Identification of novel virulence factors in Erwinia amylovora through temporal transcriptomic analysis of infected apple flowers under field conditions.

The enterobacterial pathogen Erwinia amylovora uses multiple virulence-associated traits to cause fire blight, a devastating disease of apple and pear trees. Many virulence-associated phenotypes have been studied that are critical for virulence and pathogenicity. Despite the in vitro testing that has revealed how these systems are transcriptionally regulated, information on when and where in infected tissues these genes are being expressed is lacking. Here, we used a high-throughput sequencing approach to characterize the transcriptome of E. amylovora during disease progression on apple flowers under field infection conditions. We report that type III secretion system genes and flagellar genes are strongly co-expressed. Likewise, genes involved in biosynthesis of the exopolysaccharide amylovoran and sorbitol utilization had similar expression patterns. We further identified a group of 16 genes whose expression is increased and maintained at high levels throughout disease progression across time and tissues. We chose five of these genes for mutational analysis and observed that deletion mutants lacking these genes all display reduced symptom development on apple shoots. Furthermore, these induced genes were over-represented for genes involved in sulphur metabolism and cycling, suggesting the possibility of an important role for maintenance of oxidative homeostasis during apple flower infection.