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POTATO IS THE SIXTH LARGEST CROP IN THE UNITED STATES IN TOTAL HARVEST VALUE. DISEASES OF POTATO ACCOUNT FOR BILLIONS OF DOLLARS OF LOSS IN THE UNITED STATES AND WORLD-WIDE ANNUALLY. AMONG THE MAJOR SOILBORNE BACTERIAL DISEASES OF POTATO ARE SOFT ROT, AND COMMON SCAB. THERE ARE NO EFFICACIOUS PESTICIDES OR SOIL FUMIGANTS FOR TREATING THESE DISEASES. IN THIS SEED GRANT, WE PROPOSE TO DEVELOP ALTERNATIVE ANTIMICROBIALS (LYTIC ENZYMES) TO KILL THE CAUSATIVE AGENTS OF SOFT ROT (DICKEYA SPP.) AND COMMON SCAB (STREPTOMYCES SPP.). GENOMIC SEQUENCES OF PATHOGENIC STREPTOMYCES POTATO ISOLATES FROM THE CLARKE LAB WILL BE SCREENED FOR CANDIDATE PROPHAGE ENDOLYSIN GENES. MULTIPLE REPRESENTATIVE ENDOLYSINS WILL BE EXPRESSED, PURIFIED AND SCREENED FOR IN VITRO ANTIMICROBIAL ACTIVITY, EFFICACY, AND SPECIES SPECIFICITY DETERMINED AGAINST A REPRESENTATIVE PANEL OF SCAB-CAUSING STREPTOMYCES. IN CONTRAST, DICKEYA LYTIC ENZYMES REQUIRE AN ENGINEERED APPROACH WITH A THREE DOMAIN HARBORING CONSTRUCT CONTAINING: 1) A CELL WALL TARGETING DOMAIN, 2) A PEPTIDOGLYCAN HYDROLASE DOMAIN AND 3) A CELL WALL PENETRATING DOMAIN. IN THIS SEED PROJECT, WE WILL CREATE THE CELL WALL TARGETING DOMAIN BY SCREEING CANDIDATE SCFV ANTIBODIES FOR INDIVIDUAL CLONES THAT RECOGNIZE DICKEYA. THESE ANTIBODIES WILL THEN BE USED IN THE SECOND PHASE OF THIS PROJECT (NEW PROPOSAL) TO GENERATE THE FUSION LYTIC ENZYME TO TARGET AND KILL DICKEYA. FINAL CONSTRUCTION AND TESTING OF THIS ENGINEERED FUSION ENZYME AND TESTING OF BOTH THE STREPTOMYCES AND DICKEYA ALTERNATIVE ANTIMICROBIALS IN POTATO DISEASE ASSAYS WILL BE DESCRIBED IN FUTURE PROPOSALS.THE RESULTS OF THIS PROGRAM WILL NOT ONLY ASSIST TO DEVELOP THE NEXT GENERATION OF A HIGHLY-SKILLED WORKFORCE IN AGRICULTURAL SCIENCE, BUT ALSO FURTHER ENHANCE THE GRADUATE AND UNDERGRADUATE STUDENT TRAINING CAPACITY IN RESEARCH PROGRAMS AT FLORIDA A&M UNIVERSITY.THE PROJECTWILL SYSTEMATICALLY TEACH THE GRADUATE AND UNDERGRADUATE STUDENTS INVOLVED SOPHISTICATED LABORATORY TECHNIQUES WHILE OFFERING THEM OPPORTUNITIES TO INTERACT WITH INDUSTRY/GOVERNMENT COLLABORATORS AND LEARN HOW THESE SKILLS ARE NECESSARY AND APPLICABLE TOWARDS EXCELLENCE IN AGRICULTURAL SCIENCES. THE 2 M.S STUDENTS GRADUATING FROM THIS PROJECT WILL HELP ADDRESS NATIONAL NEEDS AS HIGHLY QUALIFIED, EQUIPPED, AND WELL TRAINED SCIENTISTS.

