Screening Of Phytophthora Capsici RxLR Effectors And Functional Study Of RxLR23

In the process of Phytophthora capsici infection,pathogens can secrete a large number of RxLR effector molecules interfering with or destroying the host plant disease resistance by cooperation and functional interactions.Therefore,studying the function and protein structure of the RxLR effector is important to understand the pathogenesis of Phytophthora capsici and effective control to the infection of Phytophthora capsici.In this study,we studied the Rx LR effector combined with the relevant research foundation by using the SD33 strain of Phytophthora capsici,which was collected and isolated in our laboratory.The experimental results are as follows:1.The nucleotide and amino acid sequences of the Rx LR effector genes were screened from the genome database of Phytophthora capsici in JGI.Sequence alignment was performed by NCBI to determine which gene sequence will be screened.The Rx LR effectors were cloned from the Phytophthora capsici strain SD33 and analyzed through bioinformatics.2.Functional study of Rx LR effectors of Phytophthora capsici was performed by using pBIN-GFP2 expression vector.Four candidate effectors was transiently expressed in Nicotiana benthamiana.And it was found that the RxLR23 could induce cell death,whereas Rx LR129759 can inhibit Bax-induced cell death in Nicotiana benthamiana.3.In order to further study the function of RxLR effector molecules,four RxLR effector molecules were transiently expressed in Nicotiana benthamiana leaves and the subcellular localization of the effectors was observed by confocal microscopy.It was found that Rx LR23,Rx LR97196 and Rx LR129759 were localized in the nucleus and cytoplasm,while Rx LR101012 was located in the nucleus.4.According to the three-dimensional structure of the protein RxLR23,which was previously obtained from our laboratory,three conserved W domains of C-terminal were truncated to different degrees.Transient expression of truncated Rx LR23 in leaves of Nicotiana benthamiana using p BIN-GFP2 vector showed that there is no significant difference in the effect of three W domains on triggering ETI.5.Knockout of the effector gene Rx LR23 in Phytophthora capsici SD33.In order to further study the function of RxLR23 in Phytophthora capsici,the disruption and replacement experiment was carried out on the RxLR23 in Phytophthora capsici SD33 by CRISPR/Cas9 technique to obtain knockout transformants.Biological character analysis and pathogenicity identification of knockout transformants of Rx LR23.The results showed that knockout of Rx LR23 reduced the pathogenicity of Phytophthora capsici,but its effect on the growth of Phytophthora capsici and the distribution of nuclei in hyphae are not significant.Through this study,we found that the function of Rx LR effector molecules have the characteristics of diversity,including induction of cell death and suppression of Bax-induced cell death.Different effectors play a role in different locations of the host cell.The three conserved W domains of Rx LR23 have no significantly different in triggering ETI.The pathogenicity of the transformants was reduced by the pathogenicity identification assay of the Rx LR23 knockout transformants,indicating that Rx LR23 has a toxic function during infection.This study provides a theoretical basis for further understanding the role of effectors in the process of pathogen infection.