Promoter Analysis of the Cell Surface-abundant and Hypoviral-regulated Cryparin Gene from Cryphonectria parasitica

  • Kim, Myoung-Ju (Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University) ;
  • Kwon, Bo-Ra (Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University) ;
  • Park, Seung-Moon (Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University) ;
  • Chung, Hea-Jong (Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University) ;
  • Yang, Moon-Sik (Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University) ;
  • Churchill, Alice C.L. (Department of Plant Pathology, Cornell University) ;
  • Van Alfen, Neal K. (Department of Plant Pathology, University of California) ;
  • Kim, Dae-Hyuk (Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University)
  • Received : 2008.06.11
  • Accepted : 2008.08.29
  • Published : 2008.11.30

Abstract

Cryparin, encoded as a single copy gene (Crp) of the chestnut blight fungus Cryphonectria parasitica, is the most abundant protein produced by this fungus. However, its accumulation is decreased remarkably in C. parastica strains containing the double-stranded (ds) RNA virus Cryphonectria hypovirus 1. To characterize the transcriptional regulatory element(s) for strong expression and viral regulation, promoter analysis was conducted. Serial deletion of the Crp promoter region resulted in a step-wise decrease in promoter activity, indicating a localized distribution of genetic elements in the cryparin promoter. Promoter analysis indicated two positive and a repressive cis-acting elements. Among them, the promoter region between nt -1,282 and -907 appeared to be necessary for hypoviral-mediated down-regulation. An electrophoretic mobility shift assay (EMSA) on the corresponding promoter region (-1,282/-907) indicated two regions at (-1,257/-1,158) and (-1,107/-1,008) with the characteristic AGGAGGA-N42-GAGAGGA and its inverted repeat TCCTCTC-N54-TCCTCCT, respectively, appeared to be specific binding sites for cellular factors.

Keywords

Acknowledgement

Supported by : Korea Science and Engineering Foundation (KOSEF)

