Progress 01/01/03 to 02/28/05
Outputs
4d Progress report. This report serves to document research conducted under a Specific Cooperative Agreement between Boyce Thompson Institute, a co-investigator on a grant from the National Research Initiative and ARS. Additional details of research can be found in the report for the parent project CRIS 1907-22410-003-00D "Microbial and Genetic Resources for Biological Control and High-Value Uses" and the related project CRIS 1907-22410-003- 05T "Are destruxins virulence factors for the fungus Metarhizium anisopliae?". The overall project is directed toward defining the role of destruxins, a family of biologically active peptides, in the disease process of the insect pathogenic fungus Metarhizium anisopliae. Destruxins are cyclic depsipeptides with chemical features suggesting synthesis by a nonribosomal peptide synthetase. The research conducted through the SCA was to conduct the molecular analyses of a peptide synthetase gene predicted to play a role in the
biosynthesis of destruxins. The genetically altered mutants created were then assayed for destruxins production in vitro and in vivo and for virulence against their hosts in insect bioassays by USDA collaborators, D. Gibson and J. Vandenberg. The peptide synthetase gene fragment ma267 (expressed after 24 hr of growth of isolate 2575 (WT) on insect cuticle medium) was targeted as a putative destruxin peptide synthetase gene because: 1) ma267 detects DNA polymorphisms that correlate with levels of destruxin production in vitro and 2) ma267 gene expression is positively correlated with the timing of destruxin production. Agrobacterium tumefaciens-mediated transformation of M. anisopliae was conducted using the bar gene conferring resistance to glufosinate-ammonium as a selective marker. Transformants were screened by PCR and Southern analysis to identify isolates in which ma267 was disrupted; five transformants were identified as knockout (KO) strains and four were characterized. All
KO mutants are mitotically stable. Few detectable differences in phenotype were observed in the KO mutants compared to control strains. Three KO transformants generally exhibited growth rates, asexual sporulation, and destruxin production (done by USDA) comparable to controls; one of these mutants grew slightly but significantly slower and produced yellow pigment associated with sporulation a day earlier than the others. Otherwise, no other major differences were detected. Based on these results, we concluded that gene ma267 does not encode the destruxins NRPS. A fourth KO transformant (B1-3) has an additional uncharacterized mutation correlated with overproduction of metabolites not previously reported from Metarhizium. This mutant was further characterized in the lab of the USDA collaborator, D. Gibson. Several new NRPS gene fragments from cDNA of destruxins- producing cultures have been cloned. These will serve as targets for the next gene disruption studies to identify the
destruxins NRPS. Co-authored manuscripts summarizing the above-described studies are in preparation by our three research groups (Churchill, Gibson, and Vandenberg). 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Churchill, A.C.L. 2005. Linking ESTs to gene function and secondary metabolite discovery in Metarhizium anisopliae. Fungus Division Symposium: Emerging Genomics of Fungal Entomopathogens, 38th Annual Meeting of the Society for Invertebrate Pathology, Anchorage, AK.
Impacts
(N/A)
Publications
|
Progress 10/01/03 to 09/30/04
Outputs
4. What were the most significant accomplishments this past year? D. Progress Report: This report serves to document research conducted under a Specific Cooperative Agreement between ARS and Boyce Thompson Institute. Additional details of this research can be found in the report of the parent project 1907-22410-002-00D "Microbial and Plant Genetic Resources for Biological Control and High-Value Uses". Although fungal biological control agents can provide an effective means of controlling insect pests and reducing agriculture's dependence on chemical pesticides, our understanding of the components needed for effective biological control is still rudimentary. One of the leading candidates for broad-spectrum biological control of insects is the fungal pathogen, Metarhizium anisopliae. The fungus is known to produce a family of biologically active compounds called destruxins, which display a wide range of biological effects in laboratory tests including activity against
insects, plants, and microbes. It is not clear whether destruxins are produced during the infection process in insects and whether these compounds are necessary for causing disease in insects. Definitive proof of the role of destruxins in the biology of the fungus is required to rigorously evaluate the efficacy and safety of this fungal biocontrol agent. DNA analyses were conducted with several gene fragments potentially involved in destruxin biosynthesis to identify hybridization patterns that correlate with levels of destruxin production in liquid culture. Using a time course study, we identified biosynthetic genes that are expressed concomitantly with destruxin production in culture, along with a number of genes involved in secondary metabolism. Several independent transformants generated via an Agrobacterium-mediated transformation system are being evaluated for destruxin production in culture (see 22410-002-05T). These strains are being compared to a wild type,
destruxin-producing isolate to assess changes in their abilities to infect and kill insects under lab conditions. This research supports the development of alternatives to chemical pest controls by contributing to the knowledge base of what defines a safe and effective biological control agent.
Impacts
(N/A)
Publications
|
|