Identification and quantitative expression analysis of genes that are differentially expressed during conidial...

Net blotch, caused by Pyrenophora teres, is a common disease of barley ( Hordeum vulgareL.). 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.