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Dissecting the virulence-related functionality and cellular transcription mechanism of a conserved hypothetical protein in Xanthomonas oryzae pv. oryzae.

ABSTRACT: Hypothetical proteins without defined functions are largely distributed in all sequenced bacterial genomes. Understanding their potent functionalities is a basic demand for bacteriologists. Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight of rice, is one of the model systems for the study of molecular plant pathology. One-quarter of proteins in the genome of this bacterium are defined as hypothetical proteins, but their roles in Xoo pathogenicity are unknown. Here, we generated in-frame deletions for six hypothetical proteins selected from strain PXO99A and found that one of them (PXO_03177) is required for the full virulence of this strain. PXO_03177 is conserved in Xanthomonas, and is predicted to contain two domains relating to polysaccharide synthesis. However, we found that mutation of this gene did not affect the production or modification of extracellular polysaccharides (EPSs) and lipopolysaccharides (LPSs), two major polysaccharides produced by Xoo relating to its infection. Interestingly, we found that inactivation of PXO_03177 significantly impaired biofilm formation and tolerance to sodium dodecyl sulfate (SDS), both of which are considered to play key roles during Xoo infection in rice leaves. These findings thus enable us to define a function for PXO_03177 in the virulence of Xoo. Furthermore, we also found that the global regulator Clp controls the transcription of PXO_03177 by direct binding to its promoter region, presenting the first cellular regulatory pathway for the modulation of expression of this hypothetical protein gene. Our results provide reference information for PXO_03177 homologues in Xanthomonas.

SUBMITTER: Wang B

PROVIDER: S-EPMC6638143 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Dissecting the virulence-related functionality and cellular transcription mechanism of a conserved hypothetical protein in Xanthomonas oryzae pv. oryzae.

Wang Bo B Wu Guichun G Zhang Yuqiang Y Qian Guoliang G Liu Fengquan F

Molecular plant pathology 20180202

Hypothetical proteins without defined functions are largely distributed in all sequenced bacterial genomes. Understanding their potent functionalities is a basic demand for bacteriologists. Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight of rice, is one of the model systems for the study of molecular plant pathology. One-quarter of proteins in the genome of this bacterium are defined as hypothetical proteins, but their roles in Xoo pathogenicity are unknown. Here, ...[more]

PMID: 29392817

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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

Dissecting the virulence-related functionality and cellular transcription mechanism of a conserved hypothetical protein in Xanthomonas oryzae pv. oryzae.

Publications

Dissecting the virulence-related functionality and cellular transcription mechanism of a conserved hypothetical protein in Xanthomonas oryzae pv. oryzae.

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