Progress 09/01/02 to 02/28/05
Outputs
This project is directed toward defining the role of destruxins (DTX), a family of biologically active peptides, in the insect disease process of Metarhizium anisopliae (MA). DTX are cyclic depsipeptides with chemical features suggesting synthesis by a nonribosomal peptide synthetase (NRPS). Gene disruption. We targeted 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 DTX production in vitro and 2) ma267 gene expression is positively correlated with the timing of DTX production. We developed Agrobacterium tumefaciens-mediated transformation of MA 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. 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 DTX production comparable to controls. Based on these results, we concluded that gene ma267 does not encode the DTX 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. DTX detected in single infected insect larvae. Quantitative estimates of DTX levels in mycosed
larvae from HPLC MS/MS analyses ranged from 1 microgram-10 picograms/insect. DTX was not detectable in all cadavers and DTX 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.
Impacts
A major impediment in the development of biocontrol agents has been an incomplete understanding of the genetic factors that make them efficacious. Further studies are required to determine the roles that secondary metabolites play in the biology of MA. Two important points can be made from our studies:1) since we detected DTX in only a subset of insects killed by MA, our results suggest they may not be important virulence factors for the fungus. If they were critical, we would expect to detect them invariably in all cadavers. 2) We discovered a previously unreported group of mycotoxins, closely related to the mutagenic fusarins produced by Fusarium, in MA. Note that overproduction of these mycotoxins by two KO mutants did not contribute to increased virulence against insect hosts, suggesting that they may be dispensable for these insect-fungus interactions. Further genetic analyses are required to confirm this hypothesis. If the fusarin-like compounds, as well as DTX,
are shown genetically not to function as virulence factors for MA, this information is relevant to strain selection and development since toxin-deficient strains will be perceived as safer for insect biocontrol than those with the genetic potential to make such compounds. This research supports the development of alternatives to chemical pest controls by contributing to the knowledge base that defines a safe and effective biological control agent. Only by understanding the genetics of disease processes used by MA can we enhance its efficacy and safety.
Publications
- 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.
- Moon, Y.-S., Krasnoff, S.B., Donzelli, B.G.G., Vandenberg, J.D., Gibson, D.M., and Churchill, A.C.L. 2005. Targeted disruption of a peptide synthetase gene in Metarhizium anisopliae has no effect on destruxins production or virulence against insects. 38th Annual Meeting of the Society for Invertebrate Pathology, Anchorage, AK.
- Krasnoff, S.B., Moon, Y.-S., Donzelli, B.G.G., Churchill, A.C.L., Vandenberg, J.D., and Gibson, D.M. 2005. Antibacterial toxins are overproduced in a gene disruption mutant of Metarhizium anisopliae. 38th Annual Meeting of the Society for Invertebrate Pathology, Anchorage, AK.
- Moon, Y.-S., Krasnoff, S.B., Vandenberg, J.D., Gibson, D.M., and Churchill, A.C.L. 2005. Genetic analyses of a peptide synthetase gene from the insect pathogen Metarhizium anisopliae. XXIII Fungal Genetics Conference, Pacific Grove, CA, Fungal Genet. Newsl. 52 (Suppl):191.
- Moon, Y.-S., Churchill, A.C.L., Krasnoff, S.B., Vandenberg, J.D., and Gibson, D.M. 2004. Genetic analyses of putative virulence factors of Metarhizium anisopliae. 52nd Annual Meeting of the Entomological Society of America, Salt Lake City, UT.
- Churchill, A.C.L., Krasnoff, S.B., Moon, Y.-S., McLane, H., Williams, J., Vandenberg, J.D., and Gibson, D.M. 2003. Characterization of in vitro destruxin production, pathogenicity, and RFLP patterns of peptide synthetase genes in Metarhizium anisopliae. 36th Annual Meeting of the Society for Invertebrate Pathology, Burlington, VT.
