1887

Abstract

Insect pathogenic fungi such as and have an increasing role in the control of agricultural insect pests and vectors of human diseases. Many of the virulence factors are well studied but less is known of the metabolism of these fungi during the course of insect infection or saprobic growth. Here, we assessed enzyme activity and gene expression in the central carbon metabolic pathway, including isocitrate dehydrogenase, aconitase, citrate synthase, malate synthase (MLS) and isocitrate lyase (ICL), with particular attention to the glyoxylate cycle when and were grown under various conditions. We observed that ICL and MLS, glyoxylate cycle intermediates, were upregulated during growth on 2-carbon compounds (acetate and ethanol) as well as in insect haemolymph. We fused the promoter of the ICL gene () to a marker gene (mCherry) and showed that was upregulated when was grown in the presence of acetate. Furthermore, - was upregulated when fungi were engulfed by insect haemocytes as well as during appressorium formation. Addition of the ICL inhibitor 3-nitroproprionate delayed conidial germination and inhibited appressorium formation. These results show that these insect pathogenic fungi have a flexible metabolism that includes the glyoxylate cycle as an integral part of germination, pathogenesis and saprobic growth.

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2011-01-01
2024-03-28
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References

  1. Amaar Y. G., Moore M. M. 1998; Mapping of the nitrate-assimilation gene cluster ( crnA -– niiA niaD ) and characterization of the nitrite reductase gene ( niiA ) in the opportunistic fungal pathogen Aspergillus fumigatus . Curr Genet 33:206–215
    [Google Scholar]
  2. Araujo-Palomares C. L., Riquelme M., Castro-Longoria E. 2009; The polarisome component SPA-2 localizes at the apex of Neurospora crassa and partially colocalizes with the Spitzenkorper. Fungal Genet Biol 46:551–563
    [Google Scholar]
  3. Arrese E. L., Soulages J. L. 2010; Insect fat body: energy, metabolism, and regulation. Annu Rev Entomol 55:207–225
    [Google Scholar]
  4. Bash R., Lohr D. 2001; Yeast chromatin structure and regulation of GAL gene expression. Prog Nucleic Acid Res Mol Biol 65:197–259
    [Google Scholar]
  5. Bibbins M., Sheffield P. J., Gainey L. D., Mizote T., Connerton I. F. 1998; Promoter analysis of the acetate-inducible isocitrate lyase gene ( acu-3 ) from Neurospora crassa . Biochim Biophys Acta 1442:320–325
    [Google Scholar]
  6. Bidochka M. J., Clark D. C., Lewis M., Keyhani N. O. 2010; Could insect phagocytic avoidance by entomogenous fungi have evolved via selection against soil amoeboid predators?. Microbiology 156:2164–2171
    [Google Scholar]
  7. Chaure P. T., Connerton I. F. 1995; Derepression of the glyoxylate cycle in mutants of Neurospora crassa accelerated for growth on acetate. Microbiology 141:1315–1320
    [Google Scholar]
  8. Cho E. M., Boucias D., Keyhani N. O. 2006a; EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana . II. Fungal cells sporulating on chitin and producing oosporein. Microbiology 152:2855–2864
    [Google Scholar]
  9. Cho E. M., Liu L., Farmerie W., Keyhani N. O. I. 2006b; EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana . Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores and submerged conidia. Microbiology 152:2843–2854
    [Google Scholar]
  10. Clarkson J. M., Charnley A. K. 1996; New insights into the mechanisms of fungal pathogenesis in insects. Trends Microbiol 4:197–203
    [Google Scholar]
  11. Cubero B., Scazzocchio C. 1994; Two different, adjacent and divergent zinc finger binding sites are necessary for CREA-mediated carbon catabolite repression in the proline gene cluster of Aspergillus nidulans . EMBO J 13:407–415
    [Google Scholar]
  12. De Lucas J. R., Gregory S., Turner G. 1994; Analysis of the regulation of the Aspergillus nidulans acuD gene, encoding isocitrate lyase, by construction of a hybrid promoter. Mol Gen Genet 243:654–659
    [Google Scholar]
  13. Dunn M. F., Ramirez-Trujillo J. A., Hernandez-Lucas I. 2009; Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology 155:3166–3175
    [Google Scholar]
  14. Ebel F., Schwienbacher M., Beyer J., Heesemann J., Brakhage A. A., Brock M. 2006; Analysis of the regulation, expression, and localisation of the isocitrate lyase from Aspergillus fumigatus , a potential target for antifungal drug development. Fungal Genet Biol 43:476–489
    [Google Scholar]
  15. Fang W., Pei Y., Bidochka M. J. 2006; Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens . Can J Microbiol 52:623–626
    [Google Scholar]
  16. Fang W., Pei Y., Bidochka M. J. 2007; A regulator of a G protein signalling (RGS) gene, cag8 , from the insect-pathogenic fungus Metarhizium anisopliae is involved in conidiation, virulence and hydrophobin synthesis. Microbiology 153:1017–1025
    [Google Scholar]
  17. García I., Gonzalez R., Gomez D., Scazzocchio C. 2004; Chromatin rearrangements in the prnD prnB bidirectional promoter: dependence on transcription factors. Eukaryot Cell 3:144–156
    [Google Scholar]
