Background Plant defensins are a broadly distributed family of antimicrobial peptides which have been primarily studied for agriculturally relevant antifungal activity. Recent studies have probed defensins against Gram-negative bacteria revealing evidence for multiple mechanisms of action including membrane lysis and ribosomal inhibition. Herein, a truncated synthetic analog containing the γ-core motif of Amaranthus tricolor DEF2 (Atr-DEF2) reveals Gram-negative antibacterial activity and its mechanism of action is probed via proteomics, outer membrane permeability studies, and iron reduction/chelation assays. Results Atr-DEF2(G39-C54) demonstrated activity against two Gram-negative human bacterial pathogens, Escherichia coli and Klebsiella pneumoniae. Quantitative proteomics revealed changes in the E. coli proteome in response to treatment of sub-lethal concentrations of the truncated defensin, including bacterial outer membrane (OM) and iron acquisition/processing related proteins. Modification of OM charge is a common response of Gram-negative bacteria to membrane lytic antimicrobial peptides (AMPs) to reduce electrostatic interactions, and this mechanism of action was confirmed for Atr-DEF2(G39-C54) via an N-phenylnaphthalen-1-amine uptake assay. Additionally, in vitro assays confirmed the capacity of Atr-DEF2(G39-C54) to reduce Fe3+ and chelate Fe2+ at cell culture relevant concentrations, thus limiting the availability of essential enzymatic cofactors. Conclusions This study highlights the utility of plant defensin γ-core motif synthetic analogs for characterization of novel defensin activity. Proteomic changes in E. coli after treatment with Atr-DEF2(G39-C54) supported the hypothesis that membrane lysis is an important component of γ-core motif mediated antibacterial activity but also emphasized that other properties, such as metal sequestration, may contribute to a multifaceted mechanism of action.

Targeted proteolysis activities activated during the plant immune response catalyze the synthesis of stable endogenous peptides. Little is known about their biogenesis and biological roles. Herein, we characterize an Arabidopsis thaliana mutant top1top2 in which targeted proteolysis of immune-active peptides is drastically impaired during effector-triggered immunity (ETI). For effective ETI, the redox-sensitive thimet oligopeptidases TOP1 and TOP2 are required. Quantitative mass spectrometry-based peptidomics allowed differential peptidome profiling of wild type (WT) and top1top2 mutant at the early ETI stages. Biological processes of energy-producing and redox homeostasis were enriched, and TOPs were necessary to maintain the dynamics of ATP and NADP(H) accumulation in the plant during ETI. Subsequently, a set of novel TOPs substrates validated in vitro enabled the definition of the TOP-specific cleavage motif and informed an in-silico model of TOP proteolysis to generate bioactive peptide candidates. Several candidates, including those derived from proteins associated with redox metabolism, were confirmed in planta. The top1top2 background rescued WT’s ETI deficiency caused by treatment with peptides derived from targeted proteolysis of the negative immune regulator FBR12, the reductive enzyme APX1, the isoprenoid pathway enzyme DXR, and ATP-subunit β. These results demonstrate TOPs role in orchestrating the production and degradation of phytocytokines.