Niche-specific metabolic adaptation in biotrophic and necrotrophic oomycetes is manifested in differential use of nutrients, variation in gene content, and enzyme evolution.

The use of host nutrients to support pathogen growth is central to disease. We addressed the relationship between metabolism and trophic behavior by comparing metabolic gene expression during potato tuber colonization by two oomycetes, the hemibiotroph Phytophthora infestans and the necrotroph Pythium ultimum. Genes for several pathways including amino acid, nucleotide, and cofactor biosynthesis were expressed more by Ph. infestans during its biotrophic stage compared to Py. ultimum. In contrast, Py. ultimum had higher expression of genes for metabolizing compounds that are normally sequestered within plant cells but released to the pathogen upon plant cell lysis, such as starch and triacylglycerides. The transcription pattern of metabolic genes in Ph. infestans during late infection became more like that of Py. ultimum, consistent with the former's transition to necrotrophy. Interspecific variation in metabolic gene content was limited but included the presence of γ-amylase only in Py. ultimum. The pathogens were also found to employ strikingly distinct strategies for using nitrate. Measurements of mRNA, 15N labeling studies, enzyme assays, and immunoblotting indicated that the assimilation pathway in Ph. infestans was nitrate-insensitive but induced during amino acid and ammonium starvation. In contrast, the pathway was nitrate-induced but not amino acid-repressed in Py. ultimum. The lack of amino acid repression in Py. ultimum appears due to the absence of a transcription factor common to fungi and Phytophthora that acts as a nitrogen metabolite repressor. Evidence for functional diversification in nitrate reductase protein was also observed. Its temperature optimum was adapted to each organism's growth range, and its Km was much lower in Py. ultimum. In summary, we observed divergence in patterns of gene expression, gene content, and enzyme function which contribute to the fitness of each species in its niche.

Defining Transgene Insertion Sites and Off-Target Effects of Homology-Based Gene Silencing Informs the Application of Functional Genomics Tools in Phytophthora infestans.

DNA transformation and homology-based transcriptional silencing are frequently used to assess gene function in Phytophthora spp. Since unplanned side-effects of these tools are not well-characterized, we used P. infestans to study plasmid integration sites and whether knockdowns caused by homology-dependent silencing spread to other genes. Insertions occurred both in gene-dense and gene-sparse regions but disproportