The transcription regulator ChpA affects the global transcriptome including quorum sensing-dependent genes in Ralstonia pseudosolanacearum strain OE1-1.

The gram-negative plant-pathogenic ß-proteobacterium Ralstonia pseudosolanacearum strain OE1-1 produces methyl 3-hydroxymyristate as a quorum sensing (QS) signal through methyltransferase PhcB and senses the chemical via the sensor histidine kinase PhcS. This leads to activation of the LysR family transcription regulator PhcA, which regulates the genes (QS-dependent genes) responsible for QS-dependent phenotypes, including virulence. The transcription regulator ChpA, which possesses a response regulator receiver domain and also a hybrid sensor histidine kinase/response regulator phosphore-acceptor domain but lacks a DNA-binding domain, is reportedly involved in QS-dependent biofilm formation and virulence of R. pseudosolanacearum strain GMI1000. To explore the function of ChpA in QS of OE1-1, we generated a chpA-deletion mutant (ΔchpA) and revealed that the chpA deletion leads to significantly altered QS-dependent phenotypes. Furthermore, ΔchpA exhibited a loss in its infectivity in xylem vessels of tomato plant roots, losing virulence on tomato plants, similar to the phcA-deletion mutant (ΔphcA). Transcriptome analysis showed that the transcript levels of phcB, phcQ, phcR, and phcA in ΔchpA were comparable to those in OE1-1. However, the transcript levels of 89.9% and 88.9% of positively and negatively QS-dependent genes, respectively, were significantly altered in ΔchpA compared with OE1-1. Furthermore, the transcript levels of these genes in ΔchpA were positively correlated with those in ΔphcA. Together, our results suggest that ChpA is involved in the regulation of these QS-dependent genes, thereby contributing to the behaviour in host plant roots and virulence of OE1-1.

ß-1,4-Cellobiohydrolase is involved in full expression of phcA, contributing to the feedback loop in quorum sensing of Ralstonia pseudosolanacearum strain OE1-1.

After infecting roots of tomato plants, the gram-negative bacterium Ralstonia pseudosolanacearum strain OE1-1 activates quorum sensing (QS) to induce production of plant cell wall-degrading enzymes, such as ß-1,4-endoglucanase (Egl) and ß-1,4-cellobiohydrolase (CbhA), via the LysR family transcriptional regulator PhcA and then invades xylem vessels to exhibit virulence. The phcA-deletion mutant (ΔphcA) exhibits neither the ability to infect xylem vessels nor virulence. Compared with strain OE1-1, the egl-deletion mutant (Δegl) exhibits lower cellulose degradation activity, lower infectivity in xylem vessels, and reduced virulence. In this study, we analysed functions of CbhA other than cell wall degradation activity that are involved in the virulence of strain OE1-1. The cbhA-deletion mutant (ΔcbhA) lacked the ability to infect xylem vessels and displayed loss of virulence, similar to ΔphcA, but exhibited less reduced cellulose degradation activity compared with Δegl. Transcriptome analysis revealed that the phcA expression levels in ΔcbhA were significantly lower than in OE1-1, with significantly altered expression of more than 50% of PhcA-regulated genes. Deletion of cbhA led to a significant change in QS-dependent phenotypes, similar to the effects of phcA deletion. Complementation of ΔcbhA with native cbhA or transformation of this mutant with phcA controlled by a constitutive promoter recovered its QS-dependent phenotypes. The expression level of phcA in ΔcbhA-inoculated tomato plants was significantly lower than in strain OE1-1-inoculated plants. Our results collectively suggest that CbhA is involved in the full expression of phcA, thereby contributing to the QS feedback loop and virulence of strain OE1-1.

Complete genome sequence of a novel ophiovirus associated with chlorotic disease of pepper (Capsicum annuum L.) in Japan.

