Using an established spotted cDNA microarray platform, the nature of changes in the transcriptional profiles of 2200 unique genes from the chestnut blight fungus Cryphonectria parasitica in response to the absence of either the Gα subunit CPG-1 or the Gβ subunit CPGB-1 has been explored. It is reported that 216 transcripts were altered in accumulation in the Δcpg-1 strain and 163 in the Δcpgb-1 strain, with a considerable overlap (100 genes) that were changed in both cases. Of note, these commonly altered transcripts were changed in the same direction in every instance, thus suggesting a considerable redundancy in pathway control or extensive crosstalk. To further knowledge of the potential impact on G-protein-signalling of infection by hypovirus CHV1-EP713, the accumulation of CPG-1 and CPGB-1 was also investigated by Western analysis. It was demonstrated that both signalling components were reduced in abundance to approximately 25 % of wild-type levels, while their transcripts were slightly elevated. Comparison of a list of genes with altered expression in the presence of CHV1-EP713 to the data obtained in the absence of either G-protein subunit showed that more than one-half of all the transcripts changed by hypovirus infection were also changed in at least one G-protein mutant strain, with one-third being changed in both. Significantly, 95 % of the co-changed genes were altered in the same direction. These data provide the first evidence for modulation of Gβ protein levels as well as the Gβγ-signalling pathways by hypovirus infection, and support the hypothesis that modification of G-protein-signalling via both Gα and Gβγ provides for a significant contribution to hypovirus-mediated phenotype.
Agabian, N., Odds, F. C., Poulain, D., Soll, D. R. & White, T. C.(1994). Pathogenesis of invasive candidiasis. J Med Vet Mycol32 (Suppl. 1), 229–237.[CrossRef][Google Scholar]
Allen, T. & Nuss, D.(2004). Specific and common alterations in host gene transcript accumulation following infection of the chestnut blight fungus by mild and severe hypoviruses. J Virol78, 4145–4155.[CrossRef][Google Scholar]
Allen, T. D., Dawe, A. L. & Nuss, D. L.(2003). Use of cDNA microarrays to monitor transcriptional responses of the chestnut blight fungus Cryphonetria parasitica to infection by virulence-attenuating hypoviruses. Eukaryot Cell2, 1253–1265.[CrossRef][Google Scholar]
Anagnostakis, S. L.(1984). The mycelial biology of Endothia parasitica. I. Nuclear and cytoplasmic genes that determine morphology and virulence. In The Ecology and Physiology of the Fungal Mycelium, pp. 353–366. Edited by D. H. Jennings & A. D. M. Rayner. Cambridge: Cambridge University Press.
Blaauw, M., Knol, J. C., Kortholt, A., Roelofs, J., Ruchira Postma, M., Visser, A. J. & van Haastert, P. J.(2003). Phosducin-like proteins in Dictyostelium discoideum: implications for the phosducin family of proteins. EMBO J22, 5047–5057.[CrossRef][Google Scholar]
Bölker, M.(1998). Sex and crime: heterotrimeric G proteins in fungal mating and pathogenesis. Fungal Genet Biol25, 143–156.[CrossRef][Google Scholar]
Chen, B., Gao, S., Choi, G. H. & Nuss, D. L.(1996). Extensive alteration of fungal gene transcript accumulation and elevation of G-protein-regulated cAMP levels by a virulence-attenuating hypovirus. Proc Natl Acad Sci U S A93, 7996–8000.[CrossRef][Google Scholar]
Choi, G. H. & Nuss, D. L.(1992). Hypovirulence of chestnut blight fungus conferred by an infectious viral cDNA. Science257, 800–803.[CrossRef][Google Scholar]
Choi, G. H., Chen, B. & Nuss, D. L.(1995). Virus-mediated or transgenic suppression of a G-protein alpha subunit and attenuation of fungal virulence. Proc Natl Acad Sci U S A92, 305–309.[CrossRef][Google Scholar]
Cole, S. W., Galic, Z. & Zack, J. A.(2003). Controlling false-negative errors in microarray differential expression analysis: a PRIM approach. Bioinformatics19, 1808–1816.[CrossRef][Google Scholar]
Dawe, A. L. & Nuss, D. L.(2001). Hypoviruses and chestnut blight: exploiting viruses to understand and modulate fungal pathogenesis. Annu Rev Genet35, 1–29.[CrossRef][Google Scholar]
Dawe, A. L., McMains, V. C., Panglao, M., Kasahara, S., Chen, B. & Nuss, D. L.(2003). An ordered collection of expressed sequences from Cryphonectria parasitica and evidence of genomic microsynteny with Neurospora crassa and Magnaporthe grisea. Microbiology149, 2373–2384.[CrossRef][Google Scholar]
Durmowicz, M. C. & Maier, R. J.(1997). Roles of HoxX and HoxA in biosynthesis of hydrogenase in Bradyrhizobium japonicum. J Bacteriol179, 3676–3682.
