1887

Abstract

Hexose kinases play a central role in the initiation of sugar metabolism of living organisms and have also been implicated in carbon catabolite repression in yeasts and plants. In this study, the genes encoding glucokinase (Glk1) and hexokinase (Hxk1) from the plant-pathogenic ascomycete were isolated and functionally characterized. Glk1-deficient mutants were indistinguishable from the wild-type in all growth parameters tested. In contrast, Δ mutants lacking Hxk1 showed a pleiotropic growth defect. On artificial media, vegetative growth was retarded, and conidia formation strongly reduced. No or only marginal growth of Δ mutants was observed when fructose, galactose, sucrose or sorbitol were used as carbon sources, and fructose inhibited growth of the mutant in the presence of other carbon sources. mutants containing alleles with point mutations leading to enzymically inactive enzymes showed phenotypes similar to the Δ disruption mutant, indicating that loss of hexose phosphorylation activity of Hxk1 is solely responsible for the pleiotropic growth defect. Virulence of the Δ mutants was dependent on the plant tissue: on leaves, lesion formation was only slightly retarded compared to the wild-type, whereas only small lesions were formed on apples, strawberries and tomatoes. The low virulence of Δ mutants on fruits was correlated with their high contents of sugars, in particular fructose. Heterologous expression of Hxk1 and Glk1 in yeast allowed their enzymic characterization, revealing kinetic properties similar to other fungal hexokinases and glucokinases. Both Δ and Δ mutants showed normal glucose repression of secreted lipase 1 activity, indicating that, in contrast to yeast, hexose kinases are not involved in carbon catabolite repression.

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2007-08-01
2024-03-28
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References

  1. Ahuatzi D., Herrero P., de la Cera T., Moreno F. 2004; The glucose-regulated nuclear localization of hexokinase 2 in Saccharomyces cerevisiae is Mig1-dependent. J Biol Chem 279:14440–14446
    [Google Scholar]
  2. Ahuatzi D., Riera A., Pelaez R., Herrero P., Moreno F. 2007; Hxk2 regulates the phosphorylation state of Mig1 and therefore its nucleocytoplasmic distribution. J Biol Chem 282:4485–4493
    [Google Scholar]
  3. Arisan-Atac I., Wolschek M. F., Kubicek C. P. 1996; Trehalose-6-phosphate synthase A affects citrate accumulation by Aspergillus niger under conditions of high glycolytic flux. FEMS Microbiol Lett 140:77–83
    [Google Scholar]
  4. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. 1999 Short Protocols in Molecular Biology , 4th edn. New York: Wiley;
  5. Benito E. P., ten Have A., van't Klooster J. W., van Kan J. A. L. 1998; Fungal and plant gene expression during synchronized infection of tomato leaves by Botrytis cinerea . Eur J Plant Pathol 104:207–220
    [Google Scholar]
  6. Blakeman J. P. 1975; Germination of Botrytis cinerea in vitro in relation to nutrient conditions on leaf surfaces. Trans Br Mycol Soc 65:239–247
    [Google Scholar]
  7. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
    [Google Scholar]
  8. De Winde J. H., Crauwels M., Hohmann S., Thevelein J. M., Winderickx J. 1996; Differential requirement of the yeast sugar kinases for sugar sensing in establishing the catabolite-repressed state. Eur J Biochem 241:633–643
    [Google Scholar]
  9. Doehlemann G., Molitor F., Hahn M. 2005; Molecular and functional characterization of a fructose specific transporter from the gray mold fungus Botrytis cinerea . Fungal Genet Biol 42:601–610
