| In the heterothallic oomycete, Phytophthora infestans , mating and sexual differentiation generally involves the presence of the A1 and A2 mating types, which are believed to differ in their production of mating hormones and hormone receptors. Suppression subtractive cloning was used to identify genes responsible for mating and oospore formation. Seven genes were obtained. Two were induced early in mating before the appearance of gametangia, while others were induced later, concurrent with the formation of gametangia and oospores. Sequence analysis revealed that one of the late genes, M-90, was a member of the pumilio family of RNA-binding proteins. The 5′ end of another late gene, M-81 , resembled an extracellular glycoprotein from P. megasperma while its 3.5 kb long middle portion consisted of DNA repeats having similarity with proline-rich proteins. Each of the mating-induced genes were also expressed in a self-fertile heterokaryon. However, while in normal matings 5.1 and 4.0 kb RNAs were derived from M-81, only the 5.1 kb transcript was detected in the self-fertile strain. DNA blot analyses revealed polymorphism at the repeat region of M-81 within P. infestans isolates. In A1 strains 8811, 2.20 and 24.11 all M-81 alleles had the 3.5 kb form of the repeat. A2 isolates 510 and 618, however, were each heterozygotes, having both 3.5 kb and 2.4 kb forms of the repeats. This suggested that the 4.0 kb RNA is derived from the shorter form of the gene. Subtractive hybridization also identified a novel, self-replicating RNA from isolate 510. This replicon had little effect on growth or pathogenicity. However, a host gene, N-2C, was down-regulated in the strains containing the RNA plasmid. In addition, molecular markers linked to the mating type locus of P. parasitica were generated to study mating type determination in Phytophthora. Analysis of the segregation of DNA markers linked to mating type in P. parasitica and P. infestans revealed that mating type locus in P. parasitica displays Mendelian segregation. This is contrary to the situation in P. infestans, in which previous studies suggested that the locus displays non-Mendelian segregation. An improved model for mating-type determination is presented. |
