Functional Characterization Of Genes Related To The Pathogenicity Of Xanthomonas Axonopodis Pv.Glycines

Xanthomonas axonopodis pv.glycines(Xag)is one of the six major pathogenic bacteria of Xanthomonas that limits soybean production.Phosphoglycerate kinase(Pgk),an important metabolic enzyme in both pentose phosphate and Entner-doudoroff(ED)pathways,reversibly catalyzes 1,3-diphosphoglyceric acid to 3-glyceraldehyde.In this study,we showed that the pgk mutation significantly weakened the ability of Xag in acquiring sucrose,glucose,fructose,mannose and galactose.In addition,the pgk mutation resulted in significant reduction in ATP production,EPS biosynthesis,biofilm formation as well as in cell motility.As a result,pgk mutation reduced the virulence and growth of the mutant on soybean leaves.The mutant phenotypes could only be partially restored by the complementation with a wildtype pgk,but could not be rescued by the global regulator(Clp).qRT-PCR results showed that the six different sugars mentioned above could significantly induce the expression of pgk by over 10 folds relative to non-sugar condition.The clp mutation significantly reduced EPS biosynthesis,biofilm formation and cell motility,whereas overexpression of clp both in wild type Xag and clp mutant could greatly enhance in the cell motility.These results indicate that Clp plays a negative role in EPS synthesis and biofilm formation but a positive role in cell motility.We also provided evidence that Clp positively regulated the activities of cellulase,endo mannanase and amylase in Xag.The qRT-PCR results showed that clp mutation significantly inhibited the expression of gumB and gumC,but significantly induced the expression of other gum genes in the gum cluster.Both Rpf and HrpX/HrpG cascades positively regulated the expression of clp.Furthermore,bacterial infection experiments revealed that clp was essential in pathogenicity and reproduction of Xag on the soybean plants.Lastly,we found that clp played a positive role in inducing hypersensitive response(HR)in nonhost N.benthamiana plant.Together,these results indicated that Pgk and Clp are virulent factors,which are necessary for the pathogenicity and virulence in Xag.