POWDERY MILDEW (PM), A FUNGAL DISEASE, MAINLY CAUSED BY GOLOVINOMYCES AMBROSIAE HAS DEVASTATING IMPACTS ON INDUSTRIAL HEMP THROUGHOUT NORTH AMERICA RESULTING IN SIGNIFICANT CROP LOSS. UNFORTUNATELY, THE MOLECULAR ARCHITECTURE UNDERLYING PM RESISTANCE (PMR) IN HEMP IS ONLY POORLY UNDERSTOOD. THIS LACK OF KNOWLEDGE ABOUT PMR REGULATORS AND THE RESULTING ABSENCE OF RESISTANT GENETIC MATERIAL IS A MAJOR PROBLEM FOR HEMP PRODUCTION IN THE US. THE GOAL OF THE PROPOSED RESEARCH IS TO GENERATE A GENETIC PMR TOOLBOX WHICH WILL ACCELERATE THE DEVELOPMENT OF PM-RESISTANT HEMP STRAINS ALLOWING THE CREATION OF HEMP PLANTS WITH DESIRABLE TRAITS MORE QUICKLY THAN TRADITIONAL BREEDING METHODS. TO ACHIEVE THIS, WE WILL ILLUMINATE THE EARLY INFECTION STAGES OF THE PM-CAUSING FUNGUS GOLOVINOMYCES AMBROSIAE ON PM SUSCEPTIBLE CULTIVARS EMPLOYING VARIOUS STATE-OF-THE-ART GENOMIC TOOLS (RNA-SEQ, ATAC-SEQ) AT TISSUE AND SINGLE NUCLEI LEVEL RESOLUTION. THIS WILL LEAD TOGETHER WITH GENETIC MAPPING OF RESISTANCE GENES AND THE MILDEW LOCUS O FAMILY (MLO) GENE FAMILY TO A CANDIDATE GENE LIST OF PMR REGULATORS WHICH WILL BE DISRUPTED USING CRISPR-CAS9. DISRUPTION OF SUSCEPTIBILITY GENES WILL COMPROMISE THE ABILITY OF G. AMBROSIAE TO SUCCESSFULLY COLONIZE HEMP PLANTS WHEREAS THE CRISPR/CAS9-MEDIATED DELETION OF RESISTANCE GENES WILL CAUSE THE OPPOSITE LEADING TO INCREASED SUSCEPTIBILITY. THE RESULTING GENETIC MATERIAL WILL BE TESTED FOR PMR IN FIELD TRIALS AND PROMISING STRAINS WILL BE RAPIDLY MADE AVAILABLE TO INDUSTRY AND RESEARCH STAKEHOLDERS. THIS RESEARCH, DRIVEN BY GROWER REQUESTS, HAS THE POTENTIAL TO GREATLY BENEFIT THE HEMP INDUSTRY BY IMPROVING CROP PERFORMANCE, MITIGATING CROP LOSS AND REDUCING THE NEED FOR HARMFUL PESTICIDES.