The genetic requirements for HiVir-mediated onion necrosis by Pantoea ananatis, a necrotrophic plant pathogen

Pantoea ananatis is an unusual bacterial pathogen that lacks typical virulence determinants yet causes extensive necrosis in onion foliage and bulb tissues. The onion necrosis phenotype is dependent on the expression of a phosphonate toxin, pantaphos that is catalyzed by putative enzymes encoded by the HiVir gene cluster. The genetic contributions of individual hvr genes in HiVir-mediated onion necrosis remain largely unknown except for the first gene hvrA (phosphoenolpyruvate mutase, pepM) whose deletion resulted in the loss of onion pathogenicity. In this study, using gene deletion mutation and complementation, we report that of the ten remaining genes, hvrB-hvrF are also strictly required for the HiVir-mediated onion necrosis and in planta bacterial growth whereas hvrG-hvrJ partially contributed to these phenotypes. As the HiVir gene cluster is a common genetic feature shared among the onion-pathogenic P. ananatis strains, and as it could serve as a useful diagnostic marker of onion pathogenicity, we sought to understand the genetic basis of HiVir positive yet phenotypically deviant (non-pathogenic) strains. We identified and genetically characterized inactivating single nucleotide polymorphisms (SNPs) in essential hvr genes of six phenotypically deviant P. ananatis strains. Finally, inoculation of the cell-free spent medium of Ptac-driven HiVir strain caused P. ananatis-characteristic red onion scale necrosis (RSN) as well as cell death symptoms in tobacco. The co-inoculation of the spent medium with essential hvr mutant strains restored strains in planta populations to the wild-type level, suggesting that necrosis is important for proliferation of P. ananatis in onion tissue.