Plant defensin antibacterial mode of action against Pseudomonas species

Authors: SATHOFF, ANDREW - University Of Minnesota; LEWENZA, SHAWN - University Of Calgary; Samac, Deborah - Debby

Submitted to: BMC Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/11/2020
Publication Date: 6/23/2020
Citation: Sathoff, A.E., Lewenza, S., Samac, D.A. 2020. Plant defensin antibacterial mode of action against Pseudomonas species. BMC Microbiology. 20:173. https://doi.org/10.1186/s12866-020-01852-1.
DOI: https://doi.org/10.1186/s12866-020-01852-1

Interpretive Summary: Plant defensins, are antimicrobial peptides produced by plants for protection against disease causing organisms. Many defensins have antibacterial activity, but the means by which they inhibit bacterial growth is unknown. Bacterial strains with disruptions in genes for outer membrane protective modifications were surveyed for sensitivity to two plant defensins. Genes involved in protection from positively charged molecules were found to be involved in resistance to the defensin MtDef4. In these mutants the defensin disrupted the outer bacterial membrane and caused cell death. In contrast, the defensin MtDef5 appeared to target ribosomes inside bacterial cells to cause growth inhibition. Both defensins acted more rapidly than the antibiotic polymyxin B to inhibit bacterial growth. Therefore, plant defensins not only appear to be a resource for improving plant immunity to bacterial diseases but also for combatting human and animal bacterial pathogens.

Technical Abstract: 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 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. This indicates that MtDef4 damages the outer membrane similar to polymyxin B, which stimulates antimicrobial peptide resistance mechanisms to plant defensins. The ability for MtDef4 to damage bacterial outer membranes was also verified visually through 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, Pseudomonas syringae pv. syringae was modified through transposon mutagenesis to create mutants that are resistant to in vitro 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. Therefore, the MtDef4 antibacterial mode of action may also involve inhibition of translation.