Audrey M. V. Ah-Fong

Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans

Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world’s population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at

Signatures of adaptation to obligate biotrophy in the Hyaloperonospora arabidopsidis genome.

6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at ∼240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for ∼74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.

Gene expression profiling during asexual development of the late blight pathogen Phytophthora infestans reveals a highly dynamic transcriptome.

From Blight to Powdery Mildew Pathogenic effects of microbes on plants have widespread consequences. Witness, for example, the cultural upheavals driven by potato blight in the 1800s. A variety of microbial pathogens continue to afflict crop plants today, driving both loss of yield and incurring the increased costs of control mechanisms. Now, four reports analyze microbial genomes in order to understand better how plant pathogens function (see the Perspective by Dodds). Raffaele et al. (p. 1540) describe how the genome of the potato blight pathogen accommodates transfer to different hosts. Spanu et al. (p. 1543) analyze what it takes to be an obligate biotroph in barley powdery mildew, and Baxter et al. (p. 1549) ask a similar question for a natural pathogen of Arabidopsis. Schirawski et al. (p. 1546) compared genomes of maize pathogens to identify virulence determinants. Better knowledge of what in a genome makes a pathogen efficient and deadly is likely to be useful for improving agricultural crop management and breeding. A group of papers analyzes pathogen genomes to find the roots of virulence, opportunism, and life-style determinants. Many oomycete and fungal plant pathogens are obligate biotrophs, which extract nutrients only from living plant tissue and cannot grow apart from their hosts. Although these pathogens cause substantial crop losses, little is known about the molecular basis or evolution of obligate biotrophy. Here, we report the genome sequence of the oomycete Hyaloperonospora arabidopsidis (Hpa), an obligate biotroph and natural pathogen of Arabidopsis thaliana. In comparison with genomes of related, hemibiotrophic Phytophthora species, the Hpa genome exhibits dramatic reductions in genes encoding (i) RXLR effectors and other secreted pathogenicity proteins, (ii) enzymes for assimilation of inorganic nitrogen and sulfur, and (iii) proteins associated with zoospore formation and motility. These attributes comprise a genomic signature of evolution toward obligate biotrophy.

Optimization of transgene-mediated silencing in Phytophthora infestans and its association with small-interfering RNAs.

Much of the pathogenic success of Phytophthora infestans, the potato and tomato late blight agent, relies on its ability to generate from mycelia large amounts of sporangia, which release zoospores that encyst and form infection structures. To better understand these stages, Affymetrix GeneChips based on 15,650 unigenes were designed and used to profile the life cycle. Approximately half of P. infestans genes were found to exhibit significant differential expression between developmental transitions, with approximately (1)/(10) being stage-specific and most changes occurring during zoosporogenesis. Quantitative reverse-transcription polymerase chain reaction assays confirmed the robustness of the array results and showed that similar patterns of differential expression were obtained regardless of whether hyphae were from laboratory media or infected tomato. Differentially expressed genes encode potential cellular regulators, especially protein kinases; metabolic enzymes such as those involved in glycolysis, gluconeogenesis, or the biosynthesis of amino acids or lipids; regulators of DNA synthesis; structural proteins, including predicted flagellar proteins; and pathogenicity factors, including cell-wall-degrading enzymes, RXLR effector proteins, and enzymes protecting against plant defense responses. Curiously, some stage-specific transcripts do not appear to encode functional proteins. These findings reveal many new aspects of oomycete biology, as well as potential targets for crop protection chemicals.

The kinome of Phytophthora infestans reveals oomycete-specific innovations and links to other taxonomic groups

Methods for silencing genes in Phytophthora transformants have been demonstrated previously, but wide variation in effectiveness was reported in different studies. To optimize this important tool for functional genomics, we compared the abilities of sense, antisense, and hairpin transgenes introduced by protoplast, electroporation, and bombardment methods to silence the inf1 elicitin gene in Phytophthora infestans. A hairpin construct induced silencing three times more often than sense or antisense vectors, and protoplast transformation twice as much as electroporation. Using hairpins introduced into protoplasts, 61% of strains were silenced, and transgene copy number was positively correlated with silencing. The utility of bombardment was reduced by the occurrence of heterokaryons containing silenced and non-silenced nuclei, but silenced strains were obtainable from about 20% of primary transformants by single-nuclear purification. Most inf1-deficient strains were fully silenced, however some exhibited partial suppression. These produced inf1-derived RNAs of about 21-nt which correspond to both the sense and antisense strands of inf1, implicating an RNAi-like mechanism in silencing.

