Tobacco plants inoculated at their roots with RK5050 showed wilt symptoms sooner than the tomato plants (Fig. 2c). Although tobacco plants inoculated with RK5204 (ΔprhK) and RK5208 (ΔprhL) started to wilt at 4 dpi, they died later than the tobacco plants inoculated with RK5050, i.e. at 21 and 18 dpi, Selleck Dactolisib respectively. Tobacco plants inoculated with RK5253 (ΔprhM) showed wilt at 7 dpi, and

died at 21 dpi (Fig. 2c). The three mutants displayed different levels of pathogenesis on the two host plants – tomato and tobacco. They were severely impaired in the colonization of tomato xylem vessels (Fig. S1), but proliferated in tobacco leaves only slightly slower compared with the wild type (data not shown). Different host plants displayed different symptoms, depending upon the infecting strain (Lin et al., 2008). When a pUC7169 plasmid containing the three genes was transferred into each of the mutant strains, all three of the recombinant strains recovered pathogenicity to the wild-type level (Fig. 2d). Cell suspensions

with high cell density of the popA-lacZYA reporter strain and the derived prhKLM mutants were infiltrated into tomato leaves, and the in planta popA expression was monitored up to 24 h postinoculation (hpi). Cell numbers did not change during this period, and gene expression was normalized to cell number. In the leaves, popA expression in the wild type increased until 18 hpi, and then fell slightly until 24 hpi (Fig. 3). Throughout the experiments, expression levels were substantially repressed in the prhK, prhL, and prhM mutants NVP-BGJ398 datasheet (Fig. 3). All three genes (prhK, prhL, and prhM) of the prhK operon are well conserved among Betaproteobacteria. It is likely that in the genus Ralstonia, the operon contains three genes plus an additional two genes (RSc2168 and RSc2169) (Fig. 4). Except for Burkholderia glumae, the other three bacteria shown in Fig. 4 are not plant pathogens. This indicates that these three genes are quite common and are not specific to bacterial plant pathogens. Moreover, orthologs

of these three genes have been detected in a wide range of bacteria, including E. coli. RSc2171 and RSc2170, which are annotated as allophanate hydrolase Isotretinoin subunit 1 and 2, respectively (Salanoubat et al., 2002), are related to the urea amidolyase of Saccharomyces cerevisiae (Wang et al., 1997). In addition, KipI and KipA in Bacillus subtilis, which modulate the phosphorylation level of the two-component response regulator Spo0F, are homologs of RSc2171 and RSc2170, respectively (Wang et al., 1997). PrhK is 55% similar to the KipI C-terminal domain, which binds to the KinA histidine kinase (Jacques et al., 2008). RSc2169 is annotated as a LamB/YcsF family protein. In fungi, LamB seems to be required for the utilization of lactam rings as a nitrogen source (Wang et al., 1997).