Progress 10/01/04 to 09/30/05
Outputs
4d Progress report. This report serves to document research conducted under a Trust Fund Cooperative Agreement with 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 CRIS 1907-22410- 003-06S entitled "Genetic manipulation of destruxins in Metarhizium anisopliae". This project was directed toward defining the role of destruxins, a family of biologically active peptides, in the insect disease process of Metarhizium anisopliae (MA). Destruxins are cyclic depsipeptides with chemical features suggesting synthesis by a nonribosomal peptide synthetase (NRPS). Gene disruption. The NRPS gene fragment ma267, expressed after 24 hr of growth on insect cuticle medium (Freimoser et al. 2003), as a putative DTX NRPS in isolate 2575 (WT) 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 MA using the bar gene conferring resistance to glufosinate-ammonium as a selective marker was used to create mutants. Transformants were screened by PCR and Southern analysis to identify isolates in which ma267 was disrupted; five transformants were identified as knockout (KO) strains. KO mutant analyses. 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 comparable to controls. Based on these results, we concluded that gene ma267 does not encode the destruxin NRPS. A fourth KO transformant (B1-3) has an additional uncharacterized mutation correlated with overproduction of metabolites not previously
reported from a Metarhizium. Insect bioassays. We observed no detectable differences in pathogenicity among four ma267 KO mutants and control strains in multiple bioassays with beet armyworm and Colorado potato beetle. We conclude that the unknown NRPS gene ma267 does not encode a virulence factor for insect disease. Efforts are underway to identify the natural product synthesized by ma267. Destruxins detected in single infected insect larvae. Quantitative estimates of destruxin levels in mycosed larvae from HPLC MS/MS analyses ranged from 1 microgram-10 picograms/insect. Destruxin was not detectable in all cadavers and destruxin estimates for insects killed by KO strains did not differ from those for insects killed by WT strains. Identification of new mycotoxins. The KO mutant B1-3 exhibited a marked increase in the production of yellow pigment compared with all other isolates. Using ESI-MS and 1- and 2-D NMR, we identified two major components, NG-391 and NG-393, previously
reported from Fusarium sp. as stimulants of nerve-cell growth. These compounds are closely related to the fusarins, mutagenic Fusarium mycotoxins that contaminate corn. Detectable levels of these compounds were also found in control strains of MA; levels in KO B1-3 and a second KO mutant were 60- and 20-fold higher, respectively, than the other strains analyzed. HPLC-MS analyses are underway to determine if the fusarin-like compounds are detectable in MA-infected insect larvae. New DTX NRPS targets. Several new NRPS gene fragments have been cloned from cDNA of DTX-producing cultures. These will serve as targets for the next gene disruption studies to identify the DTX NRPS. Manuscripts describing these studies are in preparation.
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Progress 08/29/02 to 02/28/05
Outputs
Progress Report 4d Progress report. This report serves to document research conducted under a Trust Fund Cooperative Agreement with the National Research Initiative and ARS. Additional details of research can be found in the report for the parent project 1907-22410-003-00D "Microbial and genetic resources for biological control and high-value uses". This project was directed toward defining the role of destruxins, a family of biologically active peptides, in the insect disease process of Metarhizium anisopliae. Destruxins are cyclic depsipeptides with chemical features suggesting synthesis by a nonribosomal peptide synthetase (NRPS). Knockout (KO) mutants were created by disrupting a NRPS gene fragment, ma267 predicted to be involved in destruxin production via Agrobacterium tumefaciens-mediated transformation. All KO strains were mitotically stable and phenotypically similar to wild type, but all strains produced comparable levels of destruxins. Based on these
results, we concluded that gene ma267 does not encode the destruxin NRPS. There were no detectable differences in pathogenicity among the KO mutants and wild type strains in multiple bioassays with beet armyworm and Colorado potato beetle. We conclude that the unknown NRPS gene ma267 does not encode a virulence factor for insect disease. Efforts are underway to identify the natural product synthesized by ma267 since it is appears to be expressed early in the infection cycle. Quantitative estimates of destruxin levels in mycosed larvae from HPLC MS/MS analyses ranged from 1 microgram-10 picograms/insect. Destruxin was not detectable in all cadavers and destruxin estimates for insects killed by KO strains did not differ from those for insects killed by WT strains. A fourth KO transformant has an additional uncharacterized mutation evidenced by a marked increase in the production of yellow pigment. We identified two major components, NG-391 and NG-393, previously reported from Fusarium
sp. as stimulants of nerve- cell growth; these compounds have not been previously reported from a Metarhizium. These compounds are closely related to the fusarins, mutagenic Fusarium mycotoxins that contaminate corn. Detectable levels of these compounds were also found in control strains; levels in two KO mutants were 60- and 20-fold higher, respectively, than the other strains analyzed. Several new NRPS gene fragments cloned from cDNA of DTX- producing cultures will serve as targets for the next gene disruption studies. Neither destruxins nor the fusarin-like compounds appear to be required for virulence of the fungus, suggesting that they are dispensable for infection. Although further proof will be needed, this information is relevant to strain selection and development since toxin- deficient strains will be perceived as safer for insect biological control than those with the genetic potential to make such compounds.
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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 Trust Fund Cooperative Agreement between ARS and the National Research Initiative. 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" and the related project 1907- 22410-003-06S "Genetic Manipulation of Destruxins in Metarhizium Anisopliae". One of the leading fungal candidates for broad-spectrum biological insect control, Metarhizium anisopliae, produces a family of biologically active compounds called destruxins. Destruxins display a wide range of biological effects in laboratory tests including activity against insects, plants, and microbes. What is fundamentally unclear, however, is whether destruxins are produced during the infection process within insects and whether these compounds are
necessary for causing disease in insects. A set of core fungal isolates has been examined for destruxin production in culture, and we have used beet armyworm to assess their pathogenicity and virulence in laboratory bioassays. The level of destruxin produced in liquid culture was not an accurate predictor of biological control activity based on insect mortality data. An Agrobacterium-based transformation system was established via collaboration in order to produce transformants disrupted in destruxin production (see 22410-002- 06S). Studies are in progress to compare the independently-generated transformants to a wild type, destruxin-producing isolate for changes in phenotype and development, and to assess their pathogenicity and virulence against insects under lab conditions. Analytical methods have been developed for toxin quantitation in individual insect larvae, and this method is being applied to a series of instar larvae stages infected with the fungus. Results from these
experiments should provide definitive evidence of the role of destruxins in biological control of insects. 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.
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