Asunto(s)
Hongos/genética , Hongos/patogenicidad , Phytophthora/genética , Phytophthora/patogenicidad , Enfermedades de las Plantas/microbiología , Proteínas Algáceas/genética , Secuencia de Aminoácidos , Análisis por Conglomerados , Codón/genética , ADN de Algas/genética , ADN Complementario/genética , ADN de Hongos/genética , Evolución Molecular , Etiquetas de Secuencia Expresada , Hongos/metabolismo , Biblioteca de Genes , Lisina/biosíntesis , Datos de Secuencia Molecular , Filogenia , Phytophthora/metabolismo , Polisacárido Liasas/genética , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Virulencia/genéticaRESUMEN
SUMMARY Suppression Subtractive Hybridization (SSH) was used to search for genes of Phytophthora infestans that are induced during the infection of potato. To avoid having to distinguish the genes of the pathogen from the plant genes involved in defence responses and to isolate the genes involved in the early stages of interaction, mycelium of P. infestans was induced by contact with the host plant and then separated from the plant tissue. A differential cDNA library was generated by SSH that compared such induced mycelium with mycelium incubated in water. The expression of about 100 cDNA fragments from this differential cDNA library was analysed by hybridization of the arrayed PCR products with mRNA from control and induced mycelium. Twenty per cent of them showed increased transcript levels in mycelium within the first 24 h after exposure to a potato leaf. For six of these cDNA clones the elevated expression in response to the potato leaf could be proven by RNA gel blot analysis. Five of these cDNA clones have predicted amino acid sequence homologies to entries in the databases, including an amino acid transporter, a sucrose transporter, a spliceosome-associated factor, an ABC transporter, and a cell division control protein. We showed that the genes corresponding to these six cDNA clones are differentially regulated during their life. Reliable gene expression analysis of Phytophthora in infected leaf tissue is not possible until c. 48 h post-infection, but for two of the genes we identified, induction during in planta growth was detectable by RNA gel blot analysis. Therefore the SSH library that we have created provides a basis for the identification of P. infestans genes that are up-regulated during the interaction with the plant, which could be important for pathogenicity.