Vectors for fluorescent protein tagging in Phytophthora: tools for functional genomics and cell biology.

BackgroundOomycetes are a large group of economically and ecologically important species. Its most notorious member is Phytophthora infestans, the cause of the devastating potato late blight disease. The life cycle of P. infestans involves hyphae which differentiate into spores used for dispersal and host infection. Protein phosphorylation likely plays crucial roles in these stages, and to help understand this we present here a genome-wide analysis of the protein kinases of P. infestans and several relatives. The study also provides new insight into kinase evolution since oomycetes are taxonomically distant from organisms with well-characterized kinomes.ResultsBioinformatic searches of the genomes of P. infestans, P. ramorum, and P. sojae reveal they have similar kinomes, which for P. infestans contains 354 eukaryotic protein kinases (ePKs) and 18 atypical kinases (aPKs), equaling 2% of total genes. After refining gene models, most were classifiable into families seen in other eukaryotes. Some ePK families are nevertheless unusual, especially the tyrosine kinase-like (TKL) group which includes large oomycete-specific subfamilies. Also identified were two tyrosine kinases, which are rare in non-metazoans. Several ePKs bear accessory domains not identified previously on kinases, such as cyclin-dependent kinases with integral cyclin domains. Most ePKs lack accessory domains, implying that many are regulated transcriptionally. This was confirmed by mRNA expression-profiling studies that showed that two-thirds vary significantly between hyphae, sporangia, and zoospores. Comparisons to neighboring taxa (apicomplexans, ciliates, diatoms) revealed both clade-specific and conserved features, and multiple connections to plant kinases were observed. The kinome of Hyaloperonospora arabidopsidis, an oomycete with a simpler life cycle than P. infestans, was found to be one-third smaller. Some differences may be attributable to gene clustering, which facilitates subfamily expansion (or loss) through unequal crossing-over.ConclusionThe large sizes of the Phytophthora kinomes imply that phosphorylation plays major roles in their life cycles. Their kinomes also include many novel ePKs, some specific to oomycetes or shared with neighboring groups. Little experimentation to date has addressed the biological functions of oomycete kinases, but this should be stimulated by the structural, evolutionary, and expression data presented here. This may lead to targets for disease control.

Gene expression changes during asexual sporulation by the late blight agent Phytophthora infestans occur in discrete temporal stages

Fluorescent tagging has become the strategy of choice for examining the subcellular localisation of proteins. To develop a versatile community resource for this method in oomycetes, plasmids were constructed that allow the expression of either of four spectrally distinct proteins [cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and mCherry], alone or fused at their N- or C-termini, to sequences of interest. Equivalent sets of plasmids were made using neomycin or hygromycin phosphotransferases (nptII, hpt) as selectable markers, to facilitate double-labelling and aid work in diverse species. The fluorescent proteins and drug-resistance markers were fused to transcriptional regulatory sequences from the oomycete Bremia lactucae, which are known to function in diverse oomycetes, although the promoter in the fluorescence cassette (ham34) can be replaced easily by a promoter of interest. The function of each plasmid was confirmed in Phytophthora infestans. Moreover, fusion proteins were generated using targeting sequences for the endoplasmic reticulum, Golgi, mitochondria, nuclei, and peroxisomes. Studies of the distribution of the fusions in mycelia and sporangia provided insight into cellular organisation at different stages of development. This toolbox of vectors should advance studies of gene function and cell biology in Phytophthora and other oomycetes.

New role for Cdc14 phosphatase: localization to basal bodies in the oomycete phytophthora and its evolutionary coinheritance with eukaryotic flagella.

Transcriptional changes during asexual sporangia formation by the late blight pathogen Phytophthora infestans were identified using microarrays representing 15,646 genes and RNA from sporulation time-courses, purified spores, and sporulation-defective strains. Results were confirmed by reverse transcription-polymerase chain reaction analyses of sporulation on artificial media and infected tomato. During sporulation, about 12% of genes were up-regulated compared to vegetative hyphae and 5% were down-regulated. The most prevalent induced genes had functions in signal transduction, flagella assembly, cellular organization, metabolism, and molecular or vesicular transport. Distinct patterns of expression were discerned based on the kinetics of mRNA induction and their persistence in sporangia. For example, most flagella-associated transcripts were induced very early in sporulation and maintained in sporangia, while many participants in metabolism or small molecule transport were also induced early but had low levels in sporangia. Data from this study are a resource for understanding sporogenesis, which is critical to the pathogenic success of P. infestans and other oomycetes.

