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Title: Agrobacterium-mediated disruption of a nonribosomal peptide synthetase gene in the invertebrate pathogen Metarhizium anisopliae reveals a peptide spore factor

Author
item MOON, YONG-SUN - BOYCE THOMPSON INST
item Donzelli, Bruno
item Krasnoff, Stuart
item MCLANE, HEATHER - BOYCE THOMPSON INST
item Griggs, Michael
item Cooke, Peter
item Vandenberg, John
item Gibson, Donna
item CHURCHILL, ALICE C.L. - CORNELL UNIVERSITY

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/10/2008
Publication Date: 7/23/2008
Citation: Moon, Y., Donzelli, B., Krasnoff, S., Mclane, H., Griggs, M., Cooke, P.H., Vandenberg, J.D., Gibson, D.M., Churchill, A. 2008. Agrobacterium-mediated disruption of a nonribosomal peptide synthetase gene in the invertebrate pathogen Metarhizium anisopliae reveals a peptide spore factor. Applied and Environmental Microbiology. 74:4366-4380.

Interpretive Summary: Metarhizium anisopliae is at the forefront of efforts to develop insect pathogenic fungi as biocontrol agents as an alternative to chemical pesticides. We have been using a gene-knockout strategy in order to identify compounds that may be involved in the insect host-fungus interaction. This study describes the identity of a peptide synthetase responsible for the production of a family of unique spore-localized peptides (short sequences of amino acids) called serinocyclins. In a comparison of wild type to the mutant strains, we observed no effects on pathogenicity or in spore attachment, germination, and growth, although these compounds have been previously shown to have a sublethal effect on insect behavior. This work adds to our understanding of the basic biology of the fungus and its interaction with the insect host; it also contributes to our knowledge of factors for improving the safety and efficacy of this fungus as a biocontrol agent.

Technical Abstract: Numerous secondary metabolites have been isolated from the insect pathogenic fungus Metarhizium anisopliae, but the roles of these compounds as virulence factors in disease development are poorly understood. We targeted for disruption by Agrobacterium tumefaciens-mediated transformation a putative nonribosomal peptide synthetase (NPS) gene, MaNPS1, using a cDNA expressed after 24 h growth on cockroach cuticle-containing medium. Four of six gene disruption mutants identified were examined further. Chemical analyses showed the presence of serinocyclins, cyclic heptapeptides, in extracts of conidia of control strains, whereas the compounds were undetectable in 'manps1 mutants treated identically or in other developmental stages, suggesting that MaNPS1 encodes a serinocyclin synthetase. Production of the cyclic depsipeptide destruxins, M. anisopliae metabolites also predicted to be synthesized by an NPS, was similar in 'manps1 mutant and control strains, indicating that MaNPS1 does not contribute to destruxin biosynthesis. Surprisingly, a MaNPS1 fragment detected DNA polymorphisms that correlated with relative destruxin levels produced in vitro, and MaNPS1 was expressed concurrently with in vitro destruxin production. 'manps1 mutants exhibited in vitro development and responses to external stresses comparable to control strains. No detectable differences in pathogenicity of the 'manps1 mutants were observed in bioassays against beet armyworm and Colorado potato beetle in comparison with control strains. This is the first report of targeted disruption of a secondary metabolite gene in M. anisopliae, which revealed a novel cyclic peptide spore factor.