**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** THE INTERACTIONS BETWEEN MICROBES AND PLANTS IN THE SOIL ARE CRITICAL FOR THE FITNESS OF PLANTS AND CROP YIELD. THESE INTERACTIONS, TO A SIGNIFICANT EXTENT, ARE MEDIATED THROUGH SMALL ORGANIC MOLECULES KNOWN AS SECONDARY METABOLITES, SYNTHESIZED BY THE MICROBES. MANY OF THESE MOLECULES HAVE BEEN FOUND TO BE DIRECTLY BENEFICIAL OR DELETERIOUS IN THESE INTERSPECIES INTERACTIONS. DESPITE THE IMPORTANCE OF THESE MOLECULES, WE ONLY HAVE A VERY LIMITED INVENTORY OF THE MOLECULES THAT CAN PRODUCED BY THE MICROBES, AS WELL AS THEIR BIOLOGICAL ACTIVITIES. THIS IS DUE TO THE DIFFICULTIES IN DIRECTLY ISOLATING THE MOLECULES IN SOIL, AND THE INABILITY TO PRODUCE THESE MOLECULES UNDER LABORATORY CONDITIONS WHERE THE COMPLEX SOIL ENVIRONMENT CANNOT BE REPLICATED. A COMPLETE UNDERSTANDING OF THE CHEMICAL INVENTORY OF A ROOT-COLONIZING FUNGUS IS THE GOAL OF THIS PROPOSAL, THE CHANGE IN KNOWLEDGE BROUGHT FORTH BY THE FINDING IN THIS PROJECT CAN LEAD TO BETTER UNDERSTANDING OF SMALL-MOLECULE MEDIATED INTERACTIONS AND THE POTENTIAL APPLICATION OF THESE MOLECULES DIRECTLY TO BENEFIT AGRICULTURE.SPECIFICALLY, WE WILL EXAMINE THE EFFECTS OF METABOLITES THAT CAN BE PRODUCED BY TRICHODERMA HARZIANUM T-22 (THT22) ON MODEL PLANTS SUCH AS A. THALIANA AND TOMATO. THT22 IS A WIDELY USED BIOCONTROL FUNGUS THAT HAS WELL DOCUMENTED BENEFICIAL ACTIVITIES FOR PLANT HOSTS, INCLUDING INCREASED GROWTH, INCREASED IMMUNITY TOWARDS PATHOGENIC FUNGI AND INCREASED FITNESS DURING NUTRIENT STARVATION CONDITIONS. OF THE HANDFUL OF SMALL MOLECULES THAT HAVE BEEN ISOLATED FROM THT22, MOST HAVE BEEN SHOWN TO PLAY IMPORTANT ROLES IN CONFERRING THE POSITIVE TRAITS. HOWEVER, SURVEY OF THE THT22 GENOME REVEALED THE POTENTIAL TO PRODUCE AT LEAST SIXTY SECONDARY METABOLITES. MOST OF THESE PATHWAYS ARE SILENT UNDER LABORATORY CONDITIONS AND THE METABOLITES ARE UNKNOWN. WE HYPOTHESIZE THAT DURING PLANT-FUNGI INTERACTIONS, MANY OF THESE PATHWAYS WILL BE TRANSCRIPTIONALLY ACTIVE, LEADING TO THE BIOSYNTHESIS OF NEW METABOLITES THAT MAY BE IMPORTANT TO MEDIATE THE BENEFICIAL INTERACTIONS. WE WILL USE SYSTEMS AND SYNTHETIC BIOLOGY APPROACHES TO MINE THESE BIOSYNTHETIC PATHWAYS AND THE ENCODED METABOLITES; TO UNDERSTAND THE REGULATION OF THESE PATHWAYS; TO TEST THE EFFECTS OF PLANTS AND ANTAGONISTIC FUNGI TREATED WITH THESE METABOLITES; AND IDENTIFY MOLECULAR TARGETS OF THE BIOACTIVE METABOLITES.