2011 Annual Report 1a.Objectives (from AD-416) Develop molecular tools for use in evaluating secondary metabolite potential of fungal pathogens of insects using Metarhizium as a model system. 1b.Approach (from AD-416) Construct knockout mutants for analysis of secondary metabolite production using Agrobacterium-mediated transformation of insect pathogenic fungi, predominantly using Metarhizium. Perform bioinformatic analysis of transcriptome libraries and genome data. 3.Progress Report This subordinate project supports Objective 2 of the parent project: Develop molecular tools of functional genomics for use in systematics and in defining secondary metabolites and products of insect pathogenic fungi that affect biological control potential. Two gene clusters were targeted for analysis of which both were presumed to encode for nonribosomally derived peptides. One gene cluster was targeted for disruption while the other gene cluster was overexpressed by reconstruction of the transcription factor mediating its biosynthesis. In the gene disruption study, destruxins were definitively shown to be the product of the gene cluster encoding 6 complete nonribosomal peptide synthetase modules. The deletion mutants had no significant changes in virulence levels against insect larvae of and no changes in morphology and development. Gene expression was detectable at low levels during early growth phase and increased with culture time, and gene transcripts were also detectable in later stages of infected insects and in conidia. A definitive identification of the secondary metabolite induced by over-expression is underway. Monitoring of activities has been conducted by email, face-to-face meetings, and phone calls between the ADODR and the cooperator.