Phosphodiesterase EdpX1 promotes virulence, exopolysaccharide production and biofilm formation in Xanthomonas oryzae pv. oryzae

In Xanthomonas oryzae pv. oryzae (Xoo), the bacterial blight pathogen of rice, there are over twenty genes encoding GGDEF, EAL or HD-GYP domains, which are potentially involved in the metabolism of second messenger c-di-GMP. In this study, we focused on characterization of an EAL-domain protein EdpX1. Deletion of edpX1 resulted in a two-fold increase of the intracellular c-di-GMP levels, which was restored to the wild-type levels in the complemented strain Δ edpX1 (pB- edpX1 ), demonstrating EdpX1 is an active phosphodiesterase (PDE) in Xoo. In addition, colorimetric assays further confirmed the PDE activity of EdpX1 by showing that mutation of E153A at the EAL motif strongly reduced its activity. Virulence assays on the leaves of susceptible rice showed that Δ edpX1 was severely impaired in causing disease symptoms. In trans expression of wild-type edpX1 , but not edpX1 E153A , was able to complement the weakened virulence phenotype. These results indicated that an active EAL domain is required for EdpX1 to regulate the virulence of Xoo. We then demonstrated that Δ edpX1 was defective in secreting exopolysaccharide (EPS) and forming biofilm. Expression of edpX1 in Δ edpX1 , but not edpX1 E153A restored the defective phenotypes to near wild-type level. In addition, we observed that EdpX1-GFP exhibited multiple subcellular localization foci, and this pattern was dependent on its transmembrane (TM) region, which did not seem to directly contribute to the regulatory function of EdpX1. Thus, we concluded that EdpX1 exhibits PDE activity to control c-di-GMP level, and its EAL domain is necessary and sufficient for its regulation on virulence in Xoo. IMPORTANCE Bacteria utilize c-di-GMP as a second messenger to regulate various biological functions. The synthesis and degradation of c-di-GMP are catalyzed by GGDEF domains and EAL or HD-GYP domains, respectively. Multiple genes encoding these domains are often found in one bacterial strain. For example, in the genome of Xoo PXO99 A , 26 genes encoding proteins containing these domains were identified. Therefore, to fully appreciate the complexity and specificity of c-di-GMP signaling in Xoo, the enzymatic activities and regulatory functions of each GGDEF, EAL and HD-GYP domain proteins need to be elucidated. In this study, we showed the EAL-domain protein EdpX1 is a major PDE to regulate diverse virulence phenotypes through the c-di-GMP signaling pathway.