Research objectives consist of constructing binary vectors containing dominant selectable markers which will be transformed into phytopathogenic Penicillium species using A. tumefaciens. Verification of dominant selectable marker integration in Penicillium transformants will be conducted using molecular methods. Conidial germination, germ tube elongation, and organic acid production of Penicillium transformants will be examined in vitro to determine genes and pathways that can be targeted to block spore germination while additional mutant strains will also be assessed for changes in virulence on artificially inoculated apple fruit. Finally, specific mutants that are shown have a discernible virulence phenotype in vitro and or in vivo will be investigated using molecular methods to determine which genes and regulatory circuits are responsible for the non-pathogenic/ reduced virulence phenotype. Pennsylvania State University and the Agricultural Research Service (ARS) desire to enter into this Agreement for the purpose of supporting research to be carried out at ARS and Cooperator facilities. ARS desires the Cooperator to provide goods and services necessary to carry out research of mutual interest within the United States.
Antibiotic resistance cassettes will serve as dominant selectable markers that will be subcloned into binary vectors and transformed into A. tumefaciens. Co-cultivation of P. expansum conidia with A. tumefaciens containing the binary vector(s) will be executed using standard procedures. Integration of the dominant selectable markers into P. expansum will be verified via PCR. Transformants will be analyzed for mitotic stability, cultural morphology, and defects in conidial germination in vitro. Virulence of transformants defective in conidial germination will also be evaluated by inoculating apple fruit and measuring lesion diameters 4 days post inoculation. Strains defective in decay or having significantly reduced virulence, compared to wild-type strains, will be targeted for genetic analysis. Plasmid rescue will be conducted to identify the coding or non-coding genomic region(s) that were interrupted by T-DNA integration of the dominant selectable markers in non-pathogenic and or weakly virulent P. expansum transformants.