CDNA cloning and characterization of two barley peroxidase transcripts induced differentially by the powdery mildew fungus Erysiphe graminis

Abstract

A cDNA library of RNA from barley leaves inoculated with Erysiphe graminis was screened using labelled cDNA enriched for specific sequences by subtractive hybridization against RNA from non-inoculated leaves. This resulted in isolation of several clones representing pathogen induced genes. By cross-hybridization and sequence analysis, one of the cDNAs (pBT6-3) was found to be a partial clone representing a putative peroxidase, for which a full-length cDNA clone (pBH6-301) was subsequently isolated. The predicted amino acid sequence revealed a 21 amino acid signal peptide and a 294 amino acid mature protein (31 kDa) and shows 56 % amino acid identity to a basic peroxidase from turnip, 89 % to a putative peroxidase from wheat, but only 38% to the amino acid sequence derived from the cDNA clone (pcD1311) of a second putative barley peroxidase expressed in leaves. Northern blot analysis showed that the pBT6-3 (pBH6-301) transcript is elevated as early as 4 h after inoculation with E. graminis f. sp. hordei and that two maxima in transcript levels appear, which can be correlated with penetration attempts by the fungus. The amount of the pcD1311 transcript was also found to increase in inoculated leaves but at a later time point.