Development of Disease Resistant Pennycress as a Novel Oilseed Crop

Field pennycress (Thlaspi arvense L.) a plant in the mustard family is emerging as a premierwinter cover crop for biofuel and edible oil production for the Midwestern agricultural rotationsystem because of its ease of genetic manipulation high oil content winter hardiness and itsability 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 susceptibleto fungal diseases. Under intensive cultivation in the field the occurrence of fungal diseases willbe more frequent and pose a major threat to pennycress production. Among several fungaldiseases of pennycress Alternaria black spot and Rhizoctonia root and crown rot pose a majorthreat to reduce the quantity and quality of seed production. Durable disease resistance whichovercomes the limits imposed by conventional breeding and replaces unsustainable chemicalfungicides is urgently needed for adoption of pennycress as a commercially viable oilseed cropin the USA.Plants express various antimicrobial peptides for defense. Defensins and defensin-likepeptides are small cysteine-rich antifungal peptides that offer an exciting opportunity for diseasecontrol in pennycress. Although they have been studied for the last several years defensin anddefensin-like peptides that inhibit fungal growth at low micromolar or submicromolarconcentrations have been discovered only recently. These peptides also inhibit fungal growthusing different modes of action (MOA) and thus peptides with different MOA can be deployedin 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 complexagronomic traits such as growth and yield. In preliminary studies we have generated transgenicpennycress lines expressing highly potent antifungal defensin MtDef5 and a defensin-likepeptide NCR2 from a model legume Medicago truncatula. Homozygous lines expressing thesepeptides 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 ofpennycress. Based on the presence of cationic and hydrophobic amino acids we hypothesize thatsome of these peptides will have potent antifungal activity against our target pathogens. In thisproposal 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 toavoid 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 toAlternaria brassicicola and Rhizoctonia solani.2. Determine in vitro antifungal activity of pennycress defensins against field isolates ofAlternaria brassicicola and Rhizoctonia solani.3.Identify pathogen-responsive promoters for controlled expression of antifungal peptides inpennycress.The successful completion of Phase I research will identify specific antifungal peptides forcommercial development as antifungal agents. Phase II research will focus on development offungal resistance strategies including CRISPR/Cas9-mediated defensin gene optimization anddemonstrating efficacy of the selected antifungal peptides for disease control.