Sucrose and malic acid in the tobacco plant induce <i>hrp</i> regulon in a phytopathogen <i>Ralstonia pseudosolanacearum</i>

Genes encoding a type III secretion system in <i>Ralstonia pseudosolanacearum</i> are regulated by HrpB as an <i>hrp</i> regulon and induced only in plants. This study aimed to identify the plant signals that induce the <i>hrp</i> regulon and confirm the signal recognition mechanism. Signaling molecules that induce <i>hrpB</i> expression were screened using resting cells of the <i>hrpB-lacZ</i> reporter strain. Only the soluble fraction of smashed tobacco seedlings induced <i>hrpB</i> expression. The heated soluble fraction retained its <i>hrpB</i>-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 <i>hrpB</i> expression. As neutral compounds, sucrose, glucose, and fructose have been found to induce <i>hrpB</i> expression. Sucrose-induced <i>hrpB</i> expression was greatly reduced in the <i>prhA</i> mutant, indicating that the TonB-dependent receptor PrhA perceives sugars. Among the organic acids found in the acidic fractions, malic acid most efficiently induced <i>hrpB</i> expression, which was reduced by the mutation of a hybrid histidine kinase gene of a two-component system, <i>rsc1598</i>, indicating that Rsc1598 may sense malic acid. We demonstrated direct binding of Rsc1598 to malic acid using isothermal titration calorimetry.IMPORTANCESimilar to other Gram-negative plant pathogens, the type III secretion system (T3SS) is the most important virulence factor in <i>Ralstonia pseudosolanacearum</i>. The genes for the T3SS are regulated as an <i>hrp</i> 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 <i>hrp</i> operon was induced even in non-host plants, possibly because sucrose and malic acid are common in plants. We also found that <i>R. pseudosolanacearum</i> membrane proteins received sucrose and malic acid independently. As a next step, antagonists of signaling molecules can be screened.