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Deciphering the functions of the outer membrane porin OprBXo involved in virulence, motility, exopolysaccharide production, biofilm formation and stress tolerance in Xanthomonas oryzae pv. oryzae.

ABSTRACT: Xanthomonas oryzae pv. oryzae (Xoo) is a Gram-negative bacterium causing bacterial leaf blight disease in rice. Previously, proteomic analysis has shown that the outer membrane protein B in Xoo (OprBXo) is more abundant in the wildtype strain than is the outer membrane protein 1 in the Xoo (Omp1X) knockout mutant. OprBXo shows high homology with OprB, which has been well characterized as a carbohydrate-selective porin in X. citri ssp. citri and Pseudomonas species. However, the functions of OprBXo in Xoo have not yet been documented. To elucidate the functions of OprBXo, we generated the OprBXo-overexpressing mutant, Xoo(OprBXo), and the knockout mutant, Xoo?oprBXo(EV). We found that the virulence and migration of Xoo(OprBXo), but not Xoo?oprBXo(EV), were markedly reduced in rice. To postulate the mechanisms affected by OprBXo, comparative proteomic analysis was performed. Based on the results of proteomics, we employed diverse phenotypic assays to characterize the functions of OprBXo. Abnormal twitching motility and reduction in swarming motility were observed in Xoo(OprBXo). Moreover, Xoo(OprBXo) decreased, but Xoo?oprBXo(EV) enhanced, exopolysaccharide production and biofilm formation. The chemotactic ability of Xoo?oprBXo(EV) was dramatically lower than that of Xoo(EV) in the presence of glucose and xylose. Xoo(OprBXo) was resistant to sodium dodecylsulphate and hydrogen peroxide, but Xoo?oprBXo(EV) was highly sensitive compared with Xoo(EV). Thus, OprBXo is not only essential for chemotaxis and stress tolerance, but also for motility, biofilm formation and exopolysaccharide production, which may contribute to the virulence of Xoo. These results will lead to new insights into the functions of a sugar-selective porin in Xoo.

SUBMITTER: Bae N

PROVIDER: S-EPMC6638129 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Deciphering the functions of the outer membrane porin OprBXo involved in virulence, motility, exopolysaccharide production, biofilm formation and stress tolerance in Xanthomonas oryzae pv. oryzae.

Bae Nahee N Park Hye-Jee HJ Park Hanbi H Kim Minyoung M Han Sang-Wook SW

Molecular plant pathology 20181022 12

Xanthomonas oryzae pv. oryzae (Xoo) is a Gram-negative bacterium causing bacterial leaf blight disease in rice. Previously, proteomic analysis has shown that the outer membrane protein B in Xoo (OprBXo) is more abundant in the wildtype strain than is the outer membrane protein 1 in the Xoo (Omp1X) knockout mutant. OprBXo shows high homology with OprB, which has been well characterized as a carbohydrate-selective porin in X. citri ssp. citri and Pseudomonas species. However, the functions of OprB ...[more]

PMID: 30073749

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Project description:In Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, there are over 20 genes encoding GGDEF, EAL, and HD-GYP domains, which are potentially involved in the metabolism of second messenger c-di-GMP. In this study, we focused on the characterization of an EAL domain protein, EdpX1. Deletion of the edpX1 gene resulted in a 2-fold increase in the intracellular c-di-GMP levels, which were restored to the wild-type levels in the complemented ΔedpX1(pB-edpX1) strain, demonstrating that EdpX1 is an active phosphodiesterase (PDE) in X. oryzae pv. oryzae. In addition, colorimetric assays further confirmed the PDE activity of EdpX1 by showing that the E153A mutation at the EAL motif strongly reduced its activity. Virulence assays on the leaves of susceptible rice showed that the ΔedpX1 mutant 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 X. oryzae pv. oryzae. We then demonstrated that the ΔedpX1 mutant was defective in secreting exopolysaccharide (EPS) and forming biofilms. The expression of edpX1 in the ΔedpX1 mutant, but not edpX1 E153A, restored the defective phenotypes to near-wild-type levels. In addition, we observed that EdpX1-green fluorescent protein (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 levels, and its EAL domain is necessary and sufficient for its regulation of virulence in X. oryzae pv. oryzae.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 an EAL or HD-GYP domain, respectively. Multiple genes encoding these domains are often found in one bacterial strain. For example, in the genome of X. oryzae pv. oryzae PXO99A, 26 genes encoding proteins containing these domains were identified. Therefore, to fully appreciate the complexity and specificity of c-di-GMP signaling in X. oryzae pv. oryzae, the enzymatic activities and regulatory functions of each GGDEF, EAL, and HD-GYP domain protein need to be elucidated. In this study, we showed that the EAL domain protein EdpX1 is a major PDE to regulate diverse virulence phenotypes through the c-di-GMP signaling pathway.

Dataset Information

Deciphering the functions of the outer membrane porin OprBXo involved in virulence, motility, exopolysaccharide production, biofilm formation and stress tolerance in Xanthomonas oryzae pv. oryzae.

Publications

Deciphering the functions of the outer membrane porin OprBXo involved in virulence, motility, exopolysaccharide production, biofilm formation and stress tolerance in Xanthomonas oryzae pv. oryzae.

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