1.2.28
NOVEL POTATO GENES INVOLVED IN DEFENCE AGAINST PHYTOPHTHORA INFESTANS ARE IMPLICATED IN APOPTOSIS

AO AVROVA2, PRJ BIRCH1, R TOTH1, GD LYON1 and JM DUNCAN1

1Scottish Crop Research Institute, lnvergowrie, Dundee DD2 5DA, UK; 2All-Russia Institute of Plant Protection, St Petersburg-Pushkin, 189620, Russia

Background and objectives
The major constraint to the production of potato is the oomycete Phytophthora infestans, the cause of late blight. Fungicides which are currently used to control this pathogen are expensive and ecologically undesirable. Development of resistance by P. infestans to the fungicides increases the complexity of such control. As a result, late-blight resistance is a priority of most potato-breeding programmes. Genetic resistance to P. infestans in both wild and cultivated potato species is of two forms, either race-non-specific, which is believed to be polygenic and highly durable, or race-specific, characterized by the interaction between dominant R gene alleles in the host and the corresponding avirulence alleles in the pathogen. Little is known about how these two forms of resistance work in practice. The aim of this work was to determine genes which may be involved in these resistance processes.

Materials and methods
Potato cv. Stirling expresses both durable and major R gene forms of resistance to P. infestans. In contrast, cv. Bintje is completely susceptible, and does not exhibit a hypersensitive response (HR). Recently developed PCR-based methods were used to compare profiles of gene expression in both infected and uninfected cv. Stirling with those of the susceptible cv. Bintje (cDNA-AFLPs [1]) and for generating a cDNA library enriched for pathogen-induced genes specific to cv. Stirling (suppression subtractive hybridization or SSH [2]). cDNA was prepared using mRNA extracted from leaves of both cultivars uninfected and 15 h after inoculation with a race of P. infestans which is avirulent to cv. Stirling. cDNA-AFLP analysis was performed using a number of primer combinations on uninfected and infected samples, with cDNA from P. infestans grown in culture used as a control. cDNAs produced by the SSH process were cloned and 1000 clones screened with probes to eliminate remaining sequences common to both cultivars. 100 clones specific to cDNA from infected Stirling were sequenced.

Results and conclusions
Using cDNA-AFLP analysis, little difference (up to 7.0% depending on the primer combination) in gene expression between the uninfected potato cultivars was observed, whereas the infected cultivars demonstrated up to 26.7% differences in banding patterns between each other and between infected and uninfected material. No P. infestans bands could be identified in infected material, suggesting that cDNA from the pathogen is only weakly represented in the host at this time point. We were thus confident that the pathogen genes are excluded from the analysis of resistance in the host. cDNA prepared from infected leaves of cv. Bintie was used as a driver to remove all common sequences from the infected cv. Stirling cDNA via SSH. The subtracted material was amplified and cloned. After screening to remove repetitive sequences such as ribosomal RNA, 100 of the remaining clones were sequenced and compared to database sequences using both FASTA (DNA homology) and BLASTX (protein homology). 60% of the sequences were either too small to identify an open reading frame, or a clear ORF was observed but no definite match was found. The remaining 40% of sequences matched with sequences in the database. Of these, approximately 85% matched with stress-related genes, mainly of plant origin, but also some of mammalian origin such as serine palmitoyltransferase, which is involved in signalling the onset of apoptosis. Analysis of recovered sequences by hybridization to AFLP-derived total cDNA material confirmed them to be upregulated only in cv. Stirling following infection by P. infestans. Evidence suggests that apoptosis-related genes are implicated in the HR specific to cv. Stirling.

References
1. Bachem CWB, Van der Hoeven RS, de Bruijn SM, et al., 1996. Plant Journal 9, 745-753.
2. Diatchenko L, Lau Y-FC, Campbell AP et al., 1996. Proceedings of the National Academy of Sciences, USA 93, 6025-6030.