Study On The Biological Function And Mechanisms Of Ralstonia Solanacearum Type Ⅲ Effector RipAW

Tomato is one of the most important vegetables in our country’s agriculture.R.solanacearum has a wide host range and fast spread,which has great harm to tomato and is difficult to prevent and control.As with most Gram-negative pathogens,type III secretion system is a key structure for bacteria’s pathogenicity.The injected effectors can inhibit plant innate immunity and are very important for the pathogenic process of R.solanacearum.Therefore,it is urgent to study the interaction mechanism between bacterial effectors and plants.RipAW is upregulated during infection,which means RipAW plays an important role in the pathogenic process of R.solanacearum.Therefore,by analyzing the biological function of the effector and demonstrating the pathway of immune response triggered by RipAW,so can we enhance the understanding of the pathogenic process.The main results of this study are as follows:Analysis of the amino acid sequence of RipAW revealed that it contained a typical Novel E3 ligase domain(NEL domain).A vitro ubiquitination assay in E.coli demonstrated that RipAW has self-ubiquitination activity.Agrobacterium tumefaciens-mediated transient expression of RipAW on N.benthamiana leaves can inhibit flg22-triggered ROS in the early stage,and can cause hypersensitive response(HR)of leaves in the late stage.However,after mutating 177th cysteine in the NEL domain,the ability to induce HR and inhibit ROS burst was lost.We further observed RipAW localizes in the cell membrane and the nucleus.HR caused by potential NLS mutat significantly weaker than wild type protein.Construction of the RipAW knockout strain revealed a significant increase in bacterial growth and pathogenicity compared with GMI1000 during infection with tomato and A.thaliana.In order to further study the interaction mechanism between RipAW and plants,we generated transgenic Arabidopsis expressing RipAW,and It was found that shows short plants and curled leaves,and the expression of immune related genes was up-regulated.Transgenic Arabidopsis overexpressing mutants of enzymatically active sites and nuclear localization signal sites could restore the autoimmunity phenotype.The key proteins of plant immune response were selected to screen the signal elements involved in RipAWinduced autoimmunity in Arabidopsis,we overexpressed RipAW in the background of eds1,pad4,ndr1,adr1,nrg1,and helperless mutants,and found that mutations of these signaling elements cannot suppress the autoimmunity phenotype caused by RipAW.Potential target proteins that interact with RipAW in A.thaliana and N.benthamiana were identified by coimmunoprecipitation and protein mass spectrometry,and the candidate proteins that RipAW could degrade were preliminarily verified.In summary,this study found that the R.solanacearum T3SS effector RipAW negatively regulates its pathogenicity,also can trigger immune responses and cell death in a variety of plants,suggesting that there are immune receptors that recognize RipAW in plants.RipAW activates the autoimmunity signaling pathway in A.thaliana,providing new clues for further understanding of the mechanism of plant-pathogen interaction.