In July 2018, pepper plants (Capsicum annuum L.) with chlorotic leaves and fruits were observed in Kochi prefecture, Japan. High-throughput sequencing (HTS) identified the possible presence of an ophiovirus-like virus possessing three RNA segments in a chlorotic leaf. Using Sanger sequencing with primers designed based on the HTS results and a different source of RNA from the one used for HTS, the complete nucleotide sequences of three RNA segments encoding four proteins on their complementary strand were determined. The amino acid sequences of these four proteins showed similarity to those of the RNA-dependent RNA polymerase, RNA-silencing suppressor protein, movement protein, and coat protein, respectively, of ophioviruses, which are negative-sense single-stranded RNA viruses. However, the coat protein amino acid sequence of the virus found on pepper plants was no more than 61.9% identical to those of any known ophioviruses, which is lower than the species demarcation threshold of 85 %. Hence, we suggest that this virus, which we have named "pepper chlorosis associated virus" (PepCaV) should be considered a member of a new species in the genus Ophiovirus, for which we propose the name "Ophiovirus capsici". The results of phylogenetic analysis using coat protein amino acid sequences of PepCaV and other ophioviruses also supported this conclusion. PepCaV RNA was found to have conserved nucleotide sequences at both the 5' and 3' termini of the different RNA segments, and the conserved terminal nucleotide sequences were predicted to form a self-complementary double-stranded region, resulting in a panhandle structure in each of the genomic RNAs.

The behavior of Ralstonia pseudosolanacearum strain OE1-1 and morphological changes of cells in tomato roots.

The soil-borne Gram-negative ß-proteobacterium Ralstonia solanacearum species complex (RSSC) infects tomato roots through the wounds where secondary roots emerge, infecting xylem vessels. Because it is difficult to observe the behavior of RSSC by a fluorescence-based microscopic approach at high magnification, we have little information on its behavior at the root apexes in tomato roots. To analyze the infection route of a strain of phylotype I of RSSC, R. pseudosolanacearum strain OE1-1, which invades tomato roots through the root apexes, we first developed an in vitro pathosystem using 4 day-old-tomato seedlings without secondary roots co-incubated with the strain OE1-1. The microscopic observation of toluidine blue-stained longitudinal semi-thin resin sections of tomato roots allowed to detect attachment of the strain OE1-1 to surfaces of the meristematic and elongation zones in tomato roots. We then observed colonization of OE1-1 in intercellular spaces between epidermis and cortex in the elongation zone, and a detached epidermis in the elongation zone. Furthermore, we observed cortical and endodermal cells without a nucleus and with the cell membrane pulling away from the cell wall. The strain OE1-1 next invaded cell wall-degenerated cortical cells and formed mushroom-shaped biofilms to progress through intercellular spaces of the cortex and endodermis, infecting pericycle cells and xylem vessels. The deletion of egl encoding ß-1,4-endoglucanase, which is one of quorum sensing (QS)-inducible plant cell wall-degrading enzymes (PCDWEs) secreted via the type II secretion system (T2SS) led to a reduced infectivity in cortical cells. Furthermore, the QS-deficient and T2SS-deficient mutants lost their infectivity in cortical cells and the following infection in xylem vessels. Taking together, infection of OE1-1, which attaches to surfaces of the meristematic and elongation zones, in cortical cells of the elongation zone in tomato roots, dependently on QS-inducible PCDWEs secreted via the T2SS, leads to its subsequent infection in xylem vessels.

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

The gram-negative plant-pathogenic ß-proteobacterium Ralstonia pseudosolanacearum strain OE1-1 produces methyl 3-hydroxymyristate as a quorum sensing (QS) signal via the methyltransferase PhcB and senses the chemical through the sensor histidine kinase PhcS. This leads to functionalization of the LysR family transcriptional regulator PhcA, regulating QS-dependent genes responsible for the QS-dependent phenotypes including virulence. The phc operon consists of phcB, phcS, phcR, and phcQ, with the latter two encoding regulator proteins with a receiver domain and a histidine kinase domain and with a receiver domain, respectively. To elucidate the function of PhcR and PhcQ in the regulation of QS-dependent genes, we generated phcR-deletion and phcQ-deletion mutants. Though the QS-dependent phenotypes of the phcR-deletion mutant were largely unchanged, deletion of phcQ led to a significant change in the QS-dependent phenotypes. Transcriptome analysis coupled with quantitative reverse transcription-PCR and RNA-sequencing revealed that phcB, phcK, and phcA in the phcR-deletion and phcQ-deletion mutants were expressed at similar levels as in strain OE1-1. Compared with strain OE1-1, expression of 22.9% and 26.4% of positively and negatively QS-dependent genes, respectively, was significantly altered in the phcR-deletion mutant. However, expression of 96.8% and 66.9% of positively and negatively QS-dependent genes, respectively, was significantly altered in the phcQ-deletion mutant. Furthermore, a strong positive correlation of expression of these QS-dependent genes was observed between the phcQ-deletion and phcA-deletion mutants. Our results indicate that PhcQ mainly contributes to the regulation of QS-dependent genes, in which PhcR is partially involved.

