Plants produce a suite of antimicrobial peptides (AMPs) to defend against the extensive array of potential pathogens encountered in their environment. Plant AMPs are classified based on their structure and presence of disulfide bonds . With an abundance of representatives from diverse plant species, plant defensins are among the most widespread and best characterized plant AMPs . Plant defensins are cationic, cysteine-rich antimicrobial peptides that usually contain four disulfide bonds. They have a conserved three-dimensional structure, a cysteine-stabilized 𝛼𝛽 (CS𝛼𝛽) motif, with a concentration of positively charged amino acid residues on the 𝛽2- 𝛽3 loop, which is classified as the γ-core motif (GXCX3−9C). The γ-core motif alone has been shown to impart antimicrobial activity and mimic the activity of the corresponding full-length defensin . Plant defensins are promiscuous peptides, which means that a single peptide can have multiple distinct functions . Along with having antimicrobial activity, plant defensins control plant development, contribute to zinc tolerance, and act as inhibitors of digestive enzymes . In crop plants, the transgenic expression of plant defensins has been used to engineer fungal and oomycete disease resistant plants. When MsDef1, a defensin from alfalfa (Medicago sativa), was expressed in potato, field-grown potatoes displayed resistance to Verticillium dahliae . NaD1, a defensin from sweet tobacco (Nicotiana alata), provided transgenic cotton with resistance to Fusarium oxysporum f. sp. vasinfectum and V. dahliae throughout three years of field trials .
Though considered to be primarily antifungal, plant defensins have been shown to demonstrate antibacterial activity against both plant and vertebrate bacterial pathogens . Spinach defensin (So-D2) is the most frequently cited plant defensin with antibacterial activity, and transgenic sweet orange and grapefruit trees expressing So-D2 exhibited increased resistance to the bacterial diseases, citrus greening and citrus canker, caused by Candidatus Liberibacter spp. and Xanthomonas axonopodis pv. citri, respectively . Plant defensins also display in vitro antibacterial activity against human pathogens. For instance, J1-1, a defensin from bell pepper (Capsicum annum) has a minimum inhibitory concentration (MIC) value of 250 µg/mL against Pseudomonas aeruginosa . Also, PaDef, a defensin from avocado (Persea americana var. drymifolia), displays antibacterial activity against Staphylococcus aureus . 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.
A major obstacle blocking the widespread usage of plant defensins as antibacterial compounds is that their antibacterial mode of action (MOA) is poorly characterized  although their MOA against fungal pathogens is well-described [12, 13, 14]. Recently, the antibacterial activity of a defensin from Medicago truncatula, MtDef5, was characterized . MtDef5 is a bi-domain defensin with two defensin domains (MtDef5A and MtDef5B) connected by a 7-amino acid linker peptide. The cationic amino acid residues found in both γ-core motifs of MtDef5 were mutated and discovered to be essential for antibacterial activity, which were the same residues previously found to be essential for antifungal activity . Additionally, MtDef5 was shown to permeabilize the plasma membrane of Xanthomonas campestris pv. campestris, a gram-negative bacterial plant pathogen, but not the gram-positive plant pathogen Clavibacter michiganensis subsp. nebraskensis . The MtDef5 peptide binds to DNA indicating that it may kill bacterial cells by inhibiting DNA synthesis or transcription.
The MOA of human and invertebrate defensins against bacterial pathogens is well characterized [17, 18]. Vertebrate defensins interact with the negatively charged lipopolysaccharide (LPS) in the bacterial outer membrane, which leads to rapid membrane permeabilization through pore formation . For example, HNP-1, the most studied human α-defensin, has an antibacterial MOA typical of many AMPs. HNP-1 dimerization occurs, and the electrostatic interaction of dimers with the bacterial membrane causes β-sheet dimers to span the membrane forming a pore, with higher order oligomers of HNP-1 forming upon dimers when the defensin is in high concentration . Another well-studied antibacterial human defensin, human β-defensin-3 (HBD3), has been shown to inhibit bacterial cell wall biosynthesis by interacting with lipid II components, which allows for HBD3 to have widespread activity against both gram-positive and gram-negative bacteria .
In response to the electrostatic interactions between cationic AMPs and negatively charged bacterial membranes, gram-positive and gram-negative bacteria have demonstrated the ability to modify their membrane surfaces . In P. aeruginosa and many other gram-negative bacteria, the PhoPQ/PmrAB systems control various genes required for resistance to AMPs . The pmr operon (PA3552-PA3559) is controlled by both PhoPQ and PmrAB and is required for the addition of aminoarabinose to mask the phosphates of lipid A in P. aeruginosa . Upstream of PmrAB, the spermidine synthesis genes PA4773 (speD2) and PA4774 (speE2) in P. aeruginosa are required for production of this polycation on the outer surface of the bacterial membrane . These surface modifications protect bacteria from cationic AMPs through masking of the negative surface charges, which limits AMP binding to bacterial membranes [24, 25]. The mini-Tn5-luxCDABE mutant library in P. aeruginosa has been used extensively to identify antimicrobial peptide MOAs and bacterial resistance mechanisms .
Pseudomonas syringae pv. syringae is a bacterial plant pathogen that causes bacterial stem blight of alfalfa, which is an economically important disease with widespread distribution in the Western United States . Currently, there are no effective means to control bacterial stem blight of alfalfa. P. syringae pv. syringae strain ALF3, pathogenic on alfalfa and M. truncatula, has a draft genome sequence  and was shown to be sensitive to M. truncatula defensins, MtDef5 and MtDef4, with IC50 values of 0.1 and 0.4 µM, respectively . Additionally, MtDef4 displays activity against Xanthomonas alfalfae subsp. alfalfae and the gram-positive bacterium Clavibacter insidiosus, while MtDef5 displays no activity against these pathogens . There is insufficient knowledge to explain this observed specificity of plant defensin antibacterial activity. Generating tools to explore plant defensin MOA against bacterial plant pathogens is necessary for evaluating the risk of bacterial evolution towards defensin resistance and for the development of plant defensins into a spray-on peptide-based biological pesticide or transgenic expression of defensins for plant protection. Furthermore, knowing the antibacterial MOA of plant defensins allows for prediction of antibacterial activity without extensive in vitro testing.
In this study, we investigated plant defensin MOA against plant and vertebrate bacterial pathogens belonging to the genus Pseudomonas. Characterized P. aeruginosa lux-reporter strains with mutations in genes involved with cationic antimicrobial peptide resistance mechanisms were screened for sensitivity to γ-core motif plant defensin peptides. We discovered that plant defensin γ-core motif peptides exhibit potent activity against P. aeruginosa with the membrane modification mutants displaying increased sensitivity compared to the wild type. Exploiting the transcriptional lux reporter feature of these mutant strains (PA3553::lux and PA4774::lux), we found that MtDef4 induces the expression of both resistance determinants, indicating that MtDef4 likely acts on the P. aeruginosa outer membrane. MtDef4 was confirmed to cause bacterial outer membrane damage through fluorescent microscopy. Transposon insertion libraries of P. syringae pv. syringae were generated and screened for plant defensin resistance. Resistant bacterial mutants were identified, transposon insertion sites were sequenced, and interrupted genes annotated as 16S and 23S ribosomal rRNA genes were found to be involved with plant defensin resistance. This suggests that MtDef4 may also function as a protein synthesis inhibitor.