PutA Family Protein Modulates EpsA Through Two Pathways To Control The Pathogenicity Of Ralstonia Solanacearum GMI1000

Ralstonia solanacearum can cause devastating vascular disease in many plants.It usually produces gene mutation,and has a complex regulation network of pathogenicity,making it difficult to control the diseases caused by this pathogen.In order to decrease the loss in agriculture,it is essential for us to deeply study the pathogenic mechanism of R.solanacearum and find out effective methods to prevent it from causing disease.Extracellular polysaccharide(EPS),which has extremely significant research value,is the pivotal pathogenic factor of R.solanacearum.We aim to discover the regulatory mechanism of EPS synthesis-related genes to enhance our understanding on the pathogenesis of this pathogen.In the early stage,we have used Tn5 to construct the mutant library by using the PepsA-lac Z reporter system in R.solanacearum GMI1000.We obtained a new regulator,TpeA(Trifunctional transcriptional regulator;Proline dehydrogenase;regulate EPS;Proline utilization A family protein),which positively regulates the synthesis of EPS and is a kind of Put A family protein.From the transcriptomics profiles,we have selected several regulators,which are positively controlled by TpeA.Overexpression of one of them,Sdg R(Sugar metabolism;Deo R/Glp R-type transcriptional Regulator),could restore the epsA expression of the tpeA mutant to the wild-type level.In this study,we performed functional analysis of the two genes in R.solanacearum GMI1000.We studied their influences on the pathogenicity,and determined their positive regulation of EPS and their regulatory mechanisms on epsA expression.Our results have improved our understanding of the correlation between the two genes in the regulation of EPS synthesis.We further revealed an overlap of a large number of differential genes and several important phenotypes,including biofilm formation and oxidative stress sensitivity,are controlled by both TpeA and Sdg R.The results presented here provide the first evidence that TpeA could control the biosynthesis of EPS,and the expression levels of other target genes by two patterns: The first one,TpeA directly binds to epsA promoter to control the expression of EPS-related gene and achieve the direct regulation of the synthesis of EPS;the second one is indirect regulation,TpeA protein controls the expression of sdgR through directly binding to its promoter,and then Sdg R positively regulates EPS synthesis by directly binding to epsA promoter.Our findings demonstrate that TpeA exerts an important role in the pathogenesis of R.solanacearum besides its function in proline catabolism.In addition,the binding site of TpeA protein on the epsA promoter was determined as CAC TCC GAA GTA GGG AAA CGA AAT G by DNase I footprinting.It was finally predicted that the promoter sequence with the characteristics of AGG NAA ANN AA(N is any nucleotide)is the binding region of TpeA protein.