Abstract
Net blotch, caused by Pyrenophora teres, is a common disease of barley (Hordeum vulgare L.). Two PCR-based differential screening techniques, cDNA-amplified fragment length polymorphism (cDNA-AFLP) and suppression subtractive hybridisation (SSH), were employed to clone cDNA copies of transcripts that are up-regulated during conidial germination. The nucleotide sequences of 35 transcripts were analysed, and the amino acid sequences of their predicted products were compared with entries in databases. Eleven of these clones showed homology to genes from other ascomycetes coding for a transcription factor, two regulatory proteins, a putative transposase, a protein required for the biogenesis of cytochrome C oxidase, a threonine synthase, a probable subunit of a phenylalanine-tRNA synthetase, a subunit of RNA polymerase I, a cation transport protein, a vacuolar ATP synthase subunit, and an RNA processing protein. One conserved hypothetical protein was found and 23 sequences could not be functionally classified. The relative expression of five transcripts at 0, 1, 2, 3, 6, 12 and 24 h after induction of germination was determined by real-time RT-PCR using 18S rRNA as the endogenous reference sequence. All transcripts showed a significant increase in expression during early stages of germination. The maximum change in expression relative to ungerminated conidia ranged between 2.6- and 6-fold. The characterisation of genes involved in biochemical processes during the germination of conidia could be useful for target-specific development of new antifungal agents.
Similar content being viewed by others
References
Andrianopoulos A, Brones J, Davis MA, Hynes MJ (1997) The amdA regulatory gene of Aspergillus nidulans: characterisation of gain-of-function mutations and identification of binding sites for the gene product. Fungal Genet Biol 21:50–63
Bachem CWB, van der Hoeven RS, de Bruijn SM, Vreugdenhil D, Zabeau M, Visser RGF (1996) Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development. Plant J 9:745–753
Beyer K, Jiménez Jiménez S, Randall TA, Lam S, Binder A, Boller T, Collinge MA (2002) Characterization of Phytophthora infestans genes regulated during the interaction with potato. Mol Plant Pathol 3:473–485
Bonnefoy N, Kermorgant M, Groudinsky O, Dujardin G (2000) The respiratory gene OXA1 has two fission yeast orthologues which together encode a function essential for cellular viability. Mol Microbiol 35:1135–1145
Bowman EJ, Bowman BJ (2000) Cellular role of the V-ATPase in Neurospora crassa: analysis of mutants resistant to concanamycin or lacking the catalytic subunit A. J Exp Biol 203:97–106
Bowman EJ, O'Neill FJ, Bowman BJ (1997) Mutations of pma-1, the gene encoding the plasma membrane H+-ATPase of Neurospora crassa, suppress inhibition of growth by concanamycin A, a specific inhibitor of vacuolar ATPases. J Biol Chem 272:14776–14786
Brambl R (1980) Mitochondrial biogenesis during fungal spore germination: biosynthesis and assembly of cytochrome c oxidase in Botryodiplodia theobromae. J Biol Chem 255:7673–7680
Cochrane VW, Cochrane JC (1970) Chlamydospore development in the absence of protein synthesis in Fusarium solani. Dev Biol 23:345–354
Dalbey RE, Kuhn A (2000) Evolutionarily related insertion pathways of bacterial, mitochondrial, and thylakoid membrane proteins. Annu Rev Cell Dev Biol 16:51–87
Deimel H, Hoffmann GM (1991) Grundlagen der Schadwirkung der Netzfleckenkrankheit an Gerste. Z Pflkrankh Pflschutz 98:137–161
D'Enfert C (1997) Fungal spore germination: insights from the molecular genetics of Aspergillus nidulans and Neurospora crassa. Fungal Genet Biol 21:163–172
Diatchenko L, Lau YFC, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD (1996) Suppression subtractive hybridisation: a method for generating differentally regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA 93:6025–6030
Feinberg, AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13
Freeman WM, Walker SJ, Vrana KE (1999) Quantitative RT-PCR: pitfalls and potential. Biotechniques 26:112–125
Galagan JE, et al (2003) The genome sequence of the filamentous fungus Neurospora crassa. Nature 422:859–868
Garrido-Franco M, Ehlert S, Messerschmidt A, Marinković S, Huber R, Laber B, Bourenkov GP, Clausen T (2002) Structure and function of threonine synthase from yeast. J Biol Chem 277:12396–12405
Gellatly KS, Ash GJ, Taylor JL (2001) Development of a method for mRNA differential display in filamentous fungi: comparison of mRNA differential display, reverse transcription polymerase chain reaction, and cDNA amplified fragment length polymorphism in Leptosphaeria maculans. Can J Microbiol 47:955–960
