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.