Location: Sunflower and Plant Biology Research
Project Number: 3060-21220-034-020-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2021
End Date: Dec 31, 2023
1. To test two cysteine-rich peptides GMA4C and NCR455 for their antifungal activity against white mold disease when sprayed on soybean plants; 2. To test the hypothesis that Sclerotinia-inhibitory antifungal peptides potentiate the fungicidal effect of fungicides registered for use on white mold; and 3. To test the hypothesis that constitutive or pathogen-inducible expression of antifungal peptides in transgenic soybean will provide robust control of white mold.
For Objective 1, the peptides NCR455 and GMA4C will be produced in Pichia pastoris and purified. Antifungal activity of these peptides will be determined by transferring plugs of the mycelial growth into control (water) and NCR455- or GMA4C-amended potato dextrose agar (PDA) plates. Antifungal peptide (AFP) concentrations of 1.5, 3, 6 and 12 µM will be used in this assay. The radial growth of the fungus in two perpendicular directions will be measured after 48 hrs. The IC50 concentration of each peptide giving half-maximal response will be determined. Using spray application, we will test these peptide’s antifungal activity against Sclerotinia by measuring their ability to reduce white mold symptoms in soybean. For Objective 2, we will determine if one or both AFPs can increase the sensitivity of a Tetraconazole (Domark)-resistant Sclerotinia isolate to this fungicide. A Tetraconazole-sensitive Sclerotinia isolate will be used as a control. The concentration of Tetraconazole at which a clear difference in the growth of the two isolates is observed will be chosen for antifungal assays in the presence of the IC50 concentration of each peptide. The mycelial plugs of the fungicide resistant and sensitive isolates will each be transferred to PDA plates amended with Tetraconazole fungicide and an AFP. The control plates will contain fungicide alone or an AFP alone. The IC50 values of each antifungal treatment will be determined as described in Objective 1. In order to determine synergistic enhancement of antifungal activity between an AFP and Tetraconazole, standard checkerboard assays will be performed for Tetraconazole+AFP combinations using a 2-fold dilution series and to test synergistic interactions between the two agents, the Fractional Inhibitory Concentration (FIC) will be calculated. The synergistic interaction of AFP and Tetraconazole will be tested in planta using 4-week-old Williams 82 soybean plants using the petiole inoculation method. For Objective 3, chimeric gene cassettes encoding the select AFPs will be constructed using a constitutive FMV 35S promoter or Sclerotinia-inducible promoter and introduced into soybean cultivar Williams 82 using Agrobacterium tumefaciens-mediated transformation. Soybean transformation with these constructs will be performed at the University of Missouri Transformation Facility. Five independent transgenic lines will be generated for each construct. We will analyze these lines for gene copy number and expression of each peptide. Anti-peptide polyclonal antibodies will be used to determine the expression level of each peptide in the leaf tissue of the transgenic lines using Western blot analysis. Homozygous lines containing single copy of the chimeric gene and expressing each peptide at 2 ppm or higher will be tested for resistance to white mold disease.