Habu Y, Fukada-Tanaka S, Hisatomi Y, Iida S (1997) Amplified restriction fragment length polymorphism-based mRNA fingerprinting using a single restriction enzyme that recognizes a 4-bp sequence. Biochem Biophys Res Comm 234:516–521
Harris AJ, Shaddock JG, Manjanatha MG, Lisenbey JA, Casciano DA (1998) Identification of differentially expressed genes in aflatoxin B-1-treated cultured primary rat hepatocytes and Fischer 344 rats. Carcinogenesis 19:1451–1458
Hubank M, Schatz DG (1994) Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res 22:5640–5648
Jones JT, Harrower BE (1998) A comparison of the efficiency of differential display and cDNA-AFLPs as tools for the isolation of differentially expressed parasite genes. Fund Appl Nematol 21:81–88
Keon JPR, Hargreaves JA (1982) A cytological study of net blotch disease of barley caused by Pyrenophora teres. Physiol Plant Pathol 22:321–329
Lane S, Zhou S, Pan T, Dai Q, Liu H (2001) The basic helix-loop-helix transcription factor Cph2 regulates hyphal development in Candida albicans partly via Tec1. Mol Cell Biol 21:6418–6428
Liang P, Pardee AB (1992) Differential display of eukaryotic messenger-RNA by means of the polymerase chain reaction. Science 257:967–971
Lisitsyn N, Wigler M (1993) Cloning the differences between two complex genomes. Science 259:946–951
Liu H (2001) Transcriptional control of dimorphism in Candida albicans. Curr Opin Microbiol 4:728–735
Loo M (1976) Some required events in conidial germination of Neurospora crassa. Dev Biol 54:201–213
MacDiarmid CW, Gardner RC (1998) Overexpression of the Saccharomyces cerevisiae magnesium transport system confers resistance to aluminium ion. J Biol Chem 273:1727–1732
Mathre DE (ed) (1997) Compendium of barley diseases (2nd edn). APS Press, St. Paul, Minn.
Onai K, Katagiri S, Akiyama M, Nakashima H (1998) Mutation of the gene for the second-largest subunit of RNA polymerase I prolongs the period length of the circadian conidiation rhythm in Neurospora crassa. Mol Gen Genet 259:264–271
Osherov N, May GS (2000) Conidial germination in Aspergillus nidulans requires RAS signaling and protein synthesis. Genetics 155:647–650
Osherov N, May GS (2001) The molecular mechanisms of conidial germination. FEMS Microbiol Lett 199:153–160
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45
Sachs MS, Yanofsky C (1991) Developmental expression of genes involved in conidiation and amino acid biosynthesis in Neurospora crassa. Dev Biol 148:117–128
Saleh A, Collart M, Martens JA, Genereaux J, Allard S, Cote J, Brandl CJ (1998) TOM1p, a yeast hect-domain protein which mediates transcriptional regulation through the ADA/SAGA coactivator complexes. J Mol Biol 282:933–946
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Schmittgen TD, Zakrajsek BA (2000) Effects of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods 46:69–81
Shim WB, Woloshuk CP (2001) Regulation of Fumonisin B1 biosynthesis and conidiation in Fusarium verticillioides by a cyclin-like (C-type) gene, FCC1. Appl Environ Microbiol 67:1607–1612
Steffenson B J, Webster RK (1992) Pathotype diversity of Pyrenophora teres f. teres on barley. Phytopathology 82:170–177
Tabb AL, Utsugi T, Wooten-Kee CR, Sasaki T, Edling SA, Gump W, Kikuchi Y, Ellis SR (2001) Genes encoding ribosomal proteins Rps0A/B of Saccharomyces cerevisiae interact with TOM1 mutants defective in ribosome synthesis. Genetics 157:1107–1116
Tucker SL, Talbot NJ (2001) Surface attachment and pre-penetration stage development by plant pathogenic fungi. Annu Rev Phytopathol 39:385–417
Ursic D, Himmel KL, Gurley KA, Webb F, Clubertson MR (1997) The yeast SEN1 gene is required for the processing of diverse RNA classes. Nucleic Acids Res 25:4778–4785
Van der Biezen EA, Juwana H, Parker JE, Jones JDG (2000) cDNA-AFLP display for the isolation of Peronospora parasitica genes expressed during infection in Arabidopsis thaliana. Mol Plant-Microbe Interact 13:895–898
Weiland JJ, Steffenson BJ, Cartwright RD, Webster RK (1999) Identification of molecular genetic markers in Pyrenophora teres f. teres associated with low virulence on 'Harbin' barley. Phytopathology 89:176–181
Acknowledgements
We thank Bernhard Jaser for support and the preparation of P. teres isolates. This work was supported by the Bayerische Forschungsstiftung
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by C. A. M. J. J. van den Hondel
Rights and permissions
About this article
Cite this article
Dilger, M., Felsenstein, F.G. & Schwarz, G. Identification and quantitative expression analysis of genes that are differentially expressed during conidial germination in Pyrenophora teres . Mol Genet Genomics 270, 147–155 (2003). https://doi.org/10.1007/s00438-003-0910-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00438-003-0910-7