Abstract
A straightforward and easy-to-apply semi-quantitative RT-PCR method was developed to study multigenic expression in the phytopathogenic fungus Botrytis cinerea. This procedure is based on the one-step reverse transcription-amplification of a specific transcript within total RNA and product amount determination by densitometric analysis of ethidium bromide fluorescence upon gel electrophoresis. The semi-quantitative analysis is achieved, at a fixed PCR cycle-number, within a range of total RNA concentrations that stays in the exponential phase of the PCR. Co-amplification of the transcript of interest with internal controls allowed comparison between different RNA samples. Using this method, we could demonstrate a differential regulation of chitin synthase genes during fungal growth and an effect of the culture carbon source on the expression of two pectin methylesterase genes in B. cinerea. Finally, the method was shown to be applicable to plant-infected tissue, making it a useful tool to detect pathogenicity genes in B. cinerea.
References
Arellano M, Cartagena-Lirola H, Hajibagheri MAN, Duran A, Valdivieso MH (2000) Proper ascospore maturation requires the chs1+ chitin synthase gene in Schizosaccharomyces pombe. Mol Microbiol 35:79–89
Beth Din A, Specht CA, Robbins PW, Yarden O (1996) chs-4, a class IV chitin synthase gene from Neurospora crassa. Mol Gen Genet 250:214–222
Bowen AR, Chen-Wu JL, Momany M, Young R, Szaniszlo PJ, Robbins PW (1992) Classification of fungal chitin synthases. Proc Natl Acad Sci USA 89:519–523
Chigira Y, Abe K, Gomi K, Nakajima T (2002) chsZ, a gene for a novel class of chitin synthase from Aspergillus oryzae. Curr Genet 41:261–267
Coley-Smith JR, Verhoeff K, Jarvis WR (1980) The biology of Botrytis. Academic Press, New York
Cottrez F, Auriault C, Apron A, Groux H (1994) Quantitative PCR: validation of the use of multispecific internal control. Nucleic Acids Res 22:2712–2713
Ferre F (1992) Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Appl 2:1–9
Freeman WM, Walker SJ, Vrana KE (1999) Quantitative RT-PCR: pitfalls and potential. Biotechniques 26:112–115
Fujiwara M, Horiuchi H, Ohta A, Takagi M (1997) A novel fungal gene encoding chitin synthase with a myosin motor-like domain. Biochem Biophys Res Commun 236:75–78
Johnston DJ, Williamson B (1992) An immunological study of the induction of polygalacturonases in Botrytis cinerea. FEMS Microbiol Lett 97:19–24
Lanfranco L, Vallino M, Bonfante P (1999) Expression of chitin synthase genes in the arbuscular mycorrhizal fungus Gigaspora margarita. New Phytol 142:347–354
Limbach FX, Jaulhac B, Piemont Y, Kuntz JL, Monteil H, Sibilia J (1999) One-step reverse transcriptase PCR method for detection of Borrelia burgdorferi mRNA in mouse lyme arthritis tissue samples. J Clin Microb 37:2037–2039
Mellado E, Aufauvre-Brown A, Specht CA, Robbins PW, Holden DW (1995) A multigene family related to chitin synthase genes of yeast in the opportunistic pathogen Aspergillus fumigatus. Mol Gen Genet 246:353–359
Pfeffer U, Fecarotta E, Vidali G (1995) Efficient one-tube RT-PCR amplification of rare transcripts using short sequence-specific reverse transcription primers. Biotechniques 18:204–206
Reignault P, Mercier M, Bompeix G, Boccara M (1994) Pectin methylesterase from Botrytis cinerea: physiological, biochemical and immunochemical studies. Microbiology 140:3249–3255
Roncero C (2002) The genetic complexity of chitin synthesis in fungi. Curr Genet 41:367–378
Sreenivasaprasad S, Burton KS, Wood DA (2000) Cloning and characterization of a chitin synthase gene cDNA from the cultivated mushroom Agaricus bisporus and its expression during morphogenesis. FEMS Microbiol Lett 189:73–77
Vidal-Cros A, Boccara M (1998) Identification of four chitin synthase genes in the rice blast disease agent Magnaporthe grisea. FEMS Microbiol Lett 165:103–109
Wang Z, Szaniszlo P (2000) WdCHS3 a gene that encodes a class III chitin synthase in Wangiella (Exophiala) dermatitidis, is expressed differentially under stress conditions. J Bacteriol 182:874–881
Werner S, Steiner U, Becher R, Kortekamp A, Zyprian E, Deising HB (2002) Chitin synthesis during in planta growth and asexual propagation of the cellulosic oomycete and obligate biotrophic grapevine pathogen Plasmopara viticola. FEMS Microbiol Lett 208:169–173
Wubben JP, Have A ten, Kan JAL van, 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
Acknowledgements
The authors wish to express their thanks to Dr. Sophie Rome for introducing them to the RNA extraction and RT-PCR techniques, Agnès Cimmerman for her kind help in Bcpme expression studies and Alia Dellagi for providing them with the A. thaliana plants.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by U. Kück
Rights and permissions
About this article
Cite this article
Choquer, M., Boccara, M. & Vidal-Cros, A. A semi-quantitative RT-PCR method to readily compare expression levels within Botrytis cinerea multigenic families in vitro and in planta. Curr Genet 43, 303–309 (2003). https://doi.org/10.1007/s00294-003-0397-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00294-003-0397-0