Function Analysis Of Genes Related To Degradation Of Phenolic Acids In Ralstonia Solanacearum

Phenolic acids,which contain a phenolic ring and an organic carboxylic acid,are abundant in plant kingdom that are involved in structure of plant cell walls.In nature,hydroxycinnamic acids and hydroxybenzoic acids are the largest types of Phenolic acids.Hydroxycinnamic acids are important precursors for lignin biosynthesis and several lines of evidence suggest that hydroxycinnamic acids are involved in the interaction between bacteria and host plants.Salicylic acid,one classic kind of hydroxybenzoic acids,is the main approach that plant respond the bacterium and evoke the plant immune system.In general,phenolic acids are toxic to most bacteria.In order to protect themselves from the toxicity of phenolic acids,many bacterial species express the phenolic acid decarboxylase(PADse,typically as pad C gene product in Bacillus subtilis)to convert phenolic acids into less toxic 4-vinyl and 4-ethyl derivatives.Phenolic acid decarboxylase regulators are a large group of transcriptional regulators that usually promote the PADse-mediated detoxification in many bacterial species.The Pad R(PADse Regulator)regulators have been well characterized to function in various survival processes of detoxification,antibiotic biosynthesis,multidrug resistance,toxin production and carbon catabolism.Ralstonia solanacearum is a gram-negative bacterium which destroys the plant cell wall to extensive proliferate in the xylem that leads to pathogenicity.It reveals that R.solanacearum probably has abilities to overcome the toxicity of phenolic acids and disrupt the plant immune system.Phenolic acids are broadly antimicrobial,whereas the bacteria could protect itself from phenolic acids toxicity by degrading low-concentration phenolic acids.Although no PADse is annotated in numerous genomes of R.solanacearum strains,we characterized two operons,fcs operon and nag operon,which encode enzymes confer R.solanacearum on metabolization of ferulic acid(FA)and salicylic acid(SA),respectively.Moreover,we found a putative phenolic acid decarboxylase regulator(rsp0309)in the genome of R.solanacearum,which perhaps regulate the expression of fcs and nag operons.We measure the growth of R.solanacearum in different concentration of phenolic acids.We found that MIC(minimum inhibitory concentration)of ferulic acid and salicylic acid are 700 ?M and 400 ?M,respectively.And then,we found fcs and nag mutants cannot grow well in mineral medium adding corresponding phenolic acids.Besides,our results indicate the ferulic acid can induce the expression of type III secretion system through Prh A-Prh I/R-Prh J-Hrp G pathway in vitro.These results demonstrated that ferulic acid is not only a carbon source to R.solanacearum,but also an important plant signal.In the absence of rsp0309,the mutant was more sensitive to phenolic acids(SA and FA)than wild-type strains,indicating that Rsp0309 is important to promote detoxification of phenolic acids in R.solanacearum through affected nag and fcs operons.Based that Rsp0309 is a transcriptional factor,we perform the RNA-seq with both wild type and mutant.From the results of RNA-seq,revealed that(1)positively regulates expression of genes for degradation of SA and FA;(2)positively regulates expression of genes encoding type III secretion system(T3SS)and type III effectors(T3Es)both in vitro and in planta;(3)positively regulates expression of flagella,type IV pilus;(4)positively regulates expression of cell wall degradation enzymes(CWDEs).To validate the RNAseq,we constructed a subset of reporter genes to measure the activity of enzymes.The experimental results are consistent with the RNA-seq broadly.All these confirmed that rsp0309 positively regulates expression of a large set of genes involved to infection process,not only the degradation of phenolic acids.So,Rsp0309 plays an important role on interaction between R.solanacearum and plant.