PhcQ mainly contributes to the regulation of quorum sensing‐dependent genes, in which PhcR is partially involved, in <i>Ralstonia pseudosolanacearum</i> strain OE1‐1

Takemura, Chika; Senuma, Wakana; Hayashi, Kazusa; Minami, Ayaka; Terazawa, Yuki; Kaneoka, Chisaki; Sakata, Megumi; Chen, Min; Zhang, Yong; Nobori, Tatsuya; Sato, Mitsuru; Kiba, Akinori; Ohnishi, Kouhei; Tsuda, Kazuhiko; Kai, Kenji; Hikichi, Yasufumi

doi:10.1111/mpp.13124

Cited by 17 publications

(19 citation statements)

References 50 publications

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“…Similar results were obtained with transcriptome analysis that PhcR is partially involved in the regulation of QS-dependent genes in R. solanacearum strain OE1-1, such as the genes norB , lecM , and xpsR were modulated by PhcR positively, and some flagellin biosynthesis-related genes and chemotaxis-related genes were negatively PhcR-dependent genes ( Takemura et al, 2021 ). In addition, the similar regulatory patterns were also found in modulation of the biosynthesis of ralfuranone, which is a SM and virulence factor produced by R. solanacearum ( Wackler et al, 2011 ), for the ralfuranone coding gene expression and biosynthesis is regulated positively by PhcA and negatively by PhcR ( Takemura et al, 2021 ). However, it is not yet clear whether PhcA and PhcR regulate the ralfuranone gene expression in a direct way similar to what we have found for the regulation of ralsolamycin.…”

Section: Discussionsupporting

confidence: 80%

PhcA and PhcR Regulate Ralsolamycin Biosynthesis Oppositely in Ralstonia solanacearum

Cao

Zhang

et al. 2022

Front. Plant Sci.

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Ralsolamycin, one of secondary metabolites in Ralstonia solanacearum, is known to be involved in crosstalk between R. solanacearum and fungi. Ralsolamycin formation is catalyzed by two-hybrid synthetases of RmyA (non-ribosomal peptide synthetase) and RmyB (polyketide synthase). A methyltransferase PhcB catalyzes formation of 3-OH MAME or 3-OH PAME, signals for the quorum sensing (QS) in R. solanacearum, while PhcB positively modulates ralsolamycin biosynthesis. A two-component system of PhcS and PhcR can response these QS signals and activate phcA expression. Here, we experimentally demonstrated that deletion of phcA (ΔphcA) substantially impaired the ralsolamycin production and expression of rmyA and rmyB in R. solanacearum strain EP1, and failed to induce chlamydospore formation of plant fungal pathogen Fusarium oxysporum f. cubense (stran FOC4). However, deletion of phcR significantly increased ralsolamycin production and expression of rmyA and rmyB, and phcR mutants exhibited enhanced ability to induce chlamydospore formation of FOC4. Results of the electrophoretic mobility shift assay suggested that both PhcA and PhcR bind to promoter of rmy operon. Taken together, these results demonstrated that both PhcA and PhcR bind to promoter of rmy operon, but regulate ralsolamycin biosynthesis in an opposite way. It could extend our knowledge on the sophisticated regulatory networks of ralsolamycin biosynthesis in R. solanacearum.

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Section: Discussionsupporting

confidence: 80%

PhcA and PhcR Regulate Ralsolamycin Biosynthesis Oppositely in Ralstonia solanacearum

Cao

Zhang

et al. 2022

Front. Plant Sci.

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“…The production of EPS I (a major extracellular polysaccharide of RSSC) and LecM (an RS-IIL lectin) is also dependent on the activation of the phc QS system (31, 32, 37). As shown in Pseudomonas aeruginosa (41), EPS and lectin may comprise the extracellular matrix of RSSC cells.…”

Section: Resultsmentioning

confidence: 99%

“…As expected, this bacterium harbors two chitinase genes (RSp0275 and RSp0924), three glucanase genes (cbh-A, egl, and RSc0818), and two lipase (or esterase) genes (RSp0138 and RSp0161) (29). The previous RNA-Seq comparison between OE1-1 and ∆phcB showed that the expression of these genes is under the control of the phc QS system (30)(31)(32). We thus constructed these gene-deletion mutants and evaluated their invasion ability into F. oxysporum chlamydospores.…”

