cDNA cloning, in vitro transcription and partial sequence analysis of mRNAs from winter wheat (Triticum aestivum L.) with induced resistance to Erysiphe graminis f. sp. tritici

Abstract

Under normal growth conditions wheat shows 100% non-host resistance to the barley powdery mildew Erysiphe graminis f. sp. hordei (Egh.). Primary inoculation of 7-day-old wheat seedlings with this fungus induced partial (60-70%) local resistance to challenge inoculation 12 hours later with the compatible pathogen Erysiphe graminis f. sp. tritici (Egt). mRNA was isolated from induced resistant first leaves (13 hours after primary inoculation) and a cDNA library was established in lambda ZAP. Differential screening of the library with sDNA probes (from induced resistant versus non-inoculated plants) resulted in isolation of 6 cDNA clones corresponding to 6 different induced, plant-encoded mRNA species. Hybridization of in vitro transcripts derived from wheat nuclei to cDNA dot blots showed that the transcription of these genes was induced rapidly, 3 hours after inoculation with either Egt or Egh. At this time point neither fungus had formed appressorial germ tubes yet. When induced resistant first leaves were challenged with the compatible pathogen (Egt), transcription of the host genes was enhanced a second time. No difference in kinetics of induction of transcription could be observed between noninduced and induced resistant leaves. One of the cloned induced mRNAs codes for a peroxidase, as shown by cDNA derived partial peptide sequence analysis. Peroxidase activity increased in intercellular washing fluids of first leaves from 6 to 36 hours after inoculation.