T HAIZEL, M SCHNEIDER and JP METRAUX

Institute of Plant Biology, University of Fribourg, CH-1700 Fribourg, Switzerland

Background and objectives
The late blight disease of potato is responsible for great losses to the potato production industry. This disease is caused by the oomycete Phytophthora infestans. Methods such as breeding resistant varieties, fungicide treatments and phytosanitary practices are currently used to hold the disease in check. However, the development of genetically engineered, resistant potato plants seems to be a possibility to complement or eventually replace existing strategies. Our goal therefore is to identify and characterize resistance genes to generate potato plants with improved resistance to P. infestans.

The hypersensitive response (HR), a defence mechanism that involves rapid plant cell death, is associated with resistance and with the induction of systemic acquired resistance (SAR). We took advantage of the fact that, in tobacco, several genes have been described that are transcriptionally activated during HR of leaves infiltrated with an incompatible isolate of Pseudomonas solanacearum [1]. Some of these genes might be related to HR itself or to resistance mechanisms that derive from it. If expressed under appropriate inducible promoters, such genes could lead to resistance. Potato homologues of these genes were therefore isolated for further characterization.

Materials and methods
Fragments of tobacco genes activated during HR (gift from Y. Marco) were used as probes to screen a potato (cultivar Datura) cDNA library, made 24 h after infection with P. infestans (gift from G. Strittmatter, MPI, Köln). The hybridization and washing steps were done at high stringency. Northern and Southern blot analyses were also carried out at high stringency.

For fungal induction experiments, leaves of 4-week-old potato plants were sprayed with P. infestans (strain 94-28) zoospore suspension (4000-5000 spores/ml) and harvested at different time points. Control plants were water-treated and harvested.

Results and conclusions
Three potato cDNA clones belonging to two families have been isolated. The first shows a characteristic signature for cytochrome P₄₅₀ and is registered as CYP71D4. The best homology was found with CYP71A1, a putative 4-cinnamate hydroxylase from avocado.

The two other genes are 72% identical but do not show any significant homologies with any gene present in the databases. Nevertheless, the expression of these two genes is different: while the first is constitutively expressed, the second exhibits a maximum induction 14 h after inoculation with P. infestans. Results from Southern blot analysis on potato genomic DNA suggest that these two genes belong to a multigene family. The function of these genes is yet to be found. However, their corresponding genomic clones have been isolated. Several promoter-reporter gene constructs have been generated for further study of the regulation and expression of these genes. Potato plants transformed with these constructs are currently being analysed.

References
1. Marco YJ, Ragueh F, Godiard L, Froissard D, 1990. Plant Molecular Biology 15, 145-154.