The quorum-sensing (QS) system plays a crucial role in regulating the virulence of the Ralstonia solanacearum species complex (RSSC). It is controlled by the phcBSR operon and phcA genes. PhcQ may contribute to the activation of PhcA and regulates the expression of QS-dependent genes. Here, we genetically demonstrate that PhcQ functions as a key component of the QS regulatory pathway by maintaining the protein stability of PhcA in R. pseudosolanacearum OE1-1. Deletion of phcQ significantly increased the expression of genes encoding the type III secretion system but decreased the expression of genes for the synthesis of exopolysaccharide, consistent with that of PhcA. The phcQ mutants exhibited enhanced growth at early stages and retained weak virulence on tobacco plants, although they are nonvirulent on tomato plants. Using an overexpression system, we positioned PhcQ downstream of PhcB and upstream of PhcA in the QS regulatory cascade. PhcQ did not affect phcA transcription, while proteomic analysis and western blot revealed that PhcQ maintains PhcA protein stability, with PhcA protein levels reduced to approximately 41% in phcQ mutants. Further structural analysis revealed a disordered tail within the C-terminus of PhcA that is dispensable for PhcA function yet critical for protein stability. Using bimolecular fluorescence complementation and GST pull-down assay, we demonstrated that PhcQ binds directly to PhcA both in vitro and in planta, and deletion of this C-terminus tail impaired their binding affinity. These findings reveal a novel regulatory mechanism where PhcQ binds to PhcA and maintains its protein stability to ensure proper QS-dependent gene regulation in RSSC.

During the quorum-sensing (QS)-active state, the Gram-negative phytopathogenic Ralstonia pseudosolanacearum strain OE1-1 activates the transcriptional regulator PhcA, regulating the QS-dependent genes including ralfuranone production-related genes such as ralA and major exopolysaccharide EPS I production-related genes such as xpsR encoding the transcriptional regulator XpsR, which are responsible for OE1-1 virulence. Ralfuranone affects the regulation of more than 80% (QS/Ral-dependent genes) of QS-dependent genes, indicating the ralfuranone-mediated feedback regulation of QS. To elucidate the mechanisms underlying the regulation of QS/Ral-dependent genes, we analyzed the transcriptomes of phcA-deletion (ΔphcA) and ralfuranone-deficient (ΔralA) mutants, as well as strain OE1-1 by RNA-sequencing. We found a novel TetR/AcrR family transcriptional regulator (RalT)-encoding gene (ralT); the expression level of ralT reduced significantly in ΔralA but not ΔphcA relative to expression level in strain OE1-1, and RalT negatively regulated ralT. A transcriptome analysis of the ralT-deletion mutant (ΔralT) showed that the ralT-deletion reduced the expression levels of 89.4% of positively QS/Ral-dependent genes including ralA and xpsR, while enhanced 44.6% of negatively QS/Ral-dependent genes. The transcript levels of these genes were positively correlated between ΔralT and ΔphcA or ΔralA, suggesting contribution of RalT to the regulation of some QS/Ral-dependent genes. However, the ralT-deletion enhanced EPS I production, suggesting that RalT represses the XpsR-independent factor(s), which is regulated PhcA and contributes to the EPS I production. Furthermore, ΔralT exhibited enhanced virulence, compared with strain OE1-1. Collectively, results of the present genetic study suggest that RalT contributes to the exquisite fine-tuning of OE1-1 virulence.