  18. Hallsworth J. E., Magan N. 1994; Effect of carbohydrate type and concentration on polyhydroxy alcohol and trehalose content of conidia of three entomopathogenic fungi. Microbiology 140:2705–2713
    [Google Scholar]
  19. Hu G., St Leger R. J. 2002; Field studies using a recombinant mycoinsecticide ( Metarhizium anisopliae ) reveal that it is rhizosphere competent. Appl Environ Microbiol 68:6383–6387
    [Google Scholar]
  20. Idnurm A., Howlett B. J. 2002; Isocitrate lyase is essential for pathogenicity of the fungus Leptosphaeria maculans to canola ( Brassica napus . Eukaryot Cell 1:719–724
    [Google Scholar]
  21. Jarrold S. L., Moore D., Potter U., Charnley A. K. 2007; The contribution of surface waxes to pre-penetration growth of an entomopathogenic fungus on host cuticle. Mycol Res 111:240–249
    [Google Scholar]
  22. Jia Y. K., Bécam A. M., Herbert C. J. 1997; The CIT3 gene of Saccharomyces cerevisiae encodes a second mitochondrial isoform of citrate synthase. Mol Microbiol 24:53–59
    [Google Scholar]
  23. Kim K. S., Rosenkrantz M. S., Guarente L. 1986; Saccharomyces cerevisiae contains two functional citrate synthase genes. Mol Cell Biol 6:1936–1942
    [Google Scholar]
  24. Kim S.-Y., Park J.-S., Oh K.-B. 2006; Effects of isocitrate lyase inhibitors on spore germination and appressorium development in Magnaporthe grisea . J Microbiol Biotechnol 16:1158–1162
    [Google Scholar]
  25. Kumar M. J., Jamaluddin M. S., Natarajan K., Kaur D., Datta A. 2000; The inducible N -acetylglucosamine catabolic pathway gene cluster in Candida albicans : discrete N -acetylglucosamine-inducible factors interact at the promoter of NAG1. Proc Natl Acad Sci U S A 97:14218–14223
    [Google Scholar]
  26. Lorenz M. C., Fink G. R. 2001; The glyoxylate cycle is required for fungal virulence. Nature 412:83–86
    [Google Scholar]
  27. Prasad S. V., Ryan R. O., Law J. H., Wells M. A. 1986; Changes in lipoprotein composition during larval–pupal metamorphosis of an insect, Manduca sexta . J Biol Chem 261:558–562
    [Google Scholar]
  28. Pupin A. M., Messias C. L., Piedrabuena A. E., Roberts D. W. 2000; Total lipids and fatty acids of strains of Metarhizium anisopliae . Braz J Microbiol 31:121–128
    [Google Scholar]
  29. Ravagnani A., Gorfinkiel L., Langdon T., Diallinas G., Adjadj E., Demais S., Gorton D., Arst H. N. Jr, Scazzocchio C. 1997; Subtle hydrophobic interactions between the seventh residue of the zinc finger loop and the first base of an HGATAR sequence determine promoter-specific recognition by the Aspergillus nidulans GATA factor AreA. EMBO J 16:3974–3986
    [Google Scholar]
  30. Rose T. M., Henikoff J. G., Henikoff S. 2003; CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design. Nucleic Acids Res 31:3763–3766
    [Google Scholar]
  31. Schloss J. V., Cleland W. W. 1982; Inhibition of isocitrate lyase by 3-nitropropionate, a reaction-intermediate analogue. Biochemistry 21:4420–4427
    [Google Scholar]
  32. Small C. L., Bidochka M. J. 2005; Up-regulation of Prl, a subtilisin-like protease, during conidiation in the insect pathogen Metarhizium anisopliae . Mycol Res 109:307–313
    [Google Scholar]
  33. St Leger R. J. 2008; Studies on adaptations of Metarhizium anisopliae to life in the soil. J Invertebr Pathol 98:271–276
    [Google Scholar]
  34. St Leger R. J., Butt T. M., Goettel M. S., Staple R. C., Roberts D. W. 1989; Production in vitro of appressoria by the entomopathogenic fungus Metarhizium anisopliae . Exp Mycol 13:274–288
    [Google Scholar]
  35. Tan G., Gao Y., Shi M., Zhang X., He S., Chen Z., An C. 2005; SiteFinding-PCR: a simple and efficient PCR method for chromosome walking. Nucleic Acids Res 33:e122
    [Google Scholar]
  36. Thomas M. B., Read A. F. 2007; Can fungal biopesticides control malaria?. Nat Rev Microbiol 5:377–383
    [Google Scholar]
  37. Todd R. B., Andrianopoulos A., Davis M. A., Hynes M. J. 1998; FacB, the Aspergillus nidulans activator of acetate utilization genes, binds dissimilar DNA sequences. EMBO J 17:2042–2054
    [Google Scholar]
  38. Trinklein N. D., Aldred S. F., Hartman S. J., Schroeder D. I., Otillar R. P., Myers R. M. 2004; An abundance of bidirectional promoters in the human genome. Genome Res 14:62–66
    [Google Scholar]
  39. Wang C., St Leger R. J. 2006; A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses. Proc Natl Acad Sci U S A 103:6647–6652
    [Google Scholar]
  40. Wang C., St Leger R. J. 2007; The MAD1 adhesin of Metarhizium anisopliae links adhesion with blastospore production and virulence to insects, and the MAD2 adhesin enables attachment to plants. Eukaryot Cell 6:808–816
    [Google Scholar]
  41. Wang Z. Y., Thornton C. R., Kershaw M. J., Debao L., Talbot N. J. 2003; The glyoxylate cycle is required for temporal regulation of virulence by the plant pathogenic fungus Magnaporthe grisea . Mol Microbiol 47:1601–1612
    [Google Scholar]
  42. Wang C., Duan Z., St Leger R. J. 2008; MOS1 osmosensor of Metarhizium anisopliae is required for adaptation to insect host hemolymph. Eukaryot Cell 7:302–309
    [Google Scholar]
  43. Wang Q., Wan L., Li D., Zhu L., Qian M., Deng M. 2009; Searching for bidirectional promoters in Arabidopsis thaliana . BMC Bioinformatics 10:Suppl. 1S29
    [Google Scholar]
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