1.10.17
A TOOL-KIT FOR STUDYING ANTISENSE GENE SILENCING IN STAGONOSPORA NODORUM

N HALL1, SB CUTIER2, RN COOLEY3, SG FOSTER3, A RENWICK4, JA LUCAS1, CE CATEN2 and JA HARGREAVES1

1Molecular Pathology Group, IACR-Long Ashton, Long Ashton, Bristol, BS18 9AR, UK; 2School of Biological Sciences, University of Birmingham, Birmingham, B15 2TT, UK; 3AgrEvo UK Ltd, Chesterford Park, Saffron Walden, Essex CB10 1XL, UK; 4Zeneca Ltd, Jealotts Hill Research Station, Bracknell, Berkshire RG42 6ET, UK

Background and objectives
As the knowledge on the physiology and biochemistry of fungal pathogens accumulates, the number of potential target sites for chemical intervention to control diseases increases. Validation of fungicide targets is therefore becoming an increasingly important step in the discovery of new antifungal compounds. Until recently, biochemical or classical mutagenic approaches were used to verify potential targets. Targeted gene disruption offers an alternative approach that is valuable for identifying essential genes. However, the disabled strains are totally devoid of functional protein, a level of inhibition unlikely to be achieved by chemicals inhibitors in the field. It is, therefore, highly desirable to develop a system for reducing target gene expression to variable and quantifiable levels which could be correlated to growth and development of the pathogen. Antisense RNA techniques, which have been widely used in plant and mammalian systems, appear to be an ideal way of achieving this.

Materials and methods
We are developing antisense RNA methodologies for the fungal plant pathogen Stagonospora nodorum. This will require the use of constitutive and inducible promoters of varying strengths, and currently we are assessing the activity of various heterologous promoters such as the glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter from Aspergillus nidulans and native promoters such as the nitrate reductase promoter. In addition, we are screening for inducible promoters, including those induced during infection, using differential display.

Results and discussion
The suitability of this range of promoters to drive antisense expression is being examined using GUS and GFP reporter genes. Once the parameters governing antisense are better understood, attempts will be made to regulate genes that encode known fungicide targets, such as the squalene synthetase or tubulin genes which we have already cloned and sequenced.