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

Abstract

A study of the effect of inhibitors on zoospore development in Phytophthora infestans demonstrated the involvement of protein kinases and calcium and led to the discovery of a gene induced during zoosporogenesis that encoded a protein resembling Ca ؉2 -and calmodulin-regulated serine/threonine protein kinases. The calcium channel blocker verapamil and the calmodulin antagonist trifluoroperazine inhibited zoosporogenesis and encystment. The protein kinase inhibitors K-252a and KN-93 inhibited zoospore release, encystment, and cyst germination, and K-252a reduced zoospore viability. In contrast, the inhibitors had minor or no effects on sporangia directly germinating in media. Spurred by these findings, a survey of putative protein kinase genes was performed to identify any that were up-regulated during zoosporogenesis. A kinase-encoding gene was identified for which mRNA accumulation was first detected soon after chilling sporangia in water, conditions that induce sporangial cytoplasm to cleave and release zoospores. The transcript persisted in motile zoospores and in germinated cysts but was not detected in other tissues, including hyphae, hyphae placed in water, or directly germinating sporangia. The structure of the predicted protein was novel, as its C-terminal region, which binds calmodulin in related proteins, was unusually short. Concentrations of actinomycin D previously used in experiments that suggested that de novo transcription was not needed for zoosporogenesis or encystment only partially inhibited transcription of the kinase gene, probably due to poor uptake into sporangia.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...

Large-Scale Gene Discovery in the OomycetePhytophthora infestansReveals Likely Components of Phytopathogenicity Shared with True Fungi

“…Because this has been an in silico analysis, the datasets only represent a first screen for truly stage-specific genes. However, the differential expression of several predicted spore-specific genes has been confirmed (Ah Fong and Judelson 2003;Judelson and Roberts 2002).…”

Activation of Zoosporogenesis-Specific Genes in Phytophthora infestans Involves a 7-Nucleotide Promoter Motif and Cold-Induced Membrane Rigidity

“…Cultures of P. infestans isolate 1306 were maintained at 18°C, and developmental stages were obtained as described previously (25). Briefly, sporangia were purified by rubbing hyphal mats with a glass rod in water, followed by passage through 50-m mesh to remove hyphal fragments.…”

“…RNA analysis. RNA was extracted and blotted as described previously (25). Radiolabeled probes were generated from full-length cDNA clones or genespecific fragments were amplified using primer pair NIFC1SF-NIFC1SR, NIFC2SF-NIFC2SR, or NIFC2SF-NIFC2SR.…”

Sporangium-Specific Gene Expression in theOomycete Phytopathogen Phytophthorainfestans

“…Developmental stages of isolate 88069 were prepared as described previously (24). Briefly, this involved nonsporulating hyphae from 5-day-old liquid rye broth cultures; sporulating hyphae or purified sporangia from 8-to 18-day-old rye agar cultures; zoospores released from sporangia after incubation in 10°C water for 90 min; germinated zoospore cysts, prepared by vortexing zoospores for 1 min followed by 6 h of incubation in rye broth at 18°C; and hyphae from liquid cultures of defined media (52) either lacking glucose but retaining fumaric acid (carbon starvation) or with 1/10 the normal amount of ammonium sulfate (nitrogen starvation).…”