Tomato Immunity And Application Potential Mediated By Exopolygalacturonases PehC Of Ralstonia Solanacearum

Plant bacterial wilt caused by Ralstonia solanacearum is a devastating disease widely distributed in the world.It has a wide host range and can infect more than 200 species of plants in more than 50 families.After infection,the whole plant will wilt and die,and it is very difficult to prevent and control,so it is called the "cancer" of plants.In China,bacterial wilt seriously damages tomato,potato,tobacco and other Solanaceae crops.However,usable disease-resistance genes are still lacking in production.New control strategies and genetic resources for disease resistance breeding against R.solanacearum is in urgent need.Pathogen-associated molecular patterns(PAMPs)recognized by pattern-recognition receptors(PRRs)on plant cell membrane activates host PTI,a first-layer of plant immunity that provides a broad spectrum of stable and durable basal resistance.Currently,tomato’s recognition mechanism towards solanum is largely unknown.The objective of this study is to isolate and identify the potential pathogen-related molecular model of R.solanacearum and analyze the role of this immune response in the process of resistance to bacterial wilt,so as to guide the cultivation of relatively stable and durable resistant varieties and help solve the problem of tomato bacterial wilt in production.It was found that the secreted proteins of R.solanacearum GMI1000 can activate typical PTI immune responses in tomato roots,including inducing extracellular alkalization,triggering ROS outburst,activating MAPKs,and inducing the expression of immunerelated genes and callose accumulation.After further screening and identification by LCMS/MS,the abundance of exopolygalacturonase Peh C was the highest in the active segment of protein spectrum.The p GST-Peh C prokaryotic expression vector was constructed and the fusion protein was purified.It was found that GST-Peh C could trigger the typical PTI immune response after treating tomato roots.Meanwhile,Peh C could trigger the PTI immune response of various Solanaceae plants with specificity in roots,but could not trigger the immune response in leaves.Peh C triggered the immune response of Solanaceae plants depends on the N-terminal sequence and was independent of its enzymatic activity.RNA-seq transcriptome analysis showed that a large number of genes related to disease resistance and signal transduction were up-regulated in tomato roots after Peh C treatment for 1 h.Therefore,Peh C was identified as a novel PAMP in R.solanacearum GMI1000 to activate PTI immune response in tomato roots.There are three kinds of pectin hydrolases encoded by the R.solanacearum genome,and it was showed that endopolygalacturonase Peh A and exopolygalacturonase Peh B did not confer the activity for stimulating tomato immune response.Phylogenetic analysis showed that endopolygalacturonases homologous to Peh A were widely distributed in plant pathogenic bacteria,while exopolygalacturonases homologous to Peh B and Peh C were only found in the genus relella R.solanacearum,and were rare in other phytopathogenic bacteria.Compared with the wild strain,the secreted protein of ?pehc strain significantly lost the ability to induce tomato immunity,but the pathogenicity of the mutant to tomato was slightly decreased and its colonization in tomato xylem was significantly reduced,suggesting that Peh C has multiple functions in R.solanacearum infection.The results showed that Peh C,as an exopolygalacturonase,could further hydrolyze the oligogalacturonides(OGs)produced by Peh A which degrades pectin into galacturonic acid monomer(Gal A).On the one hand,the wild type R.solanacearum strain can grow in minimal medium with Gal A as the only carbon source,and increase the concentration of reducing sugar in xylem for its growth after infection of tomato.However,the ?pehc mutant strain cannot effectively produce reducing sugar after infection.This result can explain the decreased colonization ability of ?pehc mutant in plants.On the other hand,OGs produced by Peh A degradation of cell wall pectin was characterized as a damage-associated molecular patterns(DAMP),which can activate the immune response of tomato,such as ROS burst and extracellular alkalization.After further hydrolysis into Gal A monomer by Peh C,the product loses the ability to activate the immune response.So peh C can help R.solanacearum escape the tomato immune response induced by OGs.In order to clarify the role of tomato immune response triggered by Peh C in antibacterial wilt,purified Peh C protein was used to pretreat tomato seedlings or treat the roots of adult plants by root irrigation and then inoculate the seedlings with GMI1000.Compared with the control group,the colonization amount of GMI1000 in the roots and stems of tomato seedlings in the treatment group was significantly reduced.The incidence and disease index of tomato wilt in pretreatment group were significantly decreased,and the survival rate was significantly increased.Furthermore,tomato transgenic lines stably expressing Peh C protein were constructed by Agrobacterium-mediated transformation of tomato explants.After inoculation with GMI1000,it was found that the incidence and disease index of the transgenic lines were significantly lower than those of the control Moneymaker plants,and the survival rate was also improved.The results showed that Peh C transgenic tomato had enhanced resistance to bcterial wilt.At the same time,the results showed that seed germination,root length and fresh weight of seedlings,and growth state of adult plants were not significantly different from wild-type plants,suggesting that Peh C protein could be used as a potential immune inducer to improve tomato resistance to bacterial wilt.To sum up,our studies have shown that Peh C secreted by GMI1000 is a new PAMP molecule which can be detected by Solanaceae plants.Peh C plays multiple functions in GMI1000 and plant interactions: it can not only help R.solanacearum in carbon source acquisition in the xylem nutrition,but also can help R.solanacearum to avoid DAMP OG to activate immune response.To counteract,tomato acquired the ability to recognize Peh C and induce PTI during evolution,reflecting the co-evolution of pathogen and host plants.