Gene expression in germinated cysts of Phytophthora nicotianae

The life cycle of Phytophthora species contains several distinct asexual developmental stages that are important for plant infection and disease development. These include vegetative growth of filamentous hyphae, production of multinucleate sporangia, motile, uninucleate zoospores and germinated cysts, the stage at which plant colonization is initiated. To understand mechanisms regulating molecular and cellular processes in germinated cysts, differential hybridization analysis of a cDNA library was used to identify genes up-regulated after cyst germination in P. nicotianae. Arrays of 12 288 random cDNA clones derived from a germinated cyst cDNA library were screened with ³²P-labelled cDNA probes synthesized from mRNA isolated from four different developmental stages and tobacco tissues infected with P. nicotianae. The resultant expression profiles for each cDNA clone led to the identification of over 300 clones showing up-regulated expression in germinated cysts compared with the other three stages. Sequencing of the 5′ end of 382 selected clones yielded 355 sequences representing 146 putative unigenes. Sequence analysis revealed similarities to genes encoding proteins involved in energy production, protein biosynthesis, signalling, cell-wall biogenesis and transcription regulation. Novel genes putatively involved in cell adhesion, cell-wall biogenesis and transcriptional regulation were identified. Thirty-one cDNA clones were analysed by Northern blotting and for 28 the pattern of expression in the Northern blots was the same as that indicated by the macroarray screening, verifying the fidelity of the colony hybridization data.