The major goals of this project are to elucidate the modes of action of two nodule-specific antifungal peptides, NCR13 and NCR044 and test the potential of these peptides to confer resistance to gray mold disease in tomato. Aim1:we will solve 3D structure of NCR13 and determine arrangement of its three predicted disulfide bonds. Disulfide bond engineering and systematic alanine scanning mutagenesis of NCR044 and NCR13 peptides will be performed to identify sequence motifs governing membrane permeabilization, phospholipid binding, internalization and antifungal activity against Botrytis cinerea. In addition, sequence motif(s) required for nucleolus-localization of NCR044 will be elucidated. Aim2:we will identify intracellular targets of NCR044 and NCR13. These experiments will establish if these peptides inhibit protein translation by binding to ribosomal RNA and/or ribosomal protein(s). They will also reveal if these two peptides bind to different intracellular targets for fungal cell killing. The fully completed gapless and annotated 38.8 Mb genome sequence of the B. cinerea genome and its functional genomics tools will provide essential resources for the protein-protein interaction studies. Aim3:We will determine if NCR044 and NCR13 are internalized by the cells of B. cinerea through endocytosis in germlings and how spore germination is inhibited. In addition, we will perform extensive high-resolution live cell imaging to identify mechanisms of internalization, subcellular dynamics and mobility of each peptide using organelle-specific fluorescent proteins and vital dyes with confocal and super-resolution microscopy and correlative electron microscopy. Aim4:we will determine if spray-applied NCR044, NCR13 and their variants confer resistance to gray mold disease in tomato plants. In addition, experiments are proposed to gain insight into peptide-imposed changes in "spore germination transcriptome" in planta.