The Rcs phosphorelay is a signal transduction system that influences the virulence phenotype of several pathogenic bacteria. In the plant pathogen Pectobacterium carotovorum subsp. carotovorum (Pcc) the response regulator of the Rcs phosphorelay, RcsB, represses expression of plant cell wall degrading enzymes (PCWDE) and motility. The focus of this study was to identify genes directly regulated by the binding of RcsB that also regulate expression of PCWDE genes in Pcc. RcsB-binding sites within the regulatory regions of the flhDC operon and the rprA and rsmB genes were identified using DNase I protection assays, while in vivo studies using flhDC : : gusA, rsmB : : gusA and rprA : : gusA gene fusions revealed gene regulation. These experiments demonstrated that the operon flhDC, a flagellar master regulator, was repressed by RcsB, and transcription of rprA was activated by RcsB. Regulation of the rsmB promoter by RcsB is more complicated. Our results show that RcsB represses rsmB expression mainly through modulating flhDC transcription. Neverthless, direct binding of RcsB on the rsmB promoter region is possible in certain conditions. Using an rprA-negative mutant, it was further demonstrated that RprA RNA is not essential for regulating expression of PCWDE under the conditions tested, whereas overexpression of rprA increased protease expression in wild-type cells. Stationary-phase sigma factor, RpoS, is the only known target gene for RprA RNA in Escherichia coli; however, in Pcc the effect of RprA RNA was found to be rpoS-independent. Overall, our results show that the Rcs phosphorelay negatively affects expression of PCWDE by inhibiting expression of flhDC and rsmB. INTRODUCTIONThe phytopathogenicity of Pectobacterium carotovorum subsp. carotovorum (Pcc) is largely due its capacity to synthesize and secrete plant cell wall degrading enzymes (PCWDE), including pectinases, cellulases and protease Marits et al., 1999; Mäe et al., 1995;Pirhonen et al., 1991). Survival of infecting Pcc largely depends on production of PCWDE for the transition from latent infection to disease state. A number of global and specific regulatory genes contribute to expression of PCWDE in Pcc (Burr et al., 2006;Chatterjee et al., 1995;Cui et al., 1995Cui et al., , 1999Cui et al., , 2001Cui et al., , 2005Eriksson et al., 1998;Flego et al., 2000;Harris et al., 1998; Hyytiäinen et al., 2003;Liu et al., 1998Liu et al., , 1999 Sjöblom et al., 2006). The posttranscriptional Rsm system plays a key role in regulating the expression of PCWDE Cui et al., 1995;Liu et al., 1998).The Rsm system consists of two regulators in Pcc: a small protein, RsmA, and RsmB RNA. RsmA is a post-transcriptional global regulator that is thought to bind mRNAs of PCWDE genes and affect their stability, whereas RsmB RNA binds multiple molecules of RsmA, thereby neutralizing its effect (Liu et al., 1998). Recently, the Rsm system was shown to be dependent on expression of a global regulator of flagellar genes, FlhDC. FlhDC activates the expression of PCWDE in Pcc strain 7...