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Progress 10/01/03 to 09/30/04
Outputs
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. A fragment of a peptide synthetase (PS) gene identified in an EST library of M. anisopliae was expressed concurrently with destruxin production. We
developed a procedure for Agrobacterium tumefaciens-mediated transformation of M. anisopliae, which uses ungerminated spores of the fungus as the target for transformation rather than protoplasts. We used the bar gene conferring resistance to the herbicide glufosinate-ammonium as a selective marker based on its previous use for transformation of M. anisopliae. More than 180 Finale-resistant transformants were selected and screened by PCR to identify isolates in which the wild type gene PS fragment was disrupted by homologous integration of the gene replacement vector. Five transformants so far have been identified as putative knockout strains by PCR using several sets of primer pairs that amplify distinct products depending on whether the gene replacement vector has integrated ectopically or at the homologous site of the gene. Characterization of the mutants for mitotic stability, fungal morphology and development, and altered pathogencity or virulence are currently in progress and
scheduled to be completed within the next six months. Destruxins A, B, and E, as well as minor analogs, were isolated and purified from M. anisopliae fermentations by repetitive chromatography on silica gel followed by RP HPLC. Compound identities were verified by comparing 1H NMR and mass spectra (LRESIMS) with spectral data reported in the literature. Larvae, inoculated with fungal spores, were examined daily for mortality up to 5 days post-treatment, then processed for analysis. The total destruxin profile of a typical dead larva that exhibited characteristic mycotic lesions but not fungal outgrowth was characterized by LCMS. Dextruxins A and B, and E, as well as minor destruxin analogs were readily detectable at 50 pg and occurred at levels as high as 3.6 and 2.2 micrograms destruxins A and B respectively per insect. We are currently measuring sample lots of 200 individual larva to validate our quantitative method. This work will be completed within the next six months.
Impacts
Fungal biological control agents have the potential to provide an effective means of controlling insect pests and reducing dependence on chemical pesticides. However, our understanding of the genetic components needed for effective biocontrol is still rudimentary. Definitive proof of the role of the peptide toxin family of destruxins in the biology of the fungus Metarhizium anisopliae is required to rigorously evaluate the efficacy and safety of this fungal biological control agent. 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.
Publications
- Churchill, A.C.L., Krasnoff, S., Moon, Y.-S., McLane, H., Williams, J., Vandenberg, J.,Gibson, D. Characterization of in vitro destruxin production, pathogenicity, and RFLP patterns of peptide synthetase genes in Metarhizium anisopliae. Society of Invertebrate Pathology. 2003. p. 51.
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Progress 10/01/02 to 09/30/03
Outputs
One of the leading fungal candidates for broad spectrum biocontrol of insects, 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 unclear, however, is whether destruxins are produced during the disease process in insects and whether they are invariantly necessary for causing disease. A set of core 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 destruxins produced in liquid culture is not an accurate predictor of biocontrol activity based on insect mortality data. 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. Additionally, we identified biosynthetic genes that are expressed concomitantly with destruxin production in vitro. An Agrobacterium-based transformation system has been established and used in attempts to disrupt a gene predicted to be involved in destruxin production. Several independent knockout (KO) transformants have been selected for further analyses. Studies are underway to compare the KO transformants to ectopic transformants and the wild type isolate to look for changes in destruxin production, colony phenotype and development, and pathogenicity and virulence against insects under laboratory conditions.
Impacts
Fungal biological control agents have the potential to provide an effective means of controlling insect pests and reducing dependence on chemical pesticides. However, our understanding of the genetic components needed for effective biocontrol is still rudimentary. Definitive proof of the role of the peptide toxin family of destruxins in the biology of the fungus Metarhizium anisopliae is required to rigorously evaluate the efficacy and safety of this fungal biological control agent. 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.
Publications
- Churchill, A.C., Krasnoff, S.B., Moon, Y, McLane, H., Williams, J.E., Vandenberg, J.D., Gibson, D.M. Characterization of in vitro destruxin production, pathogenicity, and rflp patterns of peptide synthetase genes in Metarhizium anisopliae. Proceedings of the Society for Invertebrate Pathology. 2003. v. 36. p. 51.
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