Preliminary Study On The Function And Mechanism Of Ralstonia Solanacearum Rip67 Interfering With Plant Basal Immunity

In nature,various pathogens can infect host plants and cause plant diseases.Bacterial wilt of plants caused by bacterial pathogen Ralstonia solanacearum is considered as a highly destructive soil-borne plant disease in the world.The pathogenicity of R.solanacearum mainly depends on its large repertoire of type III effectors(T3Es).Analysis the function and mechanism of R.solanacearum T3Es in plant-R.solanacearum interactions is of great theoretical significance for establishing effective prevention and control strategies to vascular bundle diseases like the bacterial wilt.Although some previous studies have demonstrated that a handful of R.solanacearum T3Es can interfere with the physiological metabolism,growth and immunity of host plants,the functions and mechanisms of most R.solanacearum T3Es remain unclear.To this end,Agrobacterium-mediated transient expression approach was employed to analyze the role of9 T3Es from the model strain GMI1000 of R.solanacearum in interfering the plant basal immunity.Then we preliminarily explored the function and mechanism of R.solanacearum Rip67 using transgenic approach,gene knockout,yeast two-hybrid,as well as other strategies.It is expected to lay a theoretical foundation for the analysis of pathogenic mechanisms of pathogens that cause vascular bundle diseases like the bacterial wilt.The detailed results are as follows:1.Identification of R.solanacearum T3Es interfering plant basal immunity using transient gene expression approachFirstly,we cloned 9 effector genes from GMI1000 genome,and evaluated their effect on plant basal immunity using Agrobacterium-mediated transient gene expression method,and identified Rip67 that promotes the infection of Phytophthora parasitica and inhibits flg22-induced ROS burst in Nicotiana benthamiana.Therefore,we conclude that Rip67 is able to interfere with plant basal defense.2.Preliminarily study on function and mechanism of Rip67a)We generated transgenic Arabidopsis thaliana lines expressing Rip67 stably by the floral dip transformation method and evaluated the effect of Rip67 on plant basal immunity.Results showed that Rip67 could inhibit flg22-induced ROS burst and promote the infection of Phytophthora capsici in Arabidopsis thaliana,further indicating that Rip67 plays an important role in plant-pathogen interactions.b)We generated rip67 knockout mutant,and evaluated the effect of Rip67 on the pathogenicity of R.solanacearum by inoculation assay.It was found that delection of Rip67 alone did not reduce the pathogenicity of R.solanacearum significantly,which may be caused by the functional redundancy of T3Es.c)We screened 151 potential interacting proteins from the cDNA library of A.thaliana using the "bait" Rip67,and selected 26 candidate proteins to verify interaction furtherly by yeast two-hybrid system.Consequently,seven proteins interact with Rip67 in Y2 H,and four of them were verified to interact with Rip67 by another approach LCA.Futhermore,functional exploration of interacting proteins demonsrated that DI19-3 could inhibit flg22-induced ROS burst and infection of P.parasitica,DC3000 Δhop Q1-1 as well as CQPS-1 in N.benthamiana.It remains unknown how Rip67 interferes plant basal immunity by interacting with DI19-3.This study is expected to set a basis for the analysis of the mechanism of Rip67,so as to promote the analysis of the pathogenic mechanism of R.solanacearum.