The goal of the proposal is to comprehensively understand the regulation, biosynthesis and bioactivities of small molecule metabolites produced by biofertilizer fungus Trichoderma harzianum T-22 (ThT22), and their roles in mediating microbe-plant and microbe-microbe interactions in the rhizosphere. ThT22 is one of the most widely applied biocontrol agents worldwide. Its strategic deployment is highly useful in protecting crops as well as ornamental plants from biotic and abiotic stress. ThT22 represents a remarkably rich treasure trove of secondary metabolites by harboring more than fifty biosynthetic gene clusters. We will establish a complete inventory of secondary metabolites of ThT22 based on genomic information using systems and synthetic biology approaches and assess the effects of the metabolites on plant robustness, nutrient utilization, and soil compositions.The objectives of the projects are:1: Establish a broader inventory of secondary metabolites of ThT22. Only two metabolites have been connected to their biosynthetic genes in ThT22. In this objective, we will use systems and synthetic biology approaches to mine the uncharacterized and transcriptionally silent biosynthetic pathways, as well as to understand conditions that activate the transcription of the pathways.2: Assess the biological activities of metabolites. The biological activities of the ThT22 metabolites in the context of plant-fungi and fungi-fungi interactions will be assessed. We will grow plants in the presence of purified compounds and monitor the fitness and transcriptomic responses of plants. Compared to plant responses in the presence of ThT22, these data will reveal which small molecules or combinations of molecules can elicit the same responses. Antifungal activities of ThT22 metabolites will be assayed in the presence of antagonistic plant pathogens.3: Investigate the mode of action of a ThT22 metabolite. In this objective we will perform a focused study on the ThT22 metabolite harzianic acid to reveal its mode of action. We propose that harzianic acid is likely to target acetolactate synthase (ALS) to inhibit plant growth at elevated concentrations. We will fully characterize this mode of action of harzianic acid, understand ThT22 self-resistance and observe plant-responses when the compound is applied.