CarAB in Pseudomonas fluorescens SP007s Reduces Symptoms on Soybean Caused by Xanthomonas axonopodis pv. glycines that Links to The Role of Multiple Genes

Abstract

Pseudomonas fluorescens SP007s has been reported to successfully reduce symptoms of pustule disease (BP) caused by Xanthomonas axonopodis pv. glycines (Xag) with its complex metabolites. The possible role of carAB in strain SP007s that encoded carbamoylphosphate synthetase (CPSase) to degrade a diffusible signal factor (DSF) of Xag was assayed in this study. Mutation in carAB resulted in lost ability to produce CPSase and to inhibit DSF of Xag demonstrated by thin layer chromatography (TLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), high performance liquid chromatography (HPLC), and agar plate assay. Mutant SP007scarABaffected multiple gene expression encoding extracellular products that included secondary metabolites associated with biocontrol traits. Mutations in carAB caused a decreased expression of phl, plt, phzE, pch (corresponding to 2,4-DAPG, pyoluteorin, phenazine, and pyochelin biosynthesis), and rpoS; whereas rpoS mutant mediated a decreased production activity of carAB expression suggesting these gene regulation required a functional carAB genes; which the stationary-phase sigma factor, RpoS was also essential for the regulated system of CPSase biosynthesis. Mutants deficient in the biosynthesis of CPSase reduced less-severe symptoms than wildtype on soybean plant, whereas CPSaseoverexpression strain and purified CPSase displayed a better reduction of BP symptoms suggesting the control ability depended CPSase mechanism of SP007s was affected by adequate levels of this enzyme biosynthesis. Mutations in rpoS significantly reduced BP symptom compared to carAB and phl&plt, whereas a double mutant strain deficient in the biosynthesis of antimicrobial metabolites 2,4- DAPG and pyoluteorin was less BP suppression than carAB, suggesting a counteracted mechanism was the evidence of biocontrol by SP007s. Taken together, we demonstrate that carAB is required for CPSase biosynthesis that can degrade DSF of Xag resulting in symptom reduction as one of biocontrol mechanism by SP007s. Induced expression of other related-genes indicates the complexity of carABdependent CPSase biosynthesis which the additional assay remains to be experimentally elucidated.