Bacterial strains and growth media
All bacterial strains utilized in this study are listed in Table 2. Pseudomonas aeruginosa strains were obtained from Dr. Lewenza at the University of Calgary. The P. aeruginosa lux-reporter strains have inactivated lipopolysaccharide (LPS) modification genes, which are bacterial genes involved in the resistance to cationic antimicrobial peptides. PA4774::lux has an interrupted outer membrane surface spermidine synthesis gene. PA3553::lux has an interrupted lipid A modification gene, which is responsible for the addition of aminoarabinose to lipid A. When the lux-reporter bacteria produce bioluminescence, they act as a real-time reporter for the induction of the inactivated gene . PAO1 was used as the wild type strain of P. aeruginosa. The P. aeruginosa strains were cultured on Luria-Bertani (LB) agar (Difco, Sparks, MD) at 37 °C. From a glycerol stock, the sequenced bacterial strain, Pseudomonas syringae pv. syringae ALF3, originally isolated from an infected alfalfa plant near Cheyenne, WY, was cultured on nutrient broth yeast extract (NBY) agar at 30 °C . ALF3 was used as the wild type strain of P. syringae pv. syringae.
Plant defensin peptide synthesis
The g-core motif peptides derived from plant defensins, MtDef4, MtDef5A, and So-D2 [16, 52, 53] (Table 1) were chemically synthesized and purified by HPLC (LifeTein, Somerset, NJ). Lyophilized defensin peptides were rehydrated in sterile water prior to each assay.
Determination of plant defensin antibacterial activity against Pseudomonas aeruginosa
To quantify defensin antibacterial activity, a spread-plate assay was used as previously described . This assay was repeated three times for each strain of P. aeruginosa. Lawns of P. aeruginosa were grown on acidic LB (pH adjusted to 5.5 with HCl) plates for 15 h at 37 °C, conditions which induce antimicrobial peptide resistance mechanisms . The plates were flooded with sterile water to harvest the bacteria. Cultures were diluted with sterile water to an OD600 of 0.1. In microcentrifuge tubes, 200 mL of bacteria were incubated at 37 °C with shaking for 3 h with various concentrations of a g-core motif defensin peptide (0, 2.5, 5, 10, 20, or 30 mg/mL). After the defensin peptide treatment, 10-fold serial dilutions were made, and 100 mL were plated in triplicate onto LB plates. Colony forming units (CFUs) were counted for P. aeruginosa after incubation for 24 h at 37 °C. Regression of the average CFUs across experimental replications versus the defensin peptide concentration was used to create a dose response curve using Microsoft Excel 2016. From these dose response curves, the IC50 value, the amount of g-core motif defensin peptide needed to inhibit the growth of bacterial strains by 50%, was calculated. The IC50 values are presented as mean ± standard error from the three repeated experiments.
Lux-reporter gene expression assay
Lux-reporter gene expression assays, adapted from Mulcahy et al. (2008), were performed in a high-throughput manner using 96-well microplates. P. aeruginosa cultures were grown overnight in acidic LB broth adjusted to a pH of 5.5. Overnight cultures were diluted by 1000 into LB broth, and 150 µL of diluted culture medium with g-core motif defensin peptide added at a sublethal concentration (0, 5, 15, or 30 mg/mL) was added to flat clear bottom 96-well microplates (Corning, Corning, NY) and overlaid with 50 µL of mineral oil to prevent evaporation. As a positive control, the antibiotic, polymyxin B, which is known to cause high gene induction of the lux-reporter strains, was added at a sublethal concentration of 0.5 mg/mL. Samples were assayed in triplicate. Microplate cultures were incubated at 37 °C for 18 h in a Synergy H1 microplate reader (BioTek, Winooski, VT) with optical density (600 nm) and luminescence (counts per second [CPS]) readings taken every 20 min throughout the incubation period. Gene expression values were normalized to growth (CPS/OD600).
Assessment of bacterial membrane permeability through fluorescent microscopy
The PA4774::lux and PA3553::lux strains of P. aeruginosa were grown overnight in acidic LB broth adjusted to a pH of 5.5. Overnight cultures were diluted by 1000 in sterile water. In microcentrifuge tubes, 150 µL of the diluted bacterial suspension was treated with 30 µg/mL of either the MtDef4 or MtDef5 𝛾-core peptide and incubated at 37 °C for 3 hours with shaking. Defensin treated bacteria were stained using a LIVE/DEAD BacLight Bacterial Viability Kit (Thermo Fisher) following the manufacturer’s instructions. On a slide with one droplet of BacLight mounting oil, 5 µL of the stained bacterial suspension was observed using fluorescent microscopy.
Pseudomonas syringae pv. syringae transposon mutagenesis
An EZ-Tn5 <R6K𝛾ori/KAN-2> Tnp Transposome Kit (Lucigen, Middleton, WI) was used to generate mutants of Pseudomonas syringae pv. syringae strain ALF3 through random transposon insertions. The transposome was transformed into the ALF3 strain using the P. syringae pv. syringae electroporation protocol previously described by Scholz-Schroeder . The transformed bacteria were plated onto NBY agar plates with 50 mg/L kanamycin and incubated at 25 °C for 48 h. Colonies were pooled by flooding the plates with sterile water. Bacterial cultures were diluted with sterile water to an OD600 of 0.1. In microcentrifuge tubes, the MtDef4 g-core motif peptide at 80 mg/mL, approximately 10 times the IC50 concentration, was added to 200 mL of the transformed bacteria, and the cultures were incubated at 25 °C with shaking for 3 h. After the defensin treatment, 10-fold serial dilutions were made and 100 mL were plated in triplicate onto NBY plus kanamycin plates. Plates were grown at 25 °C overnight. Single colonies were selected, restreaked on NBY plus kanamycin plates, grown overnight at 25 °C, and the defensin treatment was repeated. From the recovered P. syringae pv. syringae mutants resistant to the MtDef4 g-core motif peptide, genomic DNA was extracted and digested with EcoRI (NEB, Ipswich, MA). The DNA was self-ligated using T4 DNA ligase (NEB). Electrocompetent TransforMax EC100D pir-116 E. coli (Lucigen) were electroporated with 2 mL of the ligation mix. The transformed E. coli were plated on LB agar plus 50 mg/L kanamycin and grown overnight at 37 °C. Plasmid DNA was extracted using a QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA). The plasmid DNA was Sanger sequenced on both sides of the transposon insertion at the University of Minnesota Genomics Center using the supplied primers from the Tnp Transposome kit, KAN-2 FP-1 (5’-ACCTACAACAAAGCTCTCATCAACC -3’) and R6KAN-2 RP-1 (5’- CTACCCTGTGGAACACCTACATCT-3’). The resulting DNA sequences near the transposon insertion were validated using Sequencer (Gene Codes Corporation, Ann Arbor, MI). Nucleotide BLAST searches using the Pseudomonas Genome Database  were performed on the DNA sequences near the transposon insertion site to identify the locations in the ALF3 genome of the insertions and the corresponding interrupted genes with annotations.