[Google Scholar]
Flanary, P. L., DiBello, P. R., Estrada, P. & Dohlman, H. G.(2000). Functional analysis of Plp1 and Plp2, two homologues of phosducin in yeast. J Biol Chem275, 18462–18469.[CrossRef][Google Scholar]
Gao, S. & Nuss, D. L.(1996). Distinct roles for two G protein alpha subunits in fungal virulence, morphology, and reproduction revealed by targeted gene disruption. Proc Natl Acad Sci U S A93, 14122–14127.[CrossRef][Google Scholar]
Gronover, C. S., Kasulke, D., Tudzynski, P. & Tudzynski, B.(2001). The role of G-protein alpha subunits in the infection process of the gray mold fungus Botrytis cinerea. Mol Plant–Microbe Interact14, 1293–1302.[CrossRef][Google Scholar]
Hamm, H. E.(1998). The many faces of G protein signaling. J Biol Chem273, 669–672.[CrossRef][Google Scholar]
Heintzen, C., Loros, J. J. & Dunlap, J. C.(2001). The PAS protein VIVID defines a clock-associated feedback loop that represses light input, modulates gating, and regulates clock resetting. Cell104, 453–464.[CrossRef][Google Scholar]
Hoegl, L., Ollert, M. & Korting, H. C.(1996). The role of Candida albicans secreted aspartic proteinase in the development of candidoses. J Mol Med74, 135–142.[CrossRef][Google Scholar]
Hube, B., Monod, M., Schofield, D. A., Brown, A. J. & Gow, N. A.(1994). Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans. Mol Microbiol14, 87–99.[CrossRef][Google Scholar]
Jara, P., Gilbert, S., Delmas, P., Guillemot, J. C., Kaghad, M., Ferrara, P. & Loison, G.(1996). Cloning and characterization of the eapB and eapC genes of Cryphonectria parasitica encoding two new acid proteinases, and disruption of eapC. Mol Gen Genet250, 97–105.
[Google Scholar]
Kasahara, S. & Nuss, D. L.(1997). Targeted disruption of a fungal G-protein beta subunit gene results in increased vegetative growth but reduced virulence. Mol Plant–Microbe Interact10, 984–993.[CrossRef][Google Scholar]
Kasahara, S., Wang, P. & Nuss, D. L.(2000). Identification of bdm-1, a gene involved in G protein beta-subunit function and alpha-subunit accumulation. Proc Natl Acad Sci U S A97, 412–417.[CrossRef][Google Scholar]
Lengeler, K. B., Davidson, R. C., D'Souza, C., Harashima, T., Shen, W. C., Wang, P., Pan, X., Waugh, M. & Heitman, J.(2000). Signal transduction cascades regulating fungal development and virulence. Microbiol Mol Biol Rev64, 746–785.[CrossRef][Google Scholar]
Masloff, S., Jacobsen, S., Poggeler, S. & Kuck, U.(2002). Functional analysis of the C6 zinc finger gene pro1 involved in fungal sexual development. Fungal Genet Biol36, 107–116.[CrossRef][Google Scholar]
Merkel, H. W.(1906). A deadly fungus on the American chestnut. NY Zool Soc Annu Rep10, 97–103.
[Google Scholar]
Monod, M. & Borg-von, Z. M.(2002). Secreted aspartic proteases as virulence factors of Candida species. Biol Chem383, 1087–1093.
[Google Scholar]
Park, G., Xue, C., Zheng, L., Lam, S. & Xu, J. R.(2002). MST12 regulates infectious growth but not appressorium formation in the rice blast fungus Magnaporthe grisea. Mol Plant–Microbe Interact15, 183–192.[CrossRef][Google Scholar]
Parsley, T. B., Segers, G. C., Nuss, D. L. & Dawe, A. L.(2003). Analysis of altered G-protein subunit accumulation in Cryphonectria parasitica reveals a third Galpha homologue. Curr Genet43, 24–33.
[Google Scholar]
Razanamparany, V., Jara, P., Legoux, R., Delmas, P., Msayeh, F., Kaghad, M. & Loison, G.(1992). Cloning and mutation of the gene encoding endothiapepsin from Cryphonectria parasitica. Curr Genet21, 455–461.[CrossRef][Google Scholar]
Ruchel, R., de Bernardis, F., Ray, T. L., Sullivan, P. A. & Cole, G. T.(1992).Candida acid proteinases. J Med Vet Mycol30 (Suppl. 1), 123–132.
[Google Scholar]
Schwerdtfeger, C. & Linden, H.(2003). VIVID is a flavoprotein and serves as a fungal blue light photoreceptor for photoadaptation. EMBO J22, 4846–4855.[CrossRef][Google Scholar]
The following supplementary tables are available in an
Acrobat PDF file.
Supplementary Table A. A redundant list of clones that
met the criteria for differential expression in the Δ
cpg-1 strain when compared to EP155 by microarray.
Supplementary Table B. A redundant list of clones that
met the criteria for differential expression in the Δ
cpgb-1 strain when compared to EP155 by microarray.
Supplementary Table C. A redundant list of clones that
met the criteria for differential expression in the
EP155/CHV1-EP713 strain when compared to EP155 by
microarray.
In each case the
AESTclone identification numbers, the -fold
change together with notation denoting up or down (dn)
regulation in the tested strain, the
E -value of the best
BLASTXresult (from Dawe
et al. 2003) and the gi number and description from the
NCBI database are listed.