Abstract

BackgroundThough many plant defensins exhibit antibacterial activity, little is known about their antibacterial mode of action (MOA). Antimicrobial peptides with a characterized MOA induce the expression of multiple bacterial 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. These transcriptional lux reporter strains were also evaluated for lux gene expression in response to sublethal plant defensin exposure. Also, a plant pathogen, Pseudomonas syringae pv. syringae was modified through transposon mutagenesis to create mutants that are resistant to in vitro MtDef4 treatments.ResultsPlant defensins displayed specific and potent antibacterial activity against strains of P. aeruginosa. 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 also verified visually through fluorescent microscopy. Another defensin from M. truncatula, MtDef5, failed to induce lux gene expression and limited outer membrane damage was detected with fluorescent microscopy. The transposon insertion site on MtDef4 resistant P. syringae pv. syringae mutants was sequenced, and modifications of ribosomal genes were identified to contribute to enhanced resistance to plant defensin treatments.ConclusionsMtDef4 damages the outer membrane similar to polymyxin B, which stimulates antimicrobial peptide resistance mechanisms to plant defensins. MtDef5, appears to have a different antibacterial MOA. Additionally, the MtDef4 antibacterial mode of action may also involve inhibition of translation.

Highlights

  • Though many plant defensins exhibit antibacterial activity, little is known about their antibacterial mode of action (MOA)

  • Our results suggest that the antibacterial MOA of the Medicago truncatula defensin 4 (MtDef4) y-core motif peptide may involve ribosomal targeting, and the transposon insertions in P. syringae pv. syringae rRNA encoding genes could be target site mutations leading to increased MtDef4 resistance

  • In P. aeruginosa, we propose that MtDef4 and Spinacia oleracea defensin 2 (So-D2) interact with the bacterial outer membrane and possibly create pores leading to bacterial cell death

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Summary

Introduction

Though many plant defensins exhibit antibacterial activity, little is known about their antibacterial mode of action (MOA). Antimicrobial peptides with a characterized MOA induce the expression of multiple bacterial outer membrane modifications, which are required for resistance to these membrane-targeting peptides. Mini-Tn5lux mutant strains of Pseudomonas aeruginosa with Tn insertions disrupting outer membrane protective modifications were assessed for sensitivity against plant defensin peptides. These transcriptional lux reporter strains were evaluated for lux gene expression in response to sublethal plant defensin exposure. Plants produce a suite of antimicrobial peptides (AMPs) to defend against the extensive array of potential pathogens encountered in their environment. When MsDef, a defensin from alfalfa (Medicago sativa), was expressed in potato, field-grown potatoes displayed resistance to Verticillium dahliae [6]. NaD1, a defensin from sweet tobacco (Nicotiana alata), provided transgenic cotton with resistance to Fusarium oxysporum f. sp. vasinfectum and V. dahliae throughout 3 years of field trials [7]

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