ABSTRACT Soil-borne Gram-negative bacteria in the Ralstonia solanacearum species complex (RSSC) cause bacterial wilt symptoms in diverse crop plants. Although iron is an important metal ion for most organisms, the regulatory effect of iron on soil-borne RSSC gene expression remains unknown. Thus, we identified two ferric uptake regulator (Fur) proteins, Fur1 and Fur2, in R. pseudosolanacearum strain OE1-1. All four RSSC phylotype strains had two Fur homologs, a conserved Fur (Fur1) and an alternative Fur (Fur2). We also analyzed the transcriptomes of a fur1 deletion mutant, fur2 deletion mutant, and fur1⁄fur2 double deletion mutant, revealing that Fur1 and Fur2 cooperatively repress siderophore-related gene expression under Fe2+-rich conditions. Furthermore, extracellular Fe3+-chelating activity was cooperatively controlled by Fur1 and Fur2 under Fe2+-rich conditions. Additionally, nitrate metabolism-related gene expression levels were upregulated only in the fur1⁄fur2 double deletion mutant under Fe2+-rich conditions. This double deletion mutant had a lower growth level than either single deletion mutant. Virulence assays involving tomato plants revealed that single deletions (fur1 or fur2) moderately decreased virulence, whereas the double deletion (fur1 and fur2) resulted in a significant decrease in virulence. Considered together, these results suggest that the two Fur homologs cooperatively regulate gene expression under Fe2+-rich conditions to control strain OE1-1 iron uptake, growth, and virulence.IMPORTANCEThe Ralstonia solanacearum species complex (RSSC) comprising soil-borne Gram-negative phytopathogenic bacteria causes bacterial wilt diseases of diverse crop plants. Considering that phylotype I strain OE1-1 enters iron-rich roots from iron-deficient soil during an infection of tomato plants, the mechanisms controlling strain OE1-1 gene expression in response to extracellular iron levels should be clarified. In this study, RSSC was revealed to have two ferric uptake regulator homologs (Fur1 and Fur2). Notably, Fur1 and Fur2 cooperatively repress the expression of genes related to siderophores (Fe3+-chelating compounds) as well as the extracellular Fe3+-chelating activity in the presence of sufficient amounts of extracellular Fe2+. Additionally, Fur1 and Fur2 contribute to the virulence of strain OE1-1 in tomato plants. These findings suggest that RSSC uses two Fur proteins to modulate extracellular Fe3+-chelating activities in response to extracellular iron levels to maintain virulence in crop plants.

In the quorum sensing-active state, the phytopathogenic Ralstonia pseudosolanacearum strain OE1-1 activates the 347-amino acid LysR-type transcriptional regulator PhcA, which regulates PhcA-regulated genes responsible for its virulence. To elucidate the functional role of the PhcA C-terminus, the phcA-deletion strain was transformed with a truncated phcA encoding PhcA lacking only the 20 C-terminal amino acids or a frameshift-mutated phcA carrying a single adenine deletion at position 983. The frameshift-mutation but not truncation of PhcA C-terminus affected the regulation of PhcA-regulated genes, losing virulence. These results indicate that the frameshift-derived amino acid additions at the C-terminus impair PhcA’s regulatory function.

The soil-borne Gram-negative beta-proteobacterium Ralstonia solanacearum species complex (RSSC) causes bacterial wilt, a devastating plant disease that threatens crop production and food security worldwide. In this review, we first summarize current knowledge of RSSC pathogenicity and virulence, focusing on the factors that determine its ability to infect and cause disease, including advances in resistance breeding and the genetic basis of host resistance to bacterial wilt. Next, we highlight the questions provided from the previous studies and describe our recent studies, which revealed that the phc QS network is a highly complex regulatory system that dominates global gene expression and finely tunes RSSC virulence throughout the infection process, from root epidermis invasion to colonization of xylem vessels. Finally, we identify key knowledge gaps and discuss future research directions and practical strategies for the effective management of bacterial wilt.

Lipid transfer proteins (LTPs) are small cysteine-rich soluble proteins that affect flower and seed development, cuticular wax deposition, and biotic and abiotic stress responses. We isolated an LTP-encoding gene homologous to LTPVAS in Nicotiana benthamiana and designated it LTP-VASCULAR TISSUE SIZE (NbLTPVAS). This gene was expressed in seeds, leaves, roots, and stems. Additionally, NbLTPVAS expression was induced by hypersensitive response (HR)-inducing agents. Cell death was accelerated and the phytopathogenic bacterial population decreased significantly in NbLTPVAS-silenced plants infected with the incompatible Ralstonia solanacearum strain 8107. The expression of HR marker gene hin1 in NbLTPVAS-silenced plants was markedly induced by R. solanacearum 8107, indicative of the acceleration of HR. HR cell death in NbLTPVAS-silenced plants was also promoted by the Agrobacterium-mediated expression of HR-inducing proteins including INF1, AvrA, and PopP1. Excessive production of reactive oxygen species (ROS) was detected in NbLTPVAS-silenced plants. The expression of NbrbohB (encoding a ROS-generating enzyme) also increased in NbLTPVAS-silenced plants, but the expression of the antioxidant enzyme-encoding genes NbSOD and NbAPX decreased. The silencing of both NbLTPVAS and NbrbohB adversely affected HR induction. Moreover, NbLTPVAS was secreted into the intercellular washing fluid. The transient expression of the full-length NbLTPVAS induced the expression of antioxidant genes, attenuated ROS production, and suppressed the induction of HR cell death. This is the first functional analysis of LTPVAS in plant-microbe interactions. Our study provides novel insights into the role of NbLTPVAS as a negative regulator of HR via ROS homeostasis in N. benthamiana.

