Characterization of a Repetitive DNA Sequence from Erysiphe graminis fsp. tritici A.C. Payne1, M-C. Grosjean-Cournoyer2, D.W. Hollomon1 1 Institute of Arable Crop Research-Long Ashton, Dept. of Agricultural Sciences, Univ. of Bristol, Bristol BS18 9AF, UK. 2 Rhone-Poulenc AGRO, 14, rue P. Baizet, 69009 Lyon, France Erysiphe graminis is an obligate pathogen causing powdery mildew of cereals. It adapts rapidly to new environmental factors and is able to overcome both host-plant resistance and fungicide control measures. Increased understanding of genome organization of E. graminis should aid both the development of techniques leading to the cloning and analysis of pathogenicity and fungicide resistance genes and improved disease management. Repeated DNA sequences have already been described in filamentous fungi including E. graminis fsp. hordei (Rasmussen et al. Mol. Gen. Genet., 1993, 239: 298-303). The ability of these repetitive elements to move throughout the genome may be involved in the adaptive potential of powdery mildew.
We have previously described a genomic clone from E. graminis fsp. tritici (pAT5B, Grosjean-Cournoyer et al. ECFG2, 1994, D4) which contained a repetitive element which showed some homology to pBTEG20, the clone containing the B-tubulin gene of E. graminis fsp. hordei (Sherwood and Somerville, Nucleic Acid Res., 1990, 18: 1052). We have further characterized this repetitive sequence and determined by Southern analysis, that it is at least 700 bp in size. Slot blot analysis estimated the repetitive element to be present in several hundred copies per genome. The nucleotide sequence of the cloned repetitive sequence has been determined and this showed no structural similarity with repetitive elements described in other organisms. Restriction analysis and Southern hybridization of several different genomic clones from a E. graminis fsp. tritici genomic library indicated that this repetitive element is distributed throughout the genome and has identified sequence differences within a population of repetitive elements.
We are currently working to define the exact size of the repetitive element. Sequence variation within this repetitive element family in E. graminis fsp. tritici will be investigated further by Southern analysis of restricted genomic clones. To complement these approaches a second repetitive element from the E. graminis fsp. tritici genomic library will be isolated and sequenced.

Descriptors: Hordeum vulgare; Complementary DNA; Pathogenesis-related proteins; Nucleotide sequences; Amino acid sequences; Gene expression; Messenger RNA; Genetic regulation; Fungal diseases; Erysiphe graminis f.sp. hordei; Ethylene; Salicylic acid; Jasmonic acid; Nicotinic acid; Derivatives
Abstract: A barley cDNA clone (PRb-1) corresponding to an mRNA differentially induced in resistant compared to susceptible barley cultivars by powdery mildew infection was isolated and characterised. The deduced amino acid sequence revealed 24 amino acids comprising the signal peptide and 140 amino acids of the mature peptide (15 kDa). This showed close homology to PR-1-like proteins, which have been isolated from maize, tobacco, tomato and Arabidopsis thaliana. Northern blot analysis showed accumulation of the corresponding mRNA 12 h after inoculation of resistant barley cultivars with Erysiphe graminis. Increased expression of the PRb-1 gene was also observed in resistant compared with near-isogenic susceptible barley plants following treatment with ethylene, salicylic acid, methyl jasmonate and 2,6-dichloro-isonicotinic acid.
31 NAL Call. No.: SB599.P45 cDNA cloning and characterization of two barley peroxidase transcripts induced differentially by the powdery mildew fungus Erysiphe graminis. Thordal-Christensen, H.; Brandt, J.; Cho, B.H.; Rasmussen, S.K.; Gregersen, P.L.; Smedegaard-Petersen, V.; Collinge, D.B. London : Academic Press; 1992 Jun. Physiological and molecular plant pathology v. 40 (6): p. 395-409; 1992 Jun. Includes references.
Language: English
Descriptors: Hordeum vulgare; Peroxidase; Dna libraries; Erysiphe graminis; Nucleotide sequences; Amino acid sequences; Gene expression
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.

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