Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan

Background and objectives
Phytophthora infestans is a heterothallic fungus, and oosporogenesis takes place during the interaction between A1 and A2 mating-type strains. In this study, we have isolated subtracted cDNA clones having increased expression during mating of A1 and A2 strains, using a modified representational difference analysis (cDNA-RDA) method [1, 2] to search for genes specifically involved in oosporogenesis.

Materials and methods
We used mixed media of V8 broth and rye broth for single and mating cultures. Agar disks of both strains were placed in media independently or together, and incubated at 20C. Under these conditions, oogonia were found within 3 days in mating culture, increased in number until 8 days, and reaching a plateau (2000-3000 oogonia/cm2 mycelium). The highest rate of oogonia formation per day is 4 or 5 days after incubation. The concept of subtraction was as follows: (gene transcripts in mating-cultured cells)-(gene transcripts in single-cultured cells)=(mating condition-specific transcripts). We prepared driver amplicon cDNA from 6-day-old single-cultured mycelia of E009 (A1) and TB201 (A2), respectively, and tester amplicon cDNA from 6-day-old mating-cultured mycelia of the same strains. Subtraction was done three times by RDA, and we obtained mating condition-specific differential products (DP3, the Sau3AI digests of subtracted cDNA). The subtracted cDNA library was constructed from DP3. We chose 96 clones at random, sequenced them and made DIG probes for RNA dot-blot and Northern hybridization to detect the difference in gene transcripts of single and mating cultures, and for genomic Southern hybridization to confirm the origin of the clones.

Results and conclusions
The DIG-labelled DP3 probe hybridized only with total RNA from mating-cultured cells. The 14 up-regulated clones were sequenced and compared with the DDBJ database. Clone cET33 (103 bp) showed 79.5% homology with Phaeodactylum tricornutum fcp genes (fucoxanthin-chlorophyll binding proteins). Clone cET106 (106 bp) was 76.2% homologous to Streptococcus faecium translational initiation factor IF2. And clone cET32 (300 bp) was 71.9% homologous to Homo sapiens CL100 mRNA (tyrosine phosphatase). None of the clones showed high homology (over 80%) to known genes. Patterns of genomic Southern hybridization using these clones were similar in both strains. To confirm the origin of these transcripts, it is necessary to complete the method to isolate RNA from one strain in mating culture. In this study, it was confirmed that some genes were stimulated in mating condition of A1 and A2 strains. Following characterization of full-length clones, it will be interesting to investigate the role of cET106 and cET32 in relation to gene transcription and modification of proteins, respectively.

1. Hubank M, Schatz DG, 1994. Nucleic Acids Research 22, 5640-5648.
2. Niwa H, Harrison LC, Deaizpurua HJ, Cram DS, 1997. Endocrinology 138, 1419-1426.