$172,208FY2021National Institute of Food and AgricultureUSDA

Florida A & M University

Investigators

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POTATO IS THE SIXTH LARGEST CROP IN THE UNITED STATES IN TOTAL HARVEST VALUE. DISEASES OF POTATO ACCOUNT FOR BILLIONS OF DOLLARS OF LOSS IN THE UNITED STATES AND WORLD-WIDE ANNUALLY. AMONG THE MAJOR SOILBORNE BACTERIAL DISEASES OF POTATO ARE SOFT ROT, AND COMMON SCAB. THERE ARE NO EFFICACIOUS PESTICIDES OR SOIL FUMIGANTS FOR TREATING THESE DISEASES. IN THIS SEED GRANT, WE PROPOSE TO DEVELOP ALTERNATIVE ANTIMICROBIALS (LYTIC ENZYMES) TO KILL THE CAUSATIVE AGENTS OF SOFT ROT (DICKEYA SPP.) AND COMMON SCAB (STREPTOMYCES SPP.). GENOMIC SEQUENCES OF PATHOGENIC STREPTOMYCES POTATO ISOLATES FROM THE CLARKE LAB WILL BE SCREENED FOR CANDIDATE PROPHAGE ENDOLYSIN GENES. MULTIPLE REPRESENTATIVE ENDOLYSINS WILL BE EXPRESSED, PURIFIED AND SCREENED FOR IN VITRO ANTIMICROBIAL ACTIVITY, EFFICACY, AND SPECIES SPECIFICITY DETERMINED AGAINST A REPRESENTATIVE PANEL OF SCAB-CAUSING STREPTOMYCES. IN CONTRAST, DICKEYA LYTIC ENZYMES REQUIRE AN ENGINEERED APPROACH WITH A THREE DOMAIN HARBORING CONSTRUCT CONTAINING: 1) A CELL WALL TARGETING DOMAIN, 2) A PEPTIDOGLYCAN HYDROLASE DOMAIN AND 3) A CELL WALL PENETRATING DOMAIN. IN THIS SEED PROJECT, WE WILL CREATE THE CELL WALL TARGETING DOMAIN BY SCREEING CANDIDATE SCFV ANTIBODIES FOR INDIVIDUAL CLONES THAT RECOGNIZE DICKEYA. THESE ANTIBODIES WILL THEN BE USED IN THE SECOND PHASE OF THIS PROJECT (NEW PROPOSAL) TO GENERATE THE FUSION LYTIC ENZYME TO TARGET AND KILL DICKEYA. FINAL CONSTRUCTION AND TESTING OF THIS ENGINEERED FUSION ENZYME AND TESTING OF BOTH THE STREPTOMYCES AND DICKEYA ALTERNATIVE ANTIMICROBIALS IN POTATO DISEASE ASSAYS WILL BE DESCRIBED IN FUTURE PROPOSALS.THE RESULTS OF THIS PROGRAM WILL NOT ONLY ASSIST TO DEVELOP THE NEXT GENERATION OF A HIGHLY-SKILLED WORKFORCE IN AGRICULTURAL SCIENCE, BUT ALSO FURTHER ENHANCE THE GRADUATE AND UNDERGRADUATE STUDENT TRAINING CAPACITY IN RESEARCH PROGRAMS AT FLORIDA A&M UNIVERSITY.THE PROJECTWILL SYSTEMATICALLY TEACH THE GRADUATE AND UNDERGRADUATE STUDENTS INVOLVED SOPHISTICATED LABORATORY TECHNIQUES WHILE OFFERING THEM OPPORTUNITIES TO INTERACT WITH INDUSTRY/GOVERNMENT COLLABORATORS AND LEARN HOW THESE SKILLS ARE NECESSARY AND APPLICABLE TOWARDS EXCELLENCE IN AGRICULTURAL SCIENCES. THE 2 M.S STUDENTS GRADUATING FROM THIS PROJECT WILL HELP ADDRESS NATIONAL NEEDS AS HIGHLY QUALIFIED, EQUIPPED, AND WELL TRAINED SCIENTISTS. · GrantIndex