In bacteria, gene expression is mainly regulated at the transcription initiation level and its core RNA polymerase (RNAP) requires sigma factors for promoter recognition and initiation. In Erwinia amylovora, alternative sigma factor HrpL-RNAP complex regulates the transcription of hrp-type III secretion (T3SS) genes by binding to a consensus sequence known as the hrp box in hrp gene promoters. In turn, expression of hrpL has been proposed to be positively controlled by another alternative sigma factor, sigma 54 (RpoN), and HrpS, a member of σ54 enhancer-binding proteins (EBPs). However, the function of both HrpS and RpoN has not been genetically characterized in E. amylovora. In this study, we examined an alternative sigma factor cascade with an emphasis on components of HrpS-RpoN-RNAP complex in regulating T3SS gene expression in E. amylovora. Results showed mutations in hrpS, hrpL, rpoN and yhbH, but not rpoS, another alternative sigma factor, resulted in non-pathogenic phenotype on apple and immature pear fruits, and no hypersensitive response on non-host tobacco, suggesting that HrpL, HrpS, RpoN, and YhbH, a novel ribosome-binding protein, are essential for virulence and absolutely required for T3SS gene expression in E. amylovora. We also determined the regulons of both HrpS and HrpL in vivo using oligo array. These results suggest that an alternative sigma factor cascade in E. amylovora regulates expression of T3SS and controls virulence.