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Browsing by Subject "crown rot"

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    Antibacterial Activity of Plant Defensins Against Alfalfa Crown Rot Pathogens
    (2017) Sathoff, Andrew; Velivelli, Siva; Shah, Dilip; Samac, Deborah A
    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 γ-core motif, a cluster of cationic and hydrophobic residues, which is essential for antimicrobial activity. The γ-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 γ-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.
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    Plant Defensins: An Innovative Approach to Control Alfalfa Crown Rot
    (2019-07) Sathoff, Andrew
    Crown rot is a disease complex that reduces alfalfa (Medicago sativa) stand density and causes substantial losses in productivity in all alfalfa-growing areas. To evaluate plant defensins as a potential control for alfalfa crown rot, defensins were screened for antimicrobial activity. MtDef5, a defensin from Medicago truncatula, displayed high activity against both bacterial and fungal crown rot pathogens in vitro. Agrobacterium-mediated transformation was used to create transgenic lines of alfalfa constitutively expressing MtDef5. Disease bioassays demonstrated increased resistance against fungal and bacterial crown rot pathogens in the transgenic lines expressing MtDef5. Transgenic expression of defensins could be utilized to implement an eco-friendly, protein-based strategy that could provide alfalfa with enhanced resistance against crown rot and reciprocal gains in alfalfa yield. Mini-Tn5-lux mutant strains of Pseudomonas aeruginosa with Tn insertions disrupting outer membrane protective modifications were assessed for sensitivity against plant defensin peptides. Also, these strains were evaluated for lux gene expression in response to sublethal plant defensin exposure. A defensin from M. truncatula, MtDef4, induced dose-dependent gene expression of the aminoarabinose modification of LPS and surface polycation spermidine production operons. 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. M. truncatula promoter regions of pathogenesis-related (PR) genes, PR5 and PR10, were identified as being highly up-regulated during the initial stages of infection by root and foliar pathogens. Theses promoters, along with the alfalfa homolog for PR10, were cloned into plant transformation vectors ahead of the beta-glucuronidase (gus) gene. Agrobacterium-mediated transformation was used to create transgenic lines of alfalfa. Quantitative PCR assays were utilized to evaluate pathogen-induced GUS expression. The MtPR10 promoter had greater fold amplifications and greater activity than the MsPR10 and MtPR5 promoters. The MtPR10 promoter is functional in alfalfa for expression of transgenes and up-regulates genes after infection by a wide range of alfalfa pathogens.