Dataset Information

The RpoN2-PilRX regulatory system governs type IV pilus gene transcription and is required for bacterial motility and virulence in Xanthomonas oryzae pv. oryzae.

ABSTRACT: The type IV pilus (T4P), a special class of bacterial surface filament, plays crucial roles in surface adhesion, motility, biofilm formation, and virulence in pathogenic bacteria. However, the regulatory mechanism of T4P and its relationship to bacterial virulence are still little understood in Xanthomonas oryzae pv. oryzae (Xoo), the causal pathogen of bacterial blight of rice. Our previous studies showed that the ?54 factor RpoN2 regulated bacterial virulence on rice in a flagellum-independent manner in Xoo. In this study, both yeast two-hybrid and pull-down assays revealed that RpoN2 directly and specifically interacted with PilRX, a homolog of the response regulator PilR of the two-component system PilS-PilR in the pilus gene cluster. Genomic sequence and reverse transcription PCR (RT-PCR) analysis showed 13 regulons containing 25 genes encoding T4P structural components and putative regulators. A consensus RpoN2-binding sequence GGN10 GC was identified in the promoter sequences of most T4P gene transcriptional units. Electrophoretic mobility shift assays confirmed the direct binding of RpoN2 to the promoter of the major pilin gene pilAX, the inner membrane platform protein gene pilCX, and pilRX. Promoter activity and quantitative RT-PCR assays demonstrated direct and indirect transcriptional regulation by RpoN2 of the T4P genes. In addition, individual deletions of pilAX, pilCX, and pilRX resulted in significantly reduced twitching and swimming motility, biofilm formation, and virulence in rice. Taken together, the findings from the current study suggest that the RpoN2-PilRX regulatory system controls bacterial motility and virulence by regulating T4P gene transcription in Xoo.

SUBMITTER: Yu C

PROVIDER: S-EPMC7170775 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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The RpoN2-PilRX regulatory system governs type IV pilus gene transcription and is required for bacterial motility and virulence in Xanthomonas oryzae pv. oryzae.

Yu Chao C Nguyen Doan-Phuong DP Ren Zhaoyu Z Liu Jianan J Yang Fenghuan F Tian Fang F Fan Susu S Chen Huamin H

Molecular plant pathology 20200229 5

The type IV pilus (T4P), a special class of bacterial surface filament, plays crucial roles in surface adhesion, motility, biofilm formation, and virulence in pathogenic bacteria. However, the regulatory mechanism of T4P and its relationship to bacterial virulence are still little understood in Xanthomonas oryzae pv. oryzae (Xoo), the causal pathogen of bacterial blight of rice. Our previous studies showed that the σ<sup>54</sup> factor RpoN2 regulated bacterial virulence on rice in a flagellum- ...[more]

PMID: 32112711

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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

The RpoN2-PilRX regulatory system governs type IV pilus gene transcription and is required for bacterial motility and virulence in Xanthomonas oryzae pv. oryzae.

Publications

The RpoN2-PilRX regulatory system governs type IV pilus gene transcription and is required for bacterial motility and virulence in Xanthomonas oryzae pv. oryzae.

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