Plant defensin antibacterial mode of action against Pseudomonas species

Many plant defensins exhibit antibacterial activity, but little is known about their antibacterial mode of action (MOA). Antimicrobial peptides with characterized MOAs induce the expression of multiple outer membrane modifications, which are required for resistance to these membrane-targeting peptides. Mini-Tn5-lux mutant strains of Pseudomonas aeruginosa with Tn insertions disrupting outer membrane protective modifications were assessed for sensitivity against plant defensin peptides. Defensins displayed specific and potent antibacterial activity against strains of P. aeruginosa. These transcriptional lux reporter strains were also evaluated for lux gene expression in response to sublethal plant defensin exposure. A defensin from Medicago truncatula, MtDef4, induced dose-dependent gene expression of the aminoarabinose modification of LPS and surface polycation spermidine production operons. The ability for MtDef4 to damage bacterial outer membranes was visually verified with fluorescent microscopy. Another defensin from M. truncatula, MtDef5, appears to have a different antibacterial MOA. MtDef5 treatments failed to induce lux gene expression and limited outer membrane damage was detected with fluorescent microscopy. A plant pathogen, P. syringae pv. syringae was modified through transposon mutagenesis to create mutants that are resistant to MtDef4 treatments. The transposon insertion site on defensin resistant bacterial mutants was sequenced, and modifications of ribosomal genes were identified to contribute to enhanced resistance to defensin treatments