Construction and analysis of pathogenicity island deletion mutants of Erwinia amylovora

An easy gene-knockout technique, PCR-based one-step inactivation of chromosomal genes, is widely used in Escherichia coli and related enterobacteria to construct mutants. In this study, we adapted this technique to construct genomic island and large operon deletion mutants of Erwinia amylovora, including the 33.4 kb hrp-type III secretion (T3SS) pathogenicity island (PAI1) and the 15.8 kb amylovoran biosynthesis (AMS) operon. Deletion of 2 novel T3SS pathogenicity islands (PAI2 and PAI3) and an operon encoding a type II secretion system (T2SS) demonstrated that these determinants are not involved in virulence in plants. Co-inoculation experiments demonstrated that the hrp-T3SS and AMS deletion mutants could complement each other. These results further confirmed that the one-step inactivation technique can be used to generate large deletions in E. amylovora.