1.8.16
TRAPPING PROMOTERS AND TAGGING GENES IN THE BARLEY PATHOGEN PYRENOPHORA TERES

A LÖSCH, H QUADER, W SCHÄFER and FJ MAIER

Institut für Allgemeine Botanik, Molekulare Phytopathologie und Genetik, Universität Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany

Background and objectives
The ascomycete Pyrenophora teres (anamorph Drechslera teres) is a leaf pathogen causing net blotch of barley (Hordeum vulgare). REMI (restriction enzyme-mediated integration) mutagenesis [1] will yield a genomic mutant library of P. teres. With this library we aim to tag and clone genes and promotors responsible for pathogenesis.

Materials and methods
Transformation is performed with protoplasts. A vector for REMI mutagenesis was constructed to allow transformation rates to be as high as possible, with a high frequency of single integration. Simultaneously, it permits the selection of fungal sequences with promoter activity, using two different promoterless marker genes: ß-glucuronidase (GUS) and luciferase (LUC), one on each end of the linearized vector. Tests were established for detection of both mutants altered in virulence on barley leaves, and those exhibiting plant-induced promoter activity. For histological investigations, marker genes, e.g. GUS or green fluorescent protein (GFP), were constitutively expressed in P. teres.

Results and conclusions
In the process of establishing the REMI library, the influence of various factors (e.g. transformation buffers, restriction enzymes, methods for regeneration of protoplasts) was investigated. Among the first 100 transformants tested, three showed a strong decrease in pathogenicity using a detached leaf assay. 27 showed promoter activity under different conditions on artificial media.

For the constitutive expression of genes in P. teres, we used the gpd1 of Cochliobolus heterostrophus [2]. In comparison with the heterologous gpd1 promoter, two of the trapped promoters exhibited a very strong constitutive activity. We are currently cloning these promoters and the corresponding genes. Further results on the efficiency of the 'trapping and tagging system' will be presented.

After inoculation of barley plants with transformants constitutively expressing GUS or GFP, the fungus in and on the plant becomes easily detectable. Such systems allow the histological determination of the infection behavior typical of P. teres on barley. We will present the time course and route of infection of the fungus colonizing the plant.

References
1. Schiestl RH, Petes TD, 1991. Proceedings of the National Academy of Sciences, USA 88, 7585-7589.
2. Van Wert SL, Yoder OC, 1992. Current Genetics 22, 29-35.