Ralstonia solanacearum, a plant pathogenic bacterium causing "bacterial wilt" on crops, uses a quorum sensing (QS) system consisting of phc regulatory elements to control its virulence. Methyl 3-hydroxypalmitate (3-OH PAME) was previously identified as the QS signal in strain AW1. However, 3-OH PAME has not been reportedly detected from any other strains, and this suggests that they produce another unknown QS signal. Here we identify (R)-methyl 3-hydroxymyristate [(R)-3-OH MAME] as a new QS signal that regulates the production of virulence factors and secondary metabolites. (R)-3-OH MAME was synthesized by the methyltransferase PhcB and sensed by the histidine kinase PhcS. The phylogenetic trees of these proteins from R. solanacearum strains were divided into two groups, according to their QS signal types--(R)-3-OH MAME or (R)-3-OH PAME. These results demonstrate that (R)-3-OH MAME is another crucial QS signal and highlight the unique evolution of QS systems in R. solanacearum.

Genes in the hrp regulon encode component proteins of the type III secretion system and are essential for the pathogenicity of Ralstonia solanacearum. The hrp regulon is controlled by HrpB. We isolated several genes regulating hrpB expression from the Japanese strain OE1-1 using minitransposon mutagenesis. Among them, we mainly focused on two genes, hrpG and prhG, which are the positive regulators of hrpB. Although the global virulence regulator PhcA negatively regulated hrpG expression via prhIR, it positively regulated prhG expression. We further investigated the contrasting regulation of hrpG and prhG by PhcA and speculated that R. solanacearum may switch from HrpG to PrhG for hrpB activation in a cell density-dependent manner. Although the prhG mutant proliferated similarly to the wild-type in leaf intercellular spaces and in xylem vessels of the host plants, it was less virulent than the wild-type. The expression of the popA operon, which belongs to the hrp regulon, was significantly reduced in the prhG mutant by more than half in the leaf intercellular spaces and more than two-thirds in the xylem vessels when compared with the wild-type.

The mechanism of colonization of intercellular spaces by the soil-borne and vascular plant-pathogenic bacterium Ralstonia solanacearum strain OE1-1 after invasion into host plants remains unclear. To analyse the behaviour of OE1-1 cells in intercellular spaces, tomato leaves with the lower epidermis layers excised after infiltration with OE1-1 were observed under a scanning electron microscope. OE1-1 cells formed microcolonies on the surfaces of tomato cells adjacent to intercellular spaces, and then aggregated surrounded by an extracellular matrix, forming mature biofilm structures. Furthermore, OE1-1 cells produced mushroom-type biofilms when incubated in fluids of apoplasts including intercellular spaces, but not xylem fluids from tomato plants. This is the first report of biofilm formation by R. solanacearum on host plant cells after invasion into intercellular spaces and mushroom-type biofilms produced by R. solanacearum in vitro. Sugar application led to enhanced biofilm formation by OE1-1. Mutation of lecM encoding a lectin, RS-IIL, which reportedly exhibits affinity for these sugars, led to a significant decrease in biofilm formation. Colonization in intercellular spaces was significantly decreased in the lecM mutant, leading to a loss of virulence on tomato plants. Complementation of the lecM mutant with native lecM resulted in the recovery of mushroom-type biofilms and virulence on tomato plants. Together, our findings indicate that OE1-1 produces mature biofilms on the surfaces of tomato cells after invasion into intercellular spaces. RS-IIL may contribute to biofilm formation by OE1-1, which is required for OE1-1 virulence.

In tobacco (Nicotiana tabacum), Ralstonia solanacearum OE1-1 (RsOE1-1) is pathogenic, whereas R. solanacearum 8107 (Rs8107) is nonpathogenic and induces the hypersensitive response (HR). To elucidate the molecular mechanisms of plant-R. solanacearum interactions, we used differential display to isolate a cDNA fragment, A6, regulated in tobacco by inoculation with RsOE1-1. The deduced amino acid sequence predicted from full-length A6-cDNA showed similarity to small heat shock proteins from Arabidopsis (Arabidopsis thaliana; hypothetical protein), Medicago truncatula, and Cucumis melo; we therefore designated A6 to correspond to Ntshsp17 (for tobacco small heat shock protein 17). Recombinant Ntshsp17 overproduced in Escherichia coli exhibited molecular chaperone function. Expression of Ntshsp17 was increased in tobacco leaves inoculated with both RsOE1-1 and Rs8107. Expression was induced by heat treatment and by treatment with aminocyclopropane carboxylic acid, hydrogen peroxide, methyl jasmonate, and salicylic acid. Ntshsp17 expression was induced by inoculation with a HR and pathogenicity gene mutant of Rs8107 that does not induce the HR, but not by Agrobacterium-mediated transient expression of INF1, an HR elicitor. In Nbshsp17-silenced plants (an Ntshsp17 ortholog in Nicotiana benthamiana), expression of ETHYLENE-RESPONSE ELEMENT-BINDING PROTEIN, PATHOGENESIS-RELATED1a (PR1a), and PR4 genes was compromised, but expression of ELONGATION FACTOR1a was scarcely affected. Appearance of the HR was not affected in the silenced plants. In the silenced plants, growth of Rs8107 was accelerated. Bacterial growth and wilt symptoms elicited by RsOE1-1 were also accelerated in the silenced plants. These results indicate that this small heat shock protein might have a role in HR-independent defenses in Nicotiana plants.

The transcriptional regulator HrpB activates the entire hrp regulon in the plant pathogen Ralstonia solanacearum. Through a complex multigene regulatory cascade PrhA-PrhR/PrhI-PrhJ-HrpG, expression of hrpB is induced in a hrp-inducing, nutrient-poor medium and in response to contact between the bacterium and plant cell. In this study, we analyzed the expression levels of these regulatory genes and hrpB using lacZ reporter strains grown in three different conditions: in a nutrient-rich or nutrient-poor medium and co-cultivated with Arabidopsis thaliana seedlings. We found that prhA and prhIR were expressed constitutively. Expression of prhJ and hrpG was PrhA-dependent in all three conditions. Despite the high level of hrpG expression in all cases, hrpB was induced only when the bacteria were co-cultivated with A. thaliana seedlings or grown in nutrient-poor medium. A mutation in the predicted phosphorylation site of hrpG greatly reduced the function of HrpG. From these results, we conclude that the prhA-dependent regulatory cascade controls the expression of hrpG, and a new cascade, which is induced by a signal from plant cells, activates HrpG by phosphorylation. Only when both signal cascades are effective is full expression of hrpB induced. We speculate that the metabolic status of the bacteria in the nutrient-poor medium also contributes to the second cascade.