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 Ca2+- 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.

FIG. 1.

Expression of kinase transcript in P. infestans. RNA was extracted from isolate 88069, electrophoresed, blotted, and probed with a ³²P-labeled fragment of the kinase gene (PK). Blots were then stripped and hybridized with an elongation factor 1 probe (EF1). (A) RNA from nonsporulating hyphae (HY), sporulating hyphae (SH), purified sporangia (SP), zoospores released in 10°C water (ZO), or sporangia undergoing direct germination in rye media (DG). (B) Left panel shows blot using RNA from nonsporulating hyphae (HY), sporangia freshly harvested in water (0 [min]), or RNA from sporangia held in 10°C water for 30, 60, 90, or 120 min. Right panel shows quantitation of PK mRNA and zoospore development during the same experiment. Indicated are the ratio of PK and EF1 signals, as determined by phosphorimager analysis and expressed as percentage of maximum PK abundance (open circles); percentage of sporangia showing visible cytoplasmic cleavage but not having released zoospores (triangles); percentage of empty sporangia, reflecting those that released zoospores (closed circles); and percentage of sporangia not showing any differentiation (squares). RNA was extracted from both sporangia and zoospores in the samples. (C) RNA from cleaving sporangia (60 min in 10°C water [CL]), zoospores (ZO), and cysts germinated for 8 h in 10°C water (GC). (D) RNA from cleaving sporangia (CL) and hyphae placed in nitrogen or carbon-limited media or water (NS, CS, and HS).

FIG. 2.

Detection of the kinase gene in genomic DNA of P. infestans. DNA from isolate 88069 was restriction digested, blotted, and hybridized with a 6-kb HindIII fragment of a BAC containing the protein kinase gene. Digestions employed BamHI (B), EcoRI (E), HindIII (H), or PstI (P). Size standards shown in the right margin were determined using a 1-kb ladder (Invitrogen).

FIG. 3.

Structure of the kinase gene, predicted protein, and alignment with related kinases. (A) Features of gene found on 6-kb HindIII fragment of BAC clone. Indicated are the transcription start point (+1), a 16-nt oomycete-promoter motif (OO) at −96 nt, a 50-nt 5′ untranslated leader, the start codon (ATG at +51), stop codon (TAA), 45-nt 3′ untranslated leader, positions of the 12 conserved kinase domains (1 to 5, 6a, 6b, and 7 to 11), and restriction sites for EcoRI (Eco), HindIII (Hin), and PstI (Pst). (B) Sequence of predicted protein product. Indicated are the 12 kinase domains (box), the ATP-binding domain (underlined), and the catalytic site (underline with arrowheads). (C) Alignment of kinase domains of the P. infestans protein with CaMKs from Caenorhabditis elegans (GenBank accession number BAA82674), Dictyostelium (A40811), mouse (AAC48715), Aspergillus nidulans (Q00771), the yeast Saccharomyces cerevisiae (CAA40928), Fragaria (AAB88537), and Paramecium tetraurelia (AAC13356).

FIG. 4.

Neighbor-joining tree based on kinase domains. A consensus tree was developed based on the amino acid sequence of the 12 kinase domain regions of various serine/threonine protein kinases as described in Materials and Methods. Numbers at nodes indicate the percentages of their occurrence in 500 bootstrap replicates, and the scale represents 0.1 PAM units. The types of kinases examined and their GenBank accession numbers are as follows: CaMKs were from A. nidulans (AAD38850), C. elegans (BAA82674), Dictyostelium discoideum (A40811), Glomerella cingulata (AAC62515), human (NP001212), mouse (AAC48715), and S. cerevisiae (CAA40928). CDPKs (with calmodulin-like domain) were from cucumber (AAB49984), Eimeria tenella (CAA96439), P. tetraurelia (AAC13356), tobacco (AAC25423), tomato (AAK52801), and T. gondii (AF043629). A Ca²⁺- and/or Ca²⁺- and calmodulin-regulated kinase (CCaMK) from tobacco (with visinin-like domain) (AAD28791) was also tested. Plant calcium-independent protein kinases (CRKs) were from rice (AAK54157) and maize (BAA22410). PEPCKs were from Arabidopsis (AAK84668), sorghum (AAK81871), and tomato (AAF19403). A PEPCK-related kinase (PEPRK) from Arabidopsis (T45842) was also tested. SNF1-like kinases (SNRKs) were from the algae Guillardia theta (AF165818), Mesembryanthemum crystallinum (Z26846), S. cerevisiae (M13971), Schizosaccharomyces pombe (KIN1) (m36060), and soybean (Glycine max) (AF128443). Other serine/threonine kinases tested, which are not directly regulated by calcium or calmodulin, were the DNA damage-induced DUN1 kinase of S. cerevisiae (S43941), human phosphorylase kinase (catalytic subunit [KIHUCT]), and two kinases in the metazoan AGC group (the mitogen- and stress-activated MSK1 kinase of humans, T13149, and the Gallus gallus ribosomal protein S6 kinase, M28488).

FIG. 5.

Effect of actinomycin D on expression of the kinase gene. RNA was extracted from sporangia quickly isolated from culture plates in water (0-A), sporangia isolated from culture plates in water and held at 10°C for 1 h to induce zoospore release (60-A), and sporangia isolated in 10 or 100 μg of actinomycin D/ml and held at 10°C for 1 h (60 + 10A, 60 + 100A). RNA blots were hybridized with a probe for the kinase gene (PK) and were then stripped and hybridized with a probe for elongation factor 1 (EF1). Ratios between the kinase and EF1 signals (P/E) were then determined by phosphorimager analysis.