Novel Protein Kinase Induced during Sporangial Cleavage in the Oomycete Phytophthora infestans

Dispersal by means of spores is a common feature of the life cycle of many lower eukaryotes. This includes the oomycetes, which encompass many important plant pathogens, including Phytophthora, Pythium, and the downy mildews. Oomycetes lack taxonomic affinity with the so-called true fungi (i.e., ascomycetes and basidiomycetes), instead being related to diatoms and brown algae (2, 15). The asexual sporangia of oomycetes either germinate directly through a germ tube or indirectly by releasing biflagellate zoospores. Many species germinate through either pathway, while others produce only zoospores and some germinate only directly (12, 22, 26). For species displaying both modes of germination, zoospores are considered most important for dispersal and host infection. One species exhibiting this duality in germination behavior is Phytophthora infestans, which causes late blight of potato and tomato. Sporangia form on sporangiophores that emerge from infected plant tissue or from hyphae on artificial media. As with other oomycetes, the sporangia of P. infestans are multinucleate, nondesiccated, and metabolically active. Germination becomes possible once sporangia detached from sporangiophores encounter liquid. Indirect germination predominates in the absence of nutrients and at cool temperatures, typically below 12°C (30). In contrast, direct germination is favored by higher temperatures and nutrients. Indirect germination takes about 1 h and involves the cleavage of sporangial cytoplasm into multiple zoospores. These emerge from the sporangial apex and swim, displaying several tactic behaviors (8, 20), until encystment occurs in response to chemical or physical stimulation (13). Encystment occurs within seconds, involving the detachment of flagella and cell wall deposition (13). Cysts subsequently elaborate a germ tube that can penetrate and colonize a plant host or grow into new mycelium in artificial culture. Many elegant microscopic and physiological studies of zoosporogenesis, encystment, and cyst germination have been performed. Changes in cytoskeletal organization and vesicle distribution are documented, as are movements of ions such as calcium (9, 13, 17, 21, 25, 38). In contrast, little is known of the molecular biology of zoospore development or how its stages are regulated. Cleavage, zoospore release, and encystment are reportedly insensitive to actinomycin D, which suggests the involvement of preexisting mRNA or proteins. Later steps, such as cyst germination, are sensitive to such inhibitors (7, 27). In this study, to better understand the regulation of germination and zoospore development, inhibitors of cellular processes were tested for their effects on direct and indirect germination in P. infestans. One or more stages of zoospore development were inhibited by compounds affecting protein kinases and calcium pathways. This led to a search for protein kinase genes that were differentially expressed during zoosporogenesis, in recognition that protein phosphorylation regulates cellular functions in other species (5). A kinase-encoding gene was identified from which RNA transcripts became detectable soon after placing sporangia in cool water to induce zoospore release. No transcripts were found in uninduced sporangia, directly germinating sporangia, or hyphae. The predicted product of the gene resembled Ca2+- and calmodulin-regulated protein kinases (CaMKs) but lacked an obvious C-terminal regulatory or protein-association domain typically found in such proteins. The gene was also used to address the effect of actinomycin D on transcription during germination.