    [Google Scholar]
  10. Doehlemann G., Berndt P., Hahn M. 2006a; Different signalling pathways involving a G α protein, cAMP and a MAP kinase control germination of Botrytis cinerea conidia. Mol Microbiol 59:821–835
    [Google Scholar]
  11. Doehlemann G., Berndt P., Hahn M. 2006b; Trehalose metabolism is important for heat stress tolerance and spore germination of Botrytis cinerea . Microbiology 152:2625–2634
    [Google Scholar]
  12. Droby S., Lichter A. 2004; Post-harvest Botrytis infection: etiology, development and management. In Botrytis: Biology Pathology and Control pp 349–368 Edited by Elad Y., Williamson B., Tudzynski P., Delen N. Dordrecht: Kluwer;
    [Google Scholar]
  13. Efrat S., Tal M., Lodish H. F. 1994; The pancreatic beta-cell glucose sensor. Trends Biochem Sci 19:535–538
    [Google Scholar]
  14. Entian K. D. 1980; Genetic and biochemical evidence for hexokinase PII as a key enzyme involved in carbon catabolite repression in yeast. Mol Gen Genet 178:633–637
    [Google Scholar]
  15. Fekete E., Karaffa L., Sándor E., Bányai I., Seiboth B., Gyémánt G., Sepsi A., Szentirmai A., Kubicek C. P. 2004; The alternative d-galactose degrading pathway of Aspergillus nidulans proceeds via l-sorbose. Arch Microbiol 181:35–44
    [Google Scholar]
  16. Flipphi M., van de Vondervoort P. J. I., Ruijter G. J. G., Visser J., Arst H. N., Felenbok B. 2003; Onset of carbon catabolite repression in Aspergillus nidulans – parallel involvement of hexokinase and glucokinase in sugar signaling. J Biol Chem 278:11849–11857
    [Google Scholar]
  17. Frey P. A. 1996; The Leloir pathway: a mechanistic imperative for three enzymes to change the stereochemical configuration of a single carbon in galactose. FASEB J 10:461–470
    [Google Scholar]
  18. Gancedo J. M., Clifton D., Fraenkel D. G. 1977; Yeast hexokinase mutants. J Biol Chem 252:4443–4444
    [Google Scholar]
  19. Hartl L., Seiboth B. 2005; Sequential gene deletions in Hypocrea jecorina using a single blaster cassette. Curr Genet 48:204–211
    [Google Scholar]
  20. Hohmann S., Winderickx J., de Winde J. H., Valckx D., Cobbaert P., Luyten K., de Meirsman C., Ramos J., Thevelein J. M. 1999; Novel alleles of yeast hexokinase PII with distinct effects on catalytic acitivity and catabolite repression of SUC2 . Microbiology 145:703–714
    [Google Scholar]
  21. Hult K., Veide A., Gatenbeck S. 1980; The distribution of the NADPH regenerating mannitol cycle among fungal species. Arch Microbiol 128:253–255
    [Google Scholar]
  22. Katz M. E., Masoumi A., Burrows S. R., Shirtliff C. G., Cheetham B. F. 2000; The Aspergillus nidulans xprF gene encodes a hexokinase-like protein involved in the regulation of extracellular proteases. Genetics 156:1559–1571
    [Google Scholar]
  23. Kraakman L. S., Winderickx J., Thevelein J. M., de Winde J. H. 1999; Structure-function analysis of yeast hexokinase: structural requirements for triggering cAMP signalling and catabolite repression. Biochem J 343:159–168
    [Google Scholar]
  24. Lobo Z., Maitra P. K. 1977; Physiological role of glucose-phosphorylating enzymes in Saccharomyces cerevisiae . Arch Biochem Biophys 182:639–645
    [Google Scholar]
  25. Ma H., Bloom L., Dakin S. E., Walsh C. T., Botstein D. 1989; The 15 N-terminal amino acids of hexokinase II are not required for in vivo function: analysis of a truncated form of hexokinase II in Saccharomyces cerevisiae . Proteins 5:218–223
    [Google Scholar]