RNA-seq of life stages of the oomycete Phytophthora infestans reveals dynamic changes in metabolic, signal transduction, and pathogenesis genes and a major role for calcium signaling in development

Cdc14 protein phosphatases are well known for regulating the eukaryotic cell cycle, particularly during mitosis. Here we reveal a distinctly new role for Cdc14 based on studies of the microbial eukaryote Phytophthora infestans, the Irish potato famine agent. While Cdc14 is transcribed constitutively in yeast and animal cells, the P. infestans ortholog is expressed exclusively in spore stages of the life cycle and not in vegetative hyphae where the bulk of mitosis takes place. PiCdc14 expression is first detected in nuclei at sporulation, and during zoospore formation the protein accumulates at the basal body, which is the site from which flagella develop. The association of PiCdc14 with basal bodies was supported by co-localization studies with the DIP13 basal body protein and flagellar β-tubulin, and by demonstrating the enrichment of PiCdc14 in purified flagella-basal body complexes. Overexpressing PiCdc14 did not cause defects in growth or mitosis in hyphae, but interfered with cytoplasmic partitioning during zoosporogenesis. This cytokinetic defect might relate to its ability to bind microtubules, which was shown using an in vitro cosedimentation assay. The use of gene silencing to reveal the precise function of PiCdc14 in flagella is not possible since we showed previously that silencing prevents the formation of the precursor stage, sporangia. Nevertheless, the association of Cdc14 with flagella and basal bodies is consistent with their phylogenetic distribution in eukaryotes, as species that lack the ability to produce flagella generally also lack Cdc14. An ancestral role of Cdc14 in the flagellar stage of eukaryotes is thereby proposed.

Architecture of the Sporulation-Specific Cdc14 Promoter from the Oomycete Phytophthora infestans

BackgroundThe oomycete Phytophthora infestans causes the devastating late blight diseases of potato and tomato. P. infestans uses spores for dissemination and infection, like many other filamentous eukaryotic plant pathogens. The expression of a subset of its genes during spore formation and germination were studied previously, but comprehensive genome-wide data have not been available.ResultsRNA-seq was used to profile hyphae, sporangia, sporangia undergoing zoosporogenesis, motile zoospores, and germinated cysts of P. infestans. Parallel studies of two isolates generated robust expression calls for 16,000 of 17,797 predicted genes, with about 250 transcribed in one isolate but not the other. The largest changes occurred in the transition from hyphae to sporangia, when >4200 genes were up-regulated. More than 1350 of these were induced >100-fold, accounting for 26% of total mRNA. Genes encoding calcium-binding proteins, cation channels, signaling proteins, and flagellar proteins were over-represented in genes up-regulated in sporangia. Proteins associated with pathogenicity were transcribed in waves with subclasses induced during zoosporogenesis, in zoospores, or in germinated cysts. Genes involved in most metabolic pathways were down-regulated upon sporulation and reactivated during cyst germination, although there were exceptions such as DNA replication, where transcripts peaked in zoospores. Inhibitor studies indicated that the transcription of two-thirds of genes induced during zoosporogenesis relied on calcium signaling. A sporulation-induced protein kinase was shown to bind a constitutive Gβ-like protein, which contributed to fitness based on knock-down analysis.ConclusionsSpore formation and germination involves the staged expression of a large subset of the transcriptome, commensurate with the importance of spores in the life cycle. A comparison of the RNA-seq results with the older microarray data indicated that information is now available for about twice the number of genes than before. Analyses based on function revealed dynamic changes in genes involved in pathogenicity, metabolism, and signaling, with diversity in expression observed within members of multigene families and between isolates. The effects of calcium signaling, a spore-induced protein kinase, and an interacting Gβ-like protein were also demonstrated experimentally. The results reveal aspects of oomycete biology that underly their success as pathogens and potential targets for crop protection chemicals.