1.8.47
ANTISENSE AND SENSE MEDIATED GENE SILENCING AS A TOOL TO SUPPRESS EXPRESSION OF THE INF1 GENE OF PHYTOPHTHORA INFESTANS

P VAN WEST1, S KAMOUN2, JW VAN'T KLOOSTER3 and DF GOVERS4

1Department of Phytopathology, and 2Graduate School of Experimental Plant Sciences, Agricultural University, Binnenhaven 9, 6709 PD, Wageningen, The Netherlands; 3Present address: Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Aberdeen AB25 2ZD, UK; 4 Present address: Department of Plant Pathology, Ohio State University, Wooster, OH 444691, USA

Background and objectives
Most Phytophthora and Pythium species produce 10-kDa extracellular protein elicitors, generally termed elicitins. Elicitins induce a hypersensitive response in a restricted number of plants, particularly in the genus Nicotiana within the family Solanaceae. Elicitins are thought to act as avirulence factors that restrict the host range of the pathogen by triggering plant defence responses [1]. Phytophthora infestans, the causal agent of potato late blight disease, produces an elicitin named INF1. A cDNA clone encoding INF1 was isolated and characterized [2]. The aim of this study was to engineer P. infestans mutants deficient in the production of INF1. Such mutants will help us to determine the role of elicitin in host specificity.

Results and conclusions
Since P. infestans is a diploid organism and homologous integration of plasmids has not yet been demonstrated, we adopted a gene silencing strategy to inhibit inf1 expression. P. infestans was transformed with constructs carrying strong oomycete promoters fused to the inf1 coding sequence in both antisense and sense orientations. Expression of both the integrated transgenes and the native inf1 gene was analysed and the production of extracellular INF1 protein was determined. It appeared that up to 20% of the transgenic P. infestans transformants failed to produce inf1 mRNA and, as a consequence, were INFL deficient. Genomic Southern blot analysis of the inf1 silenced transformants demonstrated that the endogenous inf1 gene was not mutated or deleted. In addition, we found that the silenced phenotype remained stable during several in vitro and in planta growth conditions in time. Therefore, these INF1-deficient transformants can be used to determine the role of the elicitin protein in host specificity, unambiguously.

To unravel the mechanism of silencing, we analysed genomic DNA of the silenced transformants for methylation, but found no hyper-methylation of the endogenous and transgene inf1 sequences. Moreover, nuclear run-on assays showed that the silencing phenomenon is not based on high inf1 mRNA turnover. Further experiments to unravel the silencing mechanism will be presented.

References
1. Yu LM, 1995. Proceedings of the National Academy of Sciences, USA 92, 4088-4094.
2. Kamoun S, van West P, de Jong AJ et al., 1997. Molecular Plant-Microbe Interactions 6, 15-25.