Technical Abstract: The exopolysaccharide amylovoran is one of the major pathgenicity factors in Erwinia amylovora, the causal agent of fire blight of apples and pears. We have previously demonstrated that the RcsBCD phosphorelay system is essential for virulence by controlling amylovoran biosynthesis. We have also found that the hybrid sensor kinase RcsC differentially regulates amylovoran production in vitro and in vivo. To further understand how the Rcs system regulates E. amylovora virulence gene expression, we conducted genome-wide microarray analyses to determine the regulons of RcsB and RcsC in liquid medium and, for the first time, on immature pear fruit. Array analyses identified a total of 648 genes differentially regulated by RcsCB in vitro and in vivo. Consistent with our previous findings, RcsB acts as a positive regulator in both conditions, while RcsC positively controls expression of amylovoran biosynthetic genes in vivo, but negatively in vitro. Besides amylovoran biosynthesis and regulatory genes, cell wall and cell envelope (membrane) as well as regulatory genes are the major components of the RcsBC regulon, including many novel genes. We have also demonstrated that transcripts of rcsA, rcsC and rcsD genes, but not those of rcsB gene, were up-regulated when bacterial cells were grown in minimal medium or following infection of pear fruits compared to those of LB medium. Furthermore, a hidden Markov model (HMM) predicted 60 genes with a candidate RcsB binding site in intergenic regions of the E. amylovora ATCC 49946 genome, and 28 of these have been identified in the microarray assay. Based on these findings as well as previous reported data, a working model is proposed to illustrate how the Rcs phosphorelay system regulates virulence gene expression in E. amylovora.