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MADS-box transcription factors play significant roles in eukaryotes, but have not yet been characterized in oomycetes. Here, we describe a MADS-box protein from Phytophthora infestans, which causes late blight of potato. P. infestans and most other oomycetes express a single MADS-box gene. PiMADS is not transcribed during vegetative growth, but is induced early during asexual sporulation. Its mRNA levels oscillate in response to light, which suppresses sporulation. The protein was not detected in nonsporulating mycelia, but was found in sporulating mycelia and spores. Both mRNA and protein levels decline upon spore germination. A similar expression pattern as well as nuclear localization was observed when the protein was expressed with a fluorescent tag from the native promoter. Gene silencing triggered by a construct expressing 478 nt of MADS sequences indicated that PiMADS is required for sporulation but not hyphal growth or plant colonization. A comparison of wild type to a silenced strain by RNA-seq indicated that PiMADS regulates about 3000 sporulation-associated genes, and acts before other genes previously shown to regulate sporulation. Analysis of the silenced strain also indicated that the native gene was not transcribed while the transgene was still expressed, which contradicts current models for homology-dependent silencing in oomycetes.

Flagellated spores play important roles in the infection of plants and animals by many eukaryotic microbes. The oomycete Phytophthora infestans, which causes potato blight, expresses two phosphagen kinases (PKs). These enzymes store energy in taurocyamine, and are hypothesized to resolve spatial and temporal imbalances between rates of ATP creation and use in zoospores. A dimeric PK is found at low levels in vegetative mycelia, but high levels in ungerminated sporangia and zoospores. In contrast, a monomeric PK protein is at similar levels in all tissues, although is transcribed primarily in mycelia. Subcellular localization studies indicate that the monomeric PK is mitochondrial. In contrast, the dimeric PK is cytoplasmic in mycelia and sporangia but is retargeted to flagellar axonemes during zoosporogenesis. This supports a model in which PKs shuttle energy from mitochondria to and through flagella. Metabolite analysis indicates that deployment of the flagellar PK is coordinated with a large increase in taurocyamine, synthesized by sporulation-induced enzymes that were lost during the evolution of zoospore-lacking oomycetes. Thus, PK function is enabled by coordination of the transcriptional, metabolic and protein targeting machinery during the life cycle. Since plants lack PKs, the enzymes may be useful targets for inhibitors of oomycete plant pathogens.

Summary Zoospores are critical in the disease cycle of Phytophthora infestans, a member of the oomycete group of fungus-like microbes and the cause of potato late blight. A protein kinase induced during zoosporogenesis, Pipkz1, was shown to interact in the yeast two-hybrid system with a putative bZIP transcription factor. This interaction was confirmed in vitro using a pull-down assay. The transcription factor gene, Pibzp1, was single copy and expressed in all tissues. Transformants of P. infestans stably silenced for Pibzp1 were generated using plasmids expressing its coding region in sense or antisense orientations. A protoplast transformation method induced silencing more efficiently than transformation by an electroporation scheme. Wild-type and silenced strains exhibited no differences in hyphal growth or morphology, mating, sporangia production or zoospore release. However, zoospores from the mutants spun in tight circles, instead of exhibiting the normal pattern of straight swimming punctuated by turns. Zoospore encystment was unaffected by silencing, but cysts germinated more efficiently than controls. Germinated cysts from the mutants failed to develop appressoria and were unable to infect plants; however, they could colonize wounded tissue. These phenotypes indicate that Pibzp1 is a key regulator of several stages of the zoospore-mediated infection pathway.

Cdc14 proteins are important regulators of mitosis and the cell cycle. These phosphatases have been studied previously only in yeasts and metazoans, which grow by fission or budding. Here we describe a homologue (piCdc14) from the oomycete Phytophthora infestans, a primitive eukaryote lacking a classical cell cycle. PiCdc14 complements a cdc14ts mutant of Saccharomyces cerevisiae and may function like other Cdc14 proteins, but displays a strikingly different pattern of expression. Whereas previously studied Cdc14 genes are constitutively transcribed, piCdc14 is not expressed during normal growth but instead only during asexual sporulation. In transformants of P. infestans expressing a fusion between the piCdc14 promoter and the -glucuronidase reporter, expression was first detected in sporangiophore initials, persisted in sporangiophores bearing immature sporangia, and later became restricted to mature sporangia. After germination, expression ended a few hours before the resumption of mitosis in hyphae emerged from the spores. Homology-dependent silencing experiments supported an essential role of piCdc14 in sporulation. It is proposed that the function of piCdc14 may be to synchronise nuclear behaviour during sporulation and maintain dormancy in spores until germination. These results help illuminate the process of sporulation in oomycetes and the evolution of the cell cycle in eukaryotes.

Summary Eight genes that are upregulated during sexual development in the heterothallic oomycete, Phytophthora infestans, were identified by suppression subtractive hybridization. Two genes showed very low but detectable expression in vegetative hyphae and became induced about 40- to >100-fold early in mating, before gametangial initials appeared. The remaining six loci were not induced until later in mating, coincident with the formation of gametangia and oospores, with induction levels ranging from 60- to >100-fold. Five genes were single copy, and three were members of families. Sequence analysis revealed that the predicted products of three of the genes had similarity to proteins that influence RNA stability, namely a ribonuclease activator, the pumilio family of RNA-binding proteins and RNase H. The products of two other mating-induced genes resembled two types of Phytophthora proteins previously shown to elicit plant defence responses. Each mating-induced gene was also expressed in a self-fertile strain, which was shown to be a heterokaryon. However, quantitative and qualitative differences existed in their expression in normal matings and in the self-fertile heterokaryon. Besides the mating-induced genes, two extrachromosomal RNA elements were identified.

Zoospores are critical in the disease cycle of Phytophthora infestans, a member of the oomycete group of fungus-like microbes and the cause of potato late blight. A protein kinase induced during zoosporogenesis, Pipkz1, was shown to interact in the yeast two-hybrid system with a putative bZIP transcription factor. This interaction was confirmed in vitro using a pull-down assay. The transcription factor gene, Pibzp1, was single copy and expressed in all tissues. Transformants of P. infestans stably silenced for Pibzp1 were generated using plasmids expressing its coding region in sense or antisense orientations. A protoplast transformation method induced silencing more efficiently than transformation by an electroporation scheme. Wild-type and silenced strains exhibited no differences in hyphal growth or morphology, mating, sporangia production or zoospore release. However, zoospores from the mutants spun in tight circles, instead of exhibiting the normal pattern of straight swimming punctuated by turns. Zoospore encystment was unaffected by silencing, but cysts germinated more efficiently than controls. Germinated cysts from the mutants failed to develop appressoria and were unable to infect plants; however, they could colonize wounded tissue. These phenotypes indicate that Pibzp1 is a key regulator of several stages of the zoospore-mediated infection pathway.