The type III secretion system (T3SS) is essential for Ralstonia pseudosolanacearum to infect host plants. Transcription of the T3SS genes was not activated in nutrient-rich casamino acid-peptone-glucose (CPG) broth, but activated in a minimal medium that might mimic metabolic signals of the host plant. CPG broth at 5

Ralstonia pseudosolanacearum strain OE1-1 secretes methyl 3-hydroxymyristate (3-OH MAME) as a quorum-sensing (QS) signal. Strain OE1-1 senses the chemical by the sensor histidine kinase PhcS, leading to the activation of the LysR family transcriptional regulator PhcA. The activated PhcA controls the expression of QS-dependent genes responsible for QS-regulated phenotypes including virulence. The autophosphorylation of the histidine at amino acid position 230 (H230-PhcS) in PhcS following the 3-OH MAME sensing is required for the PhcA activation. The alternative sensor histidine kinase PhcK is involved in the regulation of phcA, which is independent of 3-OH MAME sensing. Furthermore, the H230Q-PhcS substitution of H230-PhcS with glutamine significantly decreases phcA expression. However, how PhcK and PhcS regulate phcA expression remains unclear. To elucidate the mechanisms of the phcA regulation, we generated a phcK mutant with the H205Q-PhcK substitution of autophosphorylated histidine at amino acid position 205 of PhcK with glutamine. A transcriptome analysis using quantitative real-time polymerase chain reaction assay and RNA sequencing showed that the H230Q-PhcS substitution, but not the H205Q-PhcK substitution, significantly decreased the expression level of phcA. The H230Q-PhcS substitution led to significant changes in the expression levels of QS-dependent genes and a loss of virulence, similar to phcA or phcK deletion. It is thus thought that PhcS participates in not only the 3-OH MAME sensing-independently PhcK-mediated regulation of phcA but also the PhcA activation following 3-OH MAME sensing. Both functions of PhcS are significantly influenced by the autophosphorylation of H230-PhcS. IMPORTANCE:The soil-borne Ralstonia solanacearum species complex (RSSC) infects more than 300 plant species in over 50 families, including solanaceous plants, causing the devastating wilt disease that substantially decreases agricultural production worldwide. The cell density-dependent gene regulation system, QS, is required for RSSC virulence and involves two signaling pathways for the induction and activation of PhcA, which is the master transcriptional regulator in QS. In the present study, we describe the contribution of sensor histidine kinase PhcS to the PhcA induction, along with the alternative sensor kinase PhcK, independently of the sensing of QS signal methyl 3-hydroxymyristate in a phylotype I strain of RSSC, R. pseudosolanacearum strain OE1-1. This study further expands our knowledge of multiple networks, suggesting that several PhcS-mediated two-component systems are likely necessary for RSSC QS and virulence.

