Multiple approaches towards understanding virulence in Clavibacter michiganensis subsp. sepedonicus, causal agent of bacterial ring rot of potato.

Abstract

Recent discoveries related to pathogenesis of Clavibacter michiganensis subsp. sepedonicus (Cms), causal agent of bacterial ring rot of potato, are beginning to shed light on the virulence strategies of this species. The chromosomal homologue of pat-1, chp7, was found to be necessary for HR elicitation in tobacco and virulence of Cms in eggplant and potato. Multiple chromosomal and plasmid-encoded homologues of pat-1 (chp and php genes, respectively) are present in Cms, yet only the function of chp7 has been evaluated. The development of molecular resources and techniques for genetic manipulation of Cms are needed to facilitate studies aimed at building a more comprehensive understanding of the role of pat-1 homologues in pathogenesis. Knockout mutants chp7 and php3 of Cms were generated via targeted mutagenesis. The virulence of chp7 and php3 mutants of Cms were evaluated on eggplant and potato. Significant reductions in virulence of Cms were observed for chp7 and php3 mutants in both eggplant and potato. Mutation of chp7 also resulted in the loss of non-hostHR induction and an observed reduction in bacterial titer of infected plants. These phenotypes however, were not observed in php3 mutants of Cms. A mutant of chp8 was identified in a newly generated EzTn5 transposon library of Cms. The virulence of the chp8 mutant of Cms was evaluated in eggplant. Significant reductions in both bacterial titer of infected plant tissue and virulence of Cms were observed for the chp8 mutant. In summary, three pat-1 homologues from Cms have now been evaluated for their relative contributions to virulence, and these studies indicate that each is likely contributing differently to pathogenesis of Cms. The formation of biofilms has been demonstrated in Cms both in vitro and in planta. Development of an appropriate assay for assessing biofilm formation by Cms is a necessary first step towards addressing the role of biofilms in bacterial ring rot. Two of the biofilm assays examined in this study could be considered as potential strategies for future assessment of biofilm formation by Cms. Currently, both of these assays are still diminished in their utility, given issues with inconsistencies and reproducibility.