References

  1. Allen, T.D., and Nuss, D.L. (2004). Specific and common alterations in host gene transcript accumulation following infection of the chestnut blight fungus by mild and severe hypoviruses. J. Virol. 78, 4145-4155 https://doi.org/10.1128/JVI.78.8.4145-4155.2004
  2. Allen, T.D., Dawe, A.L., and Nuss, D.L. (2003). Use of cDNA microarrays to monitor transcriptional responses of the chestnut blight fungus Cryphonectria parasitica to infection by virulenceattenuating hypoviruses. Eukaryot. Cell 2, 1253-1265 https://doi.org/10.1128/EC.2.6.1253-1265.2003
  3. Anagnostakis, S.L. (1982). Biological control of chestnut blight. Science 215, 466-471 https://doi.org/10.1126/science.215.4532.466
  4. Carpenter, C.E., Mueller, R.J., Kazmierczak, P., Zhang, L., Villalon, D.K., and Van Alfen, N.K. (1992). Effect of a virus on accumulation of a tissue-specific cell-surface protein of the fungus Cryphonectria (Endothia) parasitica. Mol. Plant Microbe Interact. 5, 55-61 https://doi.org/10.1094/MPMI-5-055
  5. Choi, G.H., and Nuss, D.L. (1990). Nucleotide sequence of the glyceraldehyde 3-phosphate dehydrogenase gene from Cryphonectria parasitica. Nucleic Acids Res. 18, 5566 https://doi.org/10.1093/nar/18.18.5566
  6. Choi, G.H., Larson, T.G., and Nuss. D.L. (1992). Molecular analysis of the laccase gene from the chestnut blight fungus and selective suppression of its expression in an isogenic hypovirulent strain. Mol. Plant Microbe Interact. 5, 119-128 https://doi.org/10.1094/MPMI-5-119
  7. Choi, E.S., Chung, H.J., Kim, M.J., Park, S.M., Cha, B.J., Yang, M. S., and Kim, D.H. (2005). Characterization of the ERK homologue CpMK2 from the chestnut blight fungus Cryphonectria parasitica. Microbiology 151, 1349-1358 https://doi.org/10.1099/mic.0.27796-0
  8. Churchill, A.C.L. (1993). Ph.D. thesis. Transformation and gene isolation by functional complementation in Cryphonectria parasitica, the chestnut blight fungus. Utah State University, Logan
  9. Churchill, A.C.L., Ciufetti, L.M., Hansen, D.R., Van Etten, H.D., and Van Alfen, N.K. (1990). Transformation of the fungal pathogen Cryphonectria parasitica with a variety of heterologous plasmids. Curr. Genet. 17, 25-31 https://doi.org/10.1007/BF00313245
  10. Cortesi, P., McCulloch, C.E., Song, H., Lin, H., and Milgroom, M.G. (2001). Genetic control of horizontal virus transmission in the chestnut blight fungus, Cryphonectria parasitica. Genetics 159, 107-118
  11. Elliston, J.E. (1985). Characteristics of dsRNA-free and dsRNA-containing strains of Endothia parasitica in relation to hypovirulence. Phytopathology 75, 151-158 https://doi.org/10.1094/Phyto-75-151
  12. Gallarda, J.L., Foley, K.P., Yang, Z., and Engel, J.D. (1989). The beta-globin stage selector element factor is erythroid-specific promoter/enhancer binding protein NF-E4. Genes Dev. 3, 1845-1859 https://doi.org/10.1101/gad.3.12a.1845
  13. Ge, L., and Rudolph, P. (1997). Simultaneous introduction of multiple mutations using overlap extension PCR. Biotechniques 22, 28-30
  14. Havir, E.A., and Anagnostakis, S.L. (1983). Oxalate production by virulent but not by hypovirulent strains of Endothia parasitica. Physiol. Plant. Pathol. 23, 369-376 https://doi.org/10.1016/0048-4059(83)90021-8
  15. Jara, P., Delmas, P., Razanamparany, V., Olsen, L., Dupin, P., Bayol, A., Begueret, J., and Loison., G. (1995). Self-cloning in filamentous fungi: application to the construction of endothiapepsin overproducers in Cryphonectria parasitica. J. Biotechnol. 40, 111-120 https://doi.org/10.1016/0168-1656(95)00036-P
  16. Johnson, M.R., Wang, K., Smith, J.B., Heslin, M.J., and Diasio, R.B. (2000). Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction. Anal. Biochem. 278, 175-184 https://doi.org/10.1006/abio.1999.4461
  17. Johnstone, I.L., Hughes, S.G., and Clutterbuck, A.J. (1985). Cloning an Aspergillus nidulans developmental gene by transformation. EMBO J. 4, 1307-1311
  18. Kazmierczak, P., Pfeiffer, P., Zhang, L., and Van Alfen, N.K. (1996). Transcriptional repression of specific host genes by the mycovirus Cryphonectria hypovirus hypovirus CHV1. J. Virol. 70, 1137-1142
  19. Kazmierczak, P., Kim, D.H., Turina, M., and Van Alfen, N.K. (2005). A hydrophobin of the chestnut blight fungus, Cryphonectria parasitica, is required for strong stromal pustule eruption. Eukaryot. Cell 4, 931-936 https://doi.org/10.1128/EC.4.5.931-936.2005
