Expression levels of popA-lacZYA in the RSc2168 (RK5363) and RSc2167 (RK5366) deletion mutants were 260 and 281 Miller units, respectively, which was not different from the levels in BIBW2992 the wild type (RK5050, Table 2). These results indicate that these two genes do not function

in the regulation of hrp regulon. While the OE1-1 strain is pathogenic to tobacco, the Japanese isolate RS1002 (Mukaihara et al., 2004) is nonpathogenic to tobacco. Instead, it elicits a hypersensitive response (HR). We monitored the expression levels of popA operon in popA-lacZYA fusion strains of RS1002; RK10001 and the three deletion mutants of prhK, prhL, and prhM genes (Table 2). popA expression was reduced to an almost basal level in all three mutants, as was observed in the OE1-1 strain. This demonstrates that the functions of PrhK, PrhL, and PrhM are not strain-specific. Many genome-wide screens for pathogenesis-related genes in R. solanacearum have been performed, both experimentally and in silico. Examples of techniques used are transposon mutagenesis (Boucher et al., 1987; Lin et al., 2008), transposon-based screening of hrpB-dependent genes (Mukaihara et al., 2004), and in silico analysis of

secreted proteins via the twin-arginine translocation system (Gonzalez et al., 2007). These analyses Natural Product Library molecular weight have identified T3SS-related hrp and effector genes, genes for type II secretion system (T2SS), flagellar and motility genes, pilus genes, and genes for biosynthesis of exopolysaccharide. Most of these genes are pathogen-specific. Although none of the screens reached saturation, some genes were identified as virulence determinants in multiple independent screenings. It Bay 11-7085 is interesting that these three pathogenesis-related genes had not been identified, despite this long screening history. Because HrpB controls the hrp regulon (Genin et al., 1992), we examined the influence of prhK, prhL, and prhM on the expression of hrpB. We constructed deletion mutants in RK5046 (hrpB-lacZYA), which resulted in RK5206 (ΔprhK),

RK5210 (ΔprhL), and RK5255 (ΔprhM). In sucrose medium, the expression levels of hrpB were substantially reduced in the prhK, prhL, and prhM deletion mutants (Table 2). These data demonstrate that prhK, prhL, and prhM are necessary for the expression of hrpB. Expression of hrpB is activated by HrpG and PrhG (Brito et al., 1999; Plener et al., 2010). We examined the involvement of prhK, prhL, and prhM in the regulation of hrpB expression by hrpG and by prhG. We constructed deletion mutants of RK5120 (hrpG-lacZYA), which resulted in RK5264 (ΔprhK), RK5260 (ΔprhL), and RK5256 (ΔprhM), and of RK5212 (prhG-lacZYA), which resulted in RK5281 (ΔprhK), RK5262 (ΔprhL), and RK5258 (ΔprhM). Their expression levels were determined in sucrose medium.