ABSTRACT Genes encoding a type III secretion system in Ralstonia pseudosolanacearum are regulated by HrpB as an hrp regulon and induced only in plants. This study aimed to identify the plant signals that induce the hrp regulon and confirm the signal recognition mechanism. Signaling molecules that induce hrpB expression were screened using resting cells of the hrpB-lacZ reporter strain. Only the soluble fraction of smashed tobacco seedlings induced hrpB expression. The heated soluble fraction retained its hrpB -inducing activity, indicating that the signaling molecules were not proteins. When the soluble fraction was fractionated into acidic, neutral, and basic components, both the acidic and neutral fractions induced hrpB expression. As neutral compounds, sucrose, glucose, and fructose have been found to induce hrpB expression. Sucrose-induced hrpB expression was greatly reduced in the prhA mutant, indicating that the TonB-dependent receptor PrhA perceives sugars. Among the organic acids found in the acidic fractions, malic acid most efficiently induced hrpB expression, which was reduced by the mutation of a hybrid histidine kinase gene of a two-component system, rsc1598 , indicating that Rsc1598 may sense malic acid. We demonstrated direct binding of Rsc1598 to malic acid using isothermal titration calorimetry. IMPORTANCE Similar to other Gram-negative plant pathogens, the type III secretion system (T3SS) is the most important virulence factor in Ralstonia pseudosolanacearum . The genes for the T3SS are regulated as an hrp regulon, activated only when the pathogen encounters the plants, indicating that the pathogen must sense plant signals. For the first time, we identified two signaling compounds, sucrose and malic acid, that are abundantly found in tobacco roots. The hrp operon was induced even in non-host plants, possibly because sucrose and malic acid are common in plants. We also found that R. pseudosolanacearum membrane proteins received sucrose and malic acid independently. As a next step, antagonists of signaling molecules can be screened.

Ralstonia solanacearum species complex (RSSC) shows a broad host range and is classified into four phylotypes. To compare type III effectors, we have determined the complete genome sequences of several RSSC strains, especially phylotype-I strains isolated in Japan, with different host specificity.

The soil-borne phytopathogenic gram-negative bacterium Ralstonia solanacearum species complex (RSSC) produces staphyloferrin B and micacocidin as siderophores that scavenge for trivalent iron (Fe3+) in the environment, depending on the intracellular divalent iron (Fe2+) concentration. The staphyloferrin B-deficient mutant reportedly retains its virulence, but the relationship between micacocidin and virulence remains unconfirmed. To elucidate the effect of micacocidin on RSSC virulence, we generated the micacocidin productivity-deficient mutant (ΔRSc1806) that lacks RSc1806, which encodes a putative polyketide synthase/non-ribosomal peptide synthetase, using the RSSC phylotype I Ralstonia pseudosolanacearum strain OE1-1. When incubated in the condition without Fe2+, ΔRSc1806 showed significantly lower Fe3+-scavenging activity, compared with OE1-1. Until 8 days after inoculation on tomato plants, ΔRSc1806 was not virulent, similar to the mutant (ΔphcA) missing phcA, which encodes the LysR-type transcriptional regulator PhcA that regulates the expression of the genes responsible for quorum sensing (QS)-dependent phenotypes including virulence. The transcriptome analysis revealed that RSc1806 deletion significantly altered the expression of more than 80% of the PhcA-regulated genes in the mutant grown in medium with or without Fe2+. Among the PhcA-regulated genes, the transcript levels of the genes whose expression was affected by the deletion of RSc1806 were strongly and positively correlated between the ΔRSc1806 and the phcA-deletion mutant. Furthermore, the deletion of RSc1806 significantly modified QS-dependent phenotypes, similar to the effects of the deletion of phcA. Collectively, our findings suggest that the deletion of micacocidin production-related RSc1806 alters the regulation of PhcA-regulated genes responsible for QS-dependent phenotypes including virulence as well as Fe3+-scavenging activity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The soilborne Gram-negative phytopathogenic beta-proteobacterium Ralstonia pseudosolanacearum strain OE1-1 produces methyl 3-hydroxymyristate (3-OH MAME) as the quorum sensing (QS) signal by the methyltransferase PhcB and senses the chemical, activating the LysR family transcriptional regulator PhcA, which regulates the QS-dependent genes responsible for QS-dependent phenotypes including virulence. The sensor histidine kinases PhcS and VsrA are reportedly involved in the regulation of QS-dependent genes. To elucidate the function of PhcS and VsrA in the active QS, we generated the phcS-deletion and vsrA-deletion mutants, which exhibited weak changes to their QS-dependent phenotypes including virulence. The phcS and vsrA-deletion mutant (ΔphcS/vsrA) had significant changes in its QS-dependent phenotypes and was nonvirulent, similar to the phcA-deletion mutant. The mutant (PhcS-H230Q) with a substitution of histidine to glutamine at amino acid position 230 in PhcS but not the mutant (VsrA-H256Q) with a substitution of histidine to glutamine at amino acid position 256 in VsrA exhibited significant changes in QS-dependent phenotypes and lost virulence. The transcriptome analysis with RNA-sequencing revealed significant alterations to the expression of QS-dependent genes in the ΔphcS/vsrA and PhcS-H230Q but not VsrA-H256Q, similar to the phcA-deletion mutant. The exogenous 3-OH MAME application led to a significantly enhanced QS-inducible major exopolysaccharide EPS I production of the strain OE1-1 and phcB-deletion mutant but not ΔphcS/vsrA and PhcS-H230Q. Collectively, results of the present genetic study suggested that PhcS contributes to QS along with VsrA and that histidine at amino acid position 230 of PhcS is required for 3-OH MAME sensing, thereby influencing QS-dependent phenotypes including virulence of the strain OE1-1. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Raslonia solanacearum species complex (RSSC) contains a relatively large number of T3 effectors (T3Es), typically ranging from 60 to 75. There are genetic and functional redundancies among the different T3Es. This redundancy complicates the functional characterization of individual effectors. Ralstonia pseudosolanacearum strain OE1-1, belonging to RSSC phylotype-I, contains 70 T3E candidates. Fifty T3Es were deleted from OE1-1, and the resultant poly-mutant strain, OE1-1D50E, was used as an effector-depleted mutant. Twenty-one conserved T3E genes with the promoter region of OE1-1 were integrated into the OE1-1 glmS locus to construct strains with a reintroduced single T3E. These strains infiltrated eggplant and tobacco leaves, and several phenotypes were observed. None of the strains caused diseases on Nicotiana benthamiana as OE1-1D50E. Several single-T3E-reintroduced strains multiplied less than the effector-depleted strain in the leaves of eggplant and N. tabacum. The ripA5-expressing strain caused electrolyte leakage from infiltrated eggplant and N. tabacum leaves. A large amount of electrolytes leaked from N. tabacum leaves with the ripAM-expressing strain inoculation. Reactive oxygen species (ROS) production was observed in N. benthamiana leaves inoculated with the ripA5-expressing strain. These results indicate that RipA5 and RipAM of R. pseudosolanacearum are primarily involved in the induction of the host plant defense system.