  20. Kim, D.H., Rigling, D., Zhang, L., and Van Alfen, N.K. (1995). A new extracellular laccase of Cryphonectria parasitica is revealed by deletion of Lac1. Mol. Plant Microbe Interact. 8, 259-266 https://doi.org/10.1094/MPMI-8-0259
  21. Kim, M.J., Kwon, T.H., Jang, Y.S., Yang, M.S., and Kim, D.H. (2000). Expression of murine M-CSF from recombinant Aspergillus niger. J. Microbiol. Biotechnol. 10, 287-292
  22. Kim, M.J., Choi, J.W., Park, S.M., Cha, B.J., Yang, M.S., and Kim, D.H. (2002). Characterization of a fungal protein kinase from Cryphonectria parasitica and its transcriptional upregulation by hypovirus. Mol. Microbiol. 45, 933-941 https://doi.org/10.1046/j.1365-2958.2002.03079.x
  23. Morris, T.J., and Dodds, J.A. (1979). Isolation and analysis of double-stranded RNA from virus-infected plant and fungal tissue. Phytopathology 69, 854-858 https://doi.org/10.1094/Phyto-69-854
  24. Orbach, M.J., Porro, E.B., and Yanofsky, C. (1986). Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker. Mol. Cell. Biol. 6, 2452-2461 https://doi.org/10.1128/MCB.6.7.2452
  25. Park, S.M., Choi, E.S., Kim, M.J., Cha, B.J., Yang, M.S., and Kim, D.H. (2004). Characterization of HOG1 homologue, CpMK1, from Cryphonectria parasitica and evidence for hypovirusmediated perturbation of its phosphorylation in response to hypertonic stress. Mol. Microbiol. 51, 1267-1277 https://doi.org/10.1111/j.1365-2958.2004.03919.x
  26. Parsley, T.B., Chen, B., Geletka, L.M., and Nuss, D.L. (2002) Differential modulation of cellular signaling pathways by mild and severe hypovirus strains. Eukaryot. Cell 1, 401-413 https://doi.org/10.1128/EC.1.3.401-413.2002
  27. Puhalla, J.E., and Anagnostakis, S.L. (1971). Genetics and nutritional requirements of Endothia parasitica. Phytopathology 61, 169-173 https://doi.org/10.1094/Phyto-61-169
  28. Purucker, M., Bodine, D., Lin, H., McDonagh, K., and Nienhuis, A.W. (1990). Structure and function of the enhancer 3' to the ${human}^A$ globin gene. Nucleic Acids Res. 18, 7407-7415 https://doi.org/10.1093/nar/18.24.7407
  29. Rigling, D., and Van Alfen, N.K. (1991). Regulation of laccase biosynthesis in the plant-pathogenic fungus Cryphonectria parasitica by double-stranded RNA. J. Bacteriol. 173, 8000-8003 https://doi.org/10.1128/jb.173.24.8000-8003.1991
  30. Rigling, D., Heiniger, U., and Hohl, H.R. (1989). Reduction of laccase activity in dsRNA-containing hypovirulent strains of Cryphonectria (Endothia) parasitica. Phytopathology 79, 219-223 https://doi.org/10.1094/Phyto-79-219
  31. Toda, N., Ishikawa, T., Nozawa, N., Kobayashi, I., Ochiai, H., Miyamoto, K., Sumita, S., Kimura, K., and Umemura, S. (2001). Deletion analysis of the enolase gene (enoA) promoter from the filamentous fungus Aspergillus oryzae. Curr. Genet. 40, 260-267 https://doi.org/10.1007/s00294-001-0258-7
  32. Varley, D.A., Podila, G.K., and Hiremath, S.T. (1992). Cutinase in Cryphonectria parasitica, the chestnut blight fungus: suppression of cutinase gene expression in isogenic hypovirulent strains containing double-stranded RNAs. Mol. Cell. Biol. 12, 4539-4544 https://doi.org/10.1128/MCB.12.10.4539
  33. Wang, P., and Nuss, D.L. (1998). Identification of a Cryphonectria parasitica laccase gene promoter element involved in cycloheximide-inducible, hypovirus-repressible transcriptional activation. Gene 210, 79-84 https://doi.org/10.1016/S0378-1119(98)00035-3
  34. Wessels, J.G.H. (1997). Hydrophobins: proteins that change the nature of the fungal surface. Adv. Microb. Physiol. 38, 1-45
  35. Wosten, H.A.B. (2001). Hydrophobins: multipurpose proteins. Annu. Rev. Microbiol. 55, 625-646 https://doi.org/10.1146/annurev.micro.55.1.625
  36. Zhang, L., Churchill, A.C., Kazmierczak, P., Kim, D.H., and Van Alfen, N.K. (1993). Hypovirulence-associated traits induced by a mycovirus of Cryphonectria parasitica are mimicked by targeted inactivation of a host gene. Mol. Cell. Biol. 13, 7782-7792 https://doi.org/10.1128/MCB.13.12.7782
  37. Zhang, L., Villalon, D., Sun, Y., Kazmierczak, P., and Van Alfen, N.K. (1994). Virus-associated down-regulation of the gene encoding cryparin, an abundant cell-surface protein from the chestnut blight fungus, Cryphonectria parasitica. Gene 139, 59-64 https://doi.org/10.1016/0378-1119(94)90523-1