  26. Möller E. M., Bahnweg G., Sandermann H., Geiger H. H. 1992; A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissue. Nucleic Acids Res 20:6115–6116
    [Google Scholar]
  27. Moore B., Zhou L., Rolland F., Hall Q., Cheng W.-H., Liu Y.-X., Hwang I., Jones T., Sheen J. 2003; Role of the Arabidopsis glucose sensor HXK1 in nutrient, light, and hormonal signaling. Science 300:332–336
    [Google Scholar]
  28. Panneman H., Ruijter G. J. G., van den Broek H. C., Visser J. 1996; Cloning and biochemical characterization of an Aspergillus niger glucokinase – evidence for the presence of separate glucokinase and hexokinase enzymes. Eur J Biochem 240:518–525
    [Google Scholar]
  29. Panneman H., Ruijter G. J. G., van den Broek H. C., Visser J. 1998; Cloning and biochemical characterization of Aspergillus niger hexokinase – the enzyme is strongly inhibited by physiological concentrations of trehalose-6-phosphate. Eur J Biochem 258:223–232
    [Google Scholar]
  30. Reis H., Pfiffi S., Hahn M. 2005; Molecular and functional characterization of a secreted lipase from Botrytis cinerea . Mol Plant Pathol 6:257–267
    [Google Scholar]
  31. Rentsch D., Laloi M., Rouhara I., Schmelzer E., Delrot S., Frommer W. B. 1995; NTR1 encodes a high affinity oligopeptide transporter in Arabidopsis . FEBS Lett 370:264–268
    [Google Scholar]
  32. Ruijter G. J. G., Panneman H., van den Broeck H. C., Bennett J. M., Visser J. 1996; Characterization of the Aspergillus nidulans frA1 mutant: hexose phosphorylation and apparent lack of involvement of hexokinase in glucose repression. FEMS Microbiol Lett 139:223–228
    [Google Scholar]
  33. Sherman F., Fink G. R., Hicks J. B. 1986 Methods in Yeast Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  34. Seiboth B., Hartl L., Pail M., Fekete E., Karaffa L., Kubicek C. P. 2004; The galactokinase of Hypocrea jecorina is essential for cellulase induction by lactose but dispensable for growth on d-galactose. Mol Microbiol 51:1015–1025
    [Google Scholar]
  35. Thevelein J. M., Hohmann S. 1995; Trehalose synthase: guard to the gate of glycolysis in yeast?. Trends Biochem Sci 20:3–10
    [Google Scholar]
  36. Thines E., Weber R. W. E., Talbot N. J. 2000; MAP kinase and protein kinase A-dependent mobilization of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea . Plant Cell 12:1703–1718
    [Google Scholar]
  37. Thompson J. D., Higgins D. G., Gibson T. J. 1994; clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
    [Google Scholar]
  38. Van Kan J. A. L. 2006; Licensed to kill: the lifestyle of a necrotrophic plant pathogen. Trends Plant Sci 11:247–253
    [Google Scholar]
  39. Velez H., Glassbrook N. J., Daub M. E. 2007; Mannitol metabolism in the phytopathogenic fungus Alternaria alternata . Fungal Genet Biol 44:258–268
    [Google Scholar]
  40. Viaud M., Brunet-Simon A., Brygoo Y., Pradier J.-M., Levis C. 2003; Cyclophilin A and calcineurin functions investigated by gene inactivation, cyclosporin A inhibition and cDNA arrays approaches in the phytopathogenic fungus Botrytis cinerea . Mol Microbiol 50:1451–1465
    [Google Scholar]
  41. Walsh R. B., Clifton D., Horak J., Fraenkel D. G. 1991; Saccharomyces cerevisiae null mutants in glucose phosphorylation: metabolism and invertase expression. Genetics 128:521–527
    [Google Scholar]
  42. Wubben J. P., Ten Have A., Van Kan J. A. L., Visser J. 2000; Regulation of endopolygalacturonase gene expression in Botrytis cinerea by galacturonic acid, ambient pH and carbon catabolite repression. Curr Genet 37:152–157
    [Google Scholar]
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