Phospholipid signaling plays an important role in Nicotiana benthamiana immune responses to phytopathogenic bacteria. In this study, we isolated three orthologs encoding class II diacylglycerol kinase (NbDGK4-1, NbDGK4-2 and NbDGK7) in the N. benthamiana genome. In contrast to the undetectable expression of NbDGK4-2, the NbDGK4-1 and NbDGK7 expression levels increased in response to Ralstonia solanacearum and the flg22 peptide. The induction of the hypersensitive response was not affected, but bacterial growth increased and the onset of bacterial wilt symptoms was accelerated in the NbDGK4-1-silenced and NbDGK7-silenced plants challenged with R. solanacearum. The expression levels of NbPR-1 and NbPR-4 (marker genes for salicylic acid and jasmonic acid signaling, respectively) decreased in the NbDGK4-1-silenced and NbDGK7-silenced plants inoculated with R. solanacearum. The expression of pathogen-associated molecular pattern-triggered immunity (PTI) marker genes were also inhibited in the NbDGK4-1-silenced and NbDGK7-silenced plants infiltrated with the type III secretion system (T3SS)-deficient R. solanacearum mutant or flg22. Accordingly, the T3SS-deficient R. solanacearum bacterial population increased in the NbDGK4-1-silenced and NbDGK7-silenced plants. Moreover, flg22-induced resistance and callose deposition were also compromised in the NbDGK4-1-silenced and NbDGK7-silenced plants. Considered together, the study results indicate class II diacylglycerol kinases may be important for N. benthamiana basal disease resistance (e.g., PTI responses).