Quorum Sensing Inhibition Attenuates the Virulence of the Plant Pathogen Ralstonia solanacearum Species Complex.

Strains of Ralstonia solanacearum species complex (RSSC) cause "bacterial wilt" on a wide range of plant species and thus lead to marked economic losses in agriculture. Quorum sensing (QS), a bacterial cell-cell communication mechanism, controls the virulence of RSSC strains by regulating the production of extracellular polysaccharide (EPS) and secondary metabolites, biofilm formation, and cellular motility. R. solanacearum strain OE1-1 employs (R)-methyl 3-hydroxymyristate (3-OH MAME) as a QS signal, which is synthesized by the PhcB methyltransferase and sensed by the PhcS/PhcRQ two-component system. We describe the design, synthesis, and biological evaluation of inhibitors of the phc QS system. Initial screening of a small set of QS signal analogues revealed that methyl 3-hydroxy-8-phenyloctanoate, named, PQI-1 (phc quorum sensing inhibitor-1), inhibited biofilm formation by strain OE1-1. To improve its inhibitory activity, the derivatives of PQI-1 were synthesized, and their QS inhibition activities were evaluated. PQIs-2-5 evolved from PQI-1 more strongly inhibited not only biofilm formation but also the production of ralfuranone and EPS. Furthermore, RNA-Seq analysis revealed that the PQIs effectively inhibited QS-dependent gene expression and repression in strain OE1-1. On the other hand, the PQIs did not affect the canonical QS systems of the representative reporter bacteria. These antagonists, especially PQI-5, reduced wilting symptoms of the tomato plants infected with strain OE1-1. Taken together, we suggest that targeting the phc QS system has potential for the development of chemicals that protect agricultural crops from bacterial wilt disease.

The putative sensor histidine kinase PhcK is required for the full expression of phcA encoding the global transcriptional regulator to drive the quorum-sensing circuit of Ralstonia solanacearum strain OE1-1.

A gram-negative plant-pathogenic bacterium Ralstonia solanacearum strain OE1-1 produces and extracellularly secretes methyl 3-hydroxymyristate (3-OH MAME), and senses the chemical as a quorum-sensing (QS) signal, activating QS. During QS a functional global transcriptional regulator PhcA, through the 3-OH MAME-dependent two-component system, induces the production of virulence factors including a major extracellular polysaccharide EPS I and ralfuranone. To elucidate the mechanisms of phcA regulation underlying the QS system, among Tn5-mutants from the strain OE1-1, we identified a mutant of RSc1351 gene (phcK), encoding a putative sensor histidine kinase, that exhibited significantly decreased QS-dependent cell aggregation. We generated a phcK-deletion mutant (ΔphcK) that produced significantly less EPS I and ralfuranone than the wild-type strain OE1-1. Quantitative reverse transcription PCR assays showed that the phcA expression level was significantly down-regulated in the ΔphcK mutant but not in other QS mutants. The transcriptome data generated with RNA sequencing technology revealed that the expression levels of 88.2% of the PhcA-positively regulated genes were down-regulated in the ΔphcK mutant, whereas the expression levels of 85.9% of the PhcA-negatively regulated genes were up-regulated. Additionally, the native phcK-expressing complemented ΔphcK strain and the ΔphcK mutant transformed with phcA controlled by a constitutive promoter recovered their cell aggregation phenotypes. Considered together, the results of this study indicate that phcK is required for full phcA expression, thereby driving the QS circuit of R. solanacearum strain OE1-1. This is the first report of the phcA transcriptional regulation of R. solanacearum.