent transformants.The results showed that the growth rate and mycelial morphology of silent transformants did not change significantly compared with LT1534 and no-load transformer.The lesions infecting capsici and Nicotiana benthamiana by silent transformants was significantly reduced,indicating that silencing RxLR553394 significantly reduced the pathogenicity of Phytophthora capsici.RxLR553394 was transiently expressed in Nicotiana benthamiana to verify whether the pathogenicity of the silent transformants could be restored.It was found that transient expression of RxLR553394 restored the pathogenicity of the silent transformants.In this study,we found that the function of effector RxLR553394 could inhibit Baxinduced cell death and play a role in the cytoplasm and nucleus.By analyzing the pathogenicity of silent transformants RxLR553394,the ability of silent transformants to infect the host plant was decreased,which indicated that RxLR553394 played a key role in the infection of the host plant.This provided a theoretical basis for the study of the function and pathogenic mechanism of the effector of Phytophthora capsici.