The RNA-binding protein CsrA controls virulence in Erwinia amylovora by regulating RelA, RcsB, and FlhD at the posttranscriptional level

CsrA, an RNA-binding protein, binds to target transcripts and alters their translation or stability. In Erwinia amylovora, CsrA positively regulates the expression of type III secretion system (T3SS), exopolysaccharide amylovoran and motility. In this study, the global effect of CsrA and its non-coding small RNA (ncsRNA) csrB in E. amylovora was determined by RNA-seq, and potential molecular mechanisms of CsrA-dependent virulence regulation were examined. Transcriptomic analyses under the T3SS-inducing condition revealed that mutation in the csrA gene led to differential expression of more than 20% genes in the genome. Among them, T3SS genes and those required for cell growth and viability were significantly down-regulated. On the other hand, the csrB mutant exhibited significant up-regulation of most major virulence genes, suggesting antagonistic effect of csrB on CsrA targets. Direct interaction between CsrA protein and csrB was further confirmed through RNA electrophoretic mobility shift assay (REMSA). However, no direct interaction between CsrA and hrpL and hrpS transcripts was detected, suggesting that HrpL and HrpS are not targets of CsrA; whereas three CsrA targets (relA, rcsB and flhD) were identified and confirmed by REMSA, site-directed mutagenesis and LacZ reporter gene assays. These findings might partially explain how CsrA positively controls E. amylovora virulence by targeting major regulators at the posttranscriptional level.