Isolation and characterization of four genes encoding pyruvate, phosphate dikinase in the oomycete plant pathogen Phytophthora cinnamomi

The oomycete genus Phytophthora contains some of the world's most devastating plant pathogens. We report here the existence in P. cinnamomi of four genes encoding the pyrophosphate-utilizing glycolytic/gluconeogenic enzyme pyruvate, phosphate dikinase (PPDK). The coding regions of the four genes are >99% identical. At least three of the genes comprise a small gene cluster, which may have arisen through recent gene duplication and inversion events. Levels of Pdk mRNA are low in vegetative hyphae, but increase rapidly and transiently upon transfer of cultures to nutrient-free media, conditions that trigger asexual sporulation. PPDK protein and enzyme activity levels do not show a similar increase during sporulation. Assays of PPDK activity in P. cinnamomi hyphal extracts suggest that the majority of glycolytic flux in sporulating hyphae probably occurs via PPDK, rather than pyruvate kinase. This finding, combined with the existence of Phytophthora-expressed sequence tags encoding two other pyrophosphate-utilizing enzymes, indicates that pyrophosphate-based metabolism may be important in Phytophthora. The possibility that PPDK and other enzymes of pyrophosphate-based metabolism may provide targets for the development of novel control measures for Phytophthora and other oomycete pathogens is discussed

Ancient origin of elicitin gene clusters in Phytophthora genomes

The genus Phytophthora belongs to the oomycetes in the eukaryotic stramenopile lineage and is comprised of over 65 species that are all destructive plant pathogens on a wide range of dicotyledons. Phytophthora produces elicitins (ELIs), a group of extracellular elicitor proteins that cause a hypersensitive response in tobacco. Database mining revealed several new classes of elicitin-like (ELL) sequences with diverse elicitin domains in Phytophthora infestans, Phytophthora sojae, Phytophthora brassicae, and Phytophthora ramorum. ELIs and ELLs were shown to be unique to Phytophthora and Pythium species. They are ubiquitous among Phytophthora species and belong to one of the most highly conserved and complex protein families in the Phytophthora genus. Phylogeny construction with elicitin domains derived from 156 ELIs and ELLs showed that most of the diversified family members existed prior to divergence of Phytophthora species from a common ancestor. Analysis to discriminate diversifying and purifying selection showed that all 17 ELI and ELL clades are under purifying selection. Within highly similar ELI groups there was no evidence for positively selected amino acids suggesting that purifying selection contributes to the continued existence of this diverse protein family. Characteristic cysteine spacing patterns were found for each phylogenetic clade. Except for the canonical clade ELI-1, ELIs and ELLs possess C-terminal domains of variable length, many of which have a high threonine, serine, or proline content suggesting an association with the cell wall. In addition, some ELIs and ELLs have a predicted glycosylphosphatidylinositol site suggesting anchoring of the C-terminal domain to the cell membrane. The eli and ell genes belonging to different clades are clustered in the genomes. Overall, eli and ell genes are expressed at different levels and in different life cycle stages but those sharing the same phylogenetic clade appear to have similar expression pattern

Immunocytochemical comparison of peripheral vesicles in zoospores of Phytophthora and Pythium species

An ultrastructural and immunocytochemical comparison has been made of vesicles in the peripheral cytoplasm of zoospores of Phytophthora cinnamomi and species of the related Oomycete genus, Pythium. Our results give evidence of three morphologically and immunologically distinct vesicle populations in Pythium aphanidermatum and Py. butleri. Large peripheral vesicles can be recognized by their size and morphology, and by labeling with monoclonal antibody, Cpa-2 raised against a P cinnamomi antigen. They occur predominantly on the dorsal surface of the zoospores and are retained within the cell during encystment. After encystment, the large peripheral vesicles move away from the plasma membrane and become distributed throughout the cyst cytoplasm, in a manner similar to that observed in P. cinnamomi. There are also small vesicles in the Pythium zoospore cortex. Some are identified as ventral vesicles through their reaction with monoclonal antibody Vsv-1, raised against a P. cinnamomi antigen. The ventral vesicles are concentrated along the ridges of the groove on the ventral surface of the zoospores, and their contents are secreted during encystment. The secreted material remains localized on the cyst surface and marks the site of germ tube emergence, as it does in P. cinnamomi. Other small peripheral vesicles occur on the dorsal surface of the Pythium zoospores. These vesicles are not labeled by any of the three antibodies tested but disappear during encystment, suggesting that their contents are secreted. Our results give strong support to the idea that three types of peripheral vesicles are a common feature of zoospores throughout the Peronosporales, and that they have similar fates during encystment

In planta immunolabelling of three types of peripheral vesicles in cells of Phytophthora cinnamomi infecting chestnut roots

Micropropagated plantlets of an improved selection of chestnut (Castanea sativa) that was susceptible to Phytophthora root rot, were inoculated in vitro at the root tip with zoospores of Phytophthora cinnamomi. Plantlets were then incubated for up to 4 d, during which time infection was examined by clearing and staining whole roots or immunolabelling cryosections with monoclonal antibodies specific for fungal vesicular components. Hyphae had colonized entire root systems by 3 d after inoculation and plantlets were dead by 4 d after inoculation. Three types of fungal vesicles, large peripheral, dorsal and ventral vesicles, were immunofluorescently labelled in hyphae infecting roots from I d after inoculation with zoospores. Their appearance correlated spatially and temporally with the production of sporangia and chlamydospores. Vesicles were not observed in fungal cells at the advancing hyphal front, but appeared ∼5 mm behind the growing front. Large peripheral vesicles were also immunolabelled in oospores formed after 10 wk in roots of plantlets inoculated with A1 and A2 mating type isolates of P. cinnamomi, either together or alone