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Title: Antibacterial activity of plant defensins against alfalfa crown rot pathogens

item SATHOFF, ANDREW - University Of Minnesota
item VELIVELLI, SIVA - Danforth Plant Science Center
item SHAH, DILIP - Danforth Plant Science Center
item Samac, Deborah - Debby

Submitted to: American Society of Microbiologists Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 2/10/2017
Publication Date: 6/1/2017
Citation: Sathoff, A., Velivelli, S., Shah, D., Samac, D.A. 2017. Antibacterial activity of plant defensins against alfalfa crown rot pathogens. American Society of Microbiologists Annual Conference. New Orleans, Lousiana. June 1-5, 2017.

Interpretive Summary:

Technical Abstract: Alfalfa (Medicago sativa) is the fourth most widely grown crop in the United States. Alfalfa crown rot is a disease complex that severely decreases alfalfa stand density and productivity in all alfalfa-producing areas. Currently, there are no viable methods of disease control. Plant defensins are small cationic antimicrobial peptides with a conserved signature of cysteines. The in vitro and in planta antifungal activity of plant defensins has been extensively studied. However, their antibacterial activity has been less well characterized. Defensins have a gamma-core motif, a cluster of cationic and hydrophobic residues, which is essential for antimicrobial activity. The gamma-core motifs of five synthetic defensins were tested for antibacterial activity against the bacterial pathogens in the alfalfa crown rot disease complex. Full length defensins, expressed using a Pichia pastoris expression system, were tested to compare antibacterial activity. A spread plate method was used to quantify antibacterial activity of defensins. Bacteria were grown out to an OD600 value of 0.1, and a 200 µL culture was incubated with shaking for 3 hours with concentrations of defensin peptide up to 30 µg/mL. The bacteria were serially diluted, and 100 µL was plated on to NBY plates. After 48 hours of incubation, the bacterial colonies were counted. The amount of defensin needed to inhibit growth of pathogen strains by 50% (IC50) was calculated. The core motif of MtDef4 was shown to be the most effective truncated peptide with IC50 values of 3.4 µM against Pseudomonas syringae pv. syringae and 4.52 µM against Xanthomonas alfalfae. Also, the corresponding full length MtDef4 peptide was found to be active against P. syringae pv. syringae and X. alfalfae with IC50 values of 0.43 µM and 0.68 µM, respectively. These experiments show the previously overlooked high biological activity of plant defensins against bacterial pathogens. Additionally, these results indicate that the gamma-core-motif can be used to predict biological activity of the full-length defensin, and that transgenic expression of plant defensins in alfalfa has the potential to lead to improved crown rot resistance.