Pathogen genetic diversity varies in response to environmental changes. However, it remains unclear whether plant barriers to invasion could be considered a genetic bottleneck for phytopathogen populations. Here, we implement a barcoding approach to generate a pool of 90 isogenic and individually barcoded Ralstonia solanacearum strains. We used 90 of these strains to inoculate tomato plants with different degrees of physical permeability to invasion (intact roots, wounded roots and xylem inoculation) and quantify the phytopathogen population dynamics during invasion. Our results reveal that the permeability of plant roots impacts the degree of population bottleneck, genetic diversity, and composition of Ralstonia populations. We also find that selection is the main driver structuring pathogen populations when barriers to infection are less permeable, i.e., intact roots, the removal of root physical and immune barriers results in the predominance of stochasticity in population assembly. Taken together, our study suggests that plant root permeability constitutes a bottleneck for phytopathogen invasion and genetic diversity.

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 β-1,4-endoglucanase (Egl) and β-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.

The dUTPase gene of herpes simplex virus has been identified using a novel approach. The upstream regulatory sequences, or the promoter and upstream regulatory sequences, of the immediate-early gene Vmw175 (ICP4) were inserted in front of genes mapping in the region 0.69 to 0.70 map units of the virus genome to enhance transient gene expression in a transfection assay. One clone, containing a gene specifying a 1.5-kb mRNA, induced significant amounts of virus-specific dUTPase activity. The enzyme activity was abolished by insertion of a HindIII linker into the KpnI site within the coding sequences of this gene. The results show that the enzyme is virus coded and that 1.5-kb mRNA specifies the dUTPase.

Ralstonia solanacearum causes a bacterial wilt of ginger (Zingiber officinale) that hampers ginger production. Because pathogenicity tests using rhizomes are laborious and time-consuming, here we developed a new test using ginger plants that were aseptically regenerated in vitro from shoot tips using 6-benzyl adenine and 1-naphthalene acid (NAA) in Murashige–Skoog (MS) agar. The regenerated plants were then cultured in MS broth with NAA to evaluate pathogenicity after the roots were dipped into a bacterial solution. The wild-type strain (MAFF 211479) caused wilt symptoms such as leaf yellowing within 15 days after inoculation (dpi), and plants had died by 28 dpi. MAFF 301069, an avirulent strain of ginger, did not cause any symptoms. Because method was suitable for assessing virulence of R. solanacearum, we then used to test the pathogenicity of MAFF 211479 mutants that we constructed and were defective in the type III secretion system. None of the mutants were pathogenic in the regenerated ginger plants or in eggplant. The mutant cells proliferated less efficiently than the wild type in the inoculated ginger plants. We thus conclude that aseptically regenerated ginger plants can be used to elucidate the infection mechanism of R. solanacearum in ginger.

Abstract Phospholipid signaling plays important roles in plant immune responses. Here, we focused on two phospholipase C3 (PLC3) orthologs in the Nicotiana benthamiana genome, NbPLC3-1 and NbPLC3-2. We generated NbPLC3-1 and NbPLC3-2-double-silenced plants (NbPLC3s-silenced plants). In NbPLC3s-silenced plants challenged with Ralstonia solanacearum 8107, induction of hypersensitive response (HR)-related cell death and bacterial population reduction was accelerated, and the expression level of Nbhin1, a HR marker gene, was enhanced. Furthermore, the expression levels of genes involved in salicylic acid and jasmonic acid signaling drastically increased, reactive oxygen species production was accelerated, and NbMEK2-induced HR-related cell death was also enhanced. Accelerated HR-related cell death was also observed by bacterial pathogens Pseudomonas cichorii, P. syringae, bacterial AvrA, oomycete INF1, and TMGMV-CP with L1 in NbPLC3s-silenced plants. Although HR-related cell death was accelerated, the bacterial population was not reduced in double NbPLC3s and NbCoi1-suppressed plants nor in NbPLC3s-silenced NahG plants. HR-related cell death acceleration and bacterial population reduction resulting from NbPLC3s-silencing were compromised by the concomitant suppression of either NbPLC3s and NbrbohB (respiratory oxidase homolog B) or NbPLC3s and NbMEK2 (mitogen activated protein kinase kinase 2). Thus, NbPLC3s may negatively regulate both HR-related cell death and disease resistance through MAP kinase- and reactive oxygen species-dependent signaling. Disease resistance was also regulated by NbPLC3s through jasmonic acid- and salicylic acid-dependent pathways.