Section: Extracellular Enzymes and Type Ii/iii Secretion Systems Are ...mentioning

confidence: 99%

Quorum sensing-dependent invasion ofRalstonia solanacearumintoFusarium oxysporumchlamydospores

Tsumori¹,

Matsuo

Murai³

et al. 2022

Preprint

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Strains ofRalstonia solanacearumspecies complex (RSSC), though known as the causative agent of bacterial wilt disease in plants, induce the chlamydospores of many fungi species and invade them through the spores. The lipopeptide ralstonins are the chlamydospore inducers produced by RSSC and are essential for this invasion. However, no mechanistic investigation of this interaction has been conducted. In this study, we report that quorum sensing (QS), which is bacterial cell–cell communication, is important for RSSC to invade the fungusFusarium oxysporum(Fo). ∆phcB, a deletion mutant of QS signal synthase, lost the ability to both produce ralstonins and invadeFochlamydospores. The QS signal methyl 3-hydroxymyristate rescued these disabilities. In contrast, exogenous ralstonin A, while inducingFochlamydospores, failed to rescue the invasive ability. Gene-deletion and -complementation experiments revealed that the QS-dependent production of extracellular polysaccharide (EPS I) is essential for this invasion. The RSSC cells adhered toFohyphae and formed biofilms there before inducing chlamydospores. This biofilm formation was not observed in the EPS I- or the ralstonin-deficient mutant. Microscopic analysis showed that RSSC infection resulted in the death ofFochlamydospores. Altogether, we reported that the RSSC QS system is important for this lethal endoparasitism. Among the factors regulated by the QS system, ralstonins, EPS I, and biofilm are important parasitic factors.

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“…We firstly compared the plasmid-based transformation frequencies of three methods, i.e., triparental mating, electroporation and natural transformation, in R. solanacearum . Triparental mating and electroporation are the two conventionally used methods for genetic transformation and gene deletion in RSSC ( Li et al, 2017 ; Corral et al, 2020 ; Takemura et al, 2021 ). Our results showed that natural transformation offered a significantly higher level of transformation frequency than the other two methods using plasmid DNA ( Figure 2A ).…”

Section: Discussionmentioning

confidence: 99%

Markerless gene deletion in Ralstonia solanacearum based on its natural transformation competence

et al. 2022

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Ralstonia solanacearum species complex (RSSC) is a group of Gram-negative bacterial pathogen capable of infecting numerous plants and crops, causing severe vascular wilt diseases. Functional analysis of the genes associated with bacterial virulence is critical for elucidating the molecular mechanisms that govern the bacterial pathogenicity. To this end, an efficient gene deletion method would be of great help. In this study, we set to develop an efficient and simple markerless gene deletion method by exploiting its natural transformation competence and the FLP/FRT recombination system. We found that natural transformation using PCR products provided much higher transformation frequency than the plasmid-based triparental mating and electroporation. We thus generated the gene deletion fusion PCR fragments by incorporating the upstream and downstream DNA fragments of the target gene and an antibiotic resistance gene flanked by FRT sites, and delivered the PCR products into R. solanacearum cells through natural transformation. Using this method, we knocked out the epsB and phcA genes, which are associated with exopolysaccharide (EPS) biosynthesis and regulation, respectively, in several R. solanacearum strains isolated from different host plants at a frequency from 5 (1E-08) to 45 (1E-08). To remove the antibiotic marker gene, the plasmid expressing the FLP enzyme was introduced into the above knockout mutants, which enabled removal of the marker gene. The effective combination of natural transformation and the FLP/FRT recombination system thus offers a simple and efficient method for functional study of putative virulence genes and for elucidation of R. solanacearum pathogenic mechanisms.

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PhcQ mainly contributes to the regulation of quorum sensing‐dependent genes, in which PhcR is partially involved, in Ralstonia pseudosolanacearum strain OE1‐1

Cited by 17 publications

References 50 publications

PhcA and PhcR Regulate Ralsolamycin Biosynthesis Oppositely in Ralstonia solanacearum

PhcA and PhcR Regulate Ralsolamycin Biosynthesis Oppositely in Ralstonia solanacearum

Quorum sensing-dependent invasion ofRalstonia solanacearumintoFusarium oxysporumchlamydospores

Markerless gene deletion in Ralstonia solanacearum based on its natural transformation competence

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