A semi-quantitative RT-PCR method to readily compare expression levels within Botrytis cinerea multigenic families in vitro and in planta.
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.
UMR 7613 CNRS/Paris VI, 4 place Jussieu, 75005, Paris, France.
Mathias
M
Choquer
Martine
M
Boccara
Anne
A
Vidal-Cros
D020171:Botrytis / Q000235:genetics*; D002265:Carboxylic Ester Hydrolases / Q000235:genetics; D002687:Chitin Synthase / Q000235:genetics; D003720:Densitometry; D004305:Dose-Response Relationship, Drug; D004996:Ethidium / Q000494:pharmacology; D005800:Genes, Fungal; D066298:In Vitro Techniques; D007364:Intercalating Agents / Q000494:pharmacology; D010944:Plants / Q000382:microbiology*; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors; D014158:Transcription, Genetic
D007364:Intercalating Agents; D012333:RNA, Messenger; D012313:RNA; D002687:Chitin Synthase; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase; D004996:Ethidium