5.4.5
CYSTEINE PROTEINASE - A NEW DEFENCE-RELATED GENE INDUCED IN BARLEY ROOTS AFTER FUNGAL ATTACK

SCHLICHTER UHA1, ANTONIW JF2, ADAMS MJ2 and STEINBISS H-H1

1Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, 50829 Cologne, Germany; 2IACR Rothamsted, Crop and Disease Management Department, Harpenden, Herts AL5 2JQ, UK

Background and objectives
As a model system for studying root-fungus interactions in barley, the infection with the highly pathogenic Ascomycete Gaeumannomyces graminis var. tritici (Ggt) was selected. Ggt is the causal agent of the severe take-all disease of barley and wheat causing high losses in agronomy. Infected plants in the field die prematurely and exhibit white heads which contain only shrivelled, sterile grains. Brownish lesions are observed on heavily infected roots. Our interest was to investigate gene expression in barley roots upon infection with Ggt in order to identifiy up-regulated genes.

Results and conclusion
Differential screening of a cDNA library derived from heavily infected barley roots (variety Maris Otter), using first-strand cDNA probes prepared from poly(A) RNA extracted from either healthy of infected barley roots, revealed the isolation of the cDNA clone rcyp (root cysteine proteinase). From Northern blot analysis, differential gene expression of the corresponding gene rcyp was confirmed. Transcription of the gene is strongly induced after infection of barley roots with Ggt in heavily infected root tissue as well as in parts of the roots not showing any lesions, but not induced in leaves of the infected plants. After challenging barley roots with the parasite Polymyxa graminis, a fungus transmitting viruses of barley mosaic disease (i.e. barley mild mosaic bymovirus, BaMMV), an enhanced transcription level was observed. Mechanical inoculation of barley leaves with BaMMV did not stimulate the expression of the rcyp gene either in roots or in leaves. It can be concluded that the expression of the rcyp gene is strongly induced after fungal attack and is restricted to the roots; even after infection with leaf pathogens such as Erysiphe graminis and Rhynchosporium secalis, no transcription was detected in leaves.

Comparing the nucleotide sequence and the deduced amino acid sequence with sequences from the database, the cDNA clone shows homologies of max. 50% to cysteine proteinases of the papain family from the plant and animal kingdom. Cysteine proteinases are pre-pro-proteins [1]. From theoretical data it can be assumed that the RCYP protein is a pre-pro-protein which is synthesized in the endoplasmatic reticulum and finally secreted to the extracelluar space. The possible role of the RCYP protein in plant defence will be discussed.

Southern blot analysis indicates that the rcyp gene is present in the barley genome as one or two copies. Using PCR on genomic barley DNA and subsequent sequence analysis it was determined that the rcyp gene contains an intron of 350 bp, inserted in the coding region of the gene. Upstream parts of the rcyp gene have been isolated by means of RACE PCR techniques. The promoter region of the gene has been analysed. The most interesting finding is the presence of elicitor-responsive elements such as W-Boxes and a C-Box at positions similar to the ones described [2]. Experiments testing the different promoter elements will be described.

References
1. Rawlings ND, Barret AJ, 1994. Methods in Enzymology 244, 461-485.
2. Rushton PJ, Tovar Torres J, Parniske M et al., 1996. EMBO Journal 15, 5690-5700.