Field pennycress (Thlaspi arvense L.) a plant in the mustard family is emerging as a premier winter cover crop for biofuel and edible oil production for the Midwestern agricultural rotation system because of its ease of genetic manipulation high oil content winter hardiness and its ability minimize soil erosion and leeching of nitrogen into waterways. Since it is grown off- season between corn and soybean crops it does not require crop displacement and avoids the 'food-vs-fuel' debate. However like other crops in the mustard family pennycress is susceptible to fungal diseases. Under intensive cultivation in the field the occurrence of fungal diseases will be more frequent and pose a major threat to pennycress production. Among several fungal diseases of pennycress Alternaria black spot and Rhizoctonia root and crown rot pose a major threat to reduce the quantity and quality of seed production. Durable disease resistance which overcomes the limits imposed by conventional breeding and replaces unsustainable chemical fungicides is urgently needed for adoption of pennycress as a commercially viable oilseed crop in the USA. Plants express various antimicrobial peptides for defense. Defensins and defensin-like peptides are small cysteine-rich antifungal peptides that offer an exciting opportunity for disease control in pennycress. Although they have been studied for the last several years defensin and defensin-like peptides that inhibit fungal growth at low micromolar or submicromolar concentrations have been discovered only recently. These peptides also inhibit fungal growth using different modes of action (MOA) and thus peptides with different MOA can be deployed in pennycress for long-lasting disease resistance. The overall goal of this proposal is to harnessthe potential of antifungal defensins and defensin-like peptides for control of Alternaria blackspot and Rhizoctonia root and crown rot in pennycress without compromising complex agronomic traits such as growth and yield. In preliminary studies we have generated transgenic pennycress lines expressing highly potent antifungal defensin MtDef5 and a defensin-likepeptide NCR2 from a model legume Medicago truncatula. Homozygous lines expressing these peptides will be tested for resistance to our target pathogens in the greenhouse. In addition insilico analysis has revealed the presence of 29 genes encoding defensin in the genome of pennycress. Based on the presence of cationic and hydrophobic amino acids we hypothesize that some of these peptides will have potent antifungal activity against our target pathogens. In this proposal we will characterize the antifungal activity of these defensins. In addition pathogen- responsive promoters will be identified and used for expression of the lead antifungal peptides to avoid any deleterious effects on complex agronomic traits such as yield and winter hardiness. Genes encoding potent antifungal defensins will also be amenable to gene-editing using theCRISPR-Cas9 approach in future. Specific objectives of this Phase I proposal are: 1. Characterize transgenic pennycress lines expressing MtDef5 and NCR2 for resistance to Alternaria brassicicola and Rhizoctonia solani.2. Determine in vitro antifungal activity of pennycress defensins against field isolates of Alternaria brassicicola and Rhizoctonia solani.3.Identify pathogen-responsive promoters for controlled expression of antifungal peptides in pennycress. The successful completion of Phase I research will identify specific antifungal peptides for commercial development as antifungal agents. Phase II research will focus on development of fungal resistance strategies including CRISPR/Cas9-mediated defensin gene optimization and demonstrating efficacy of the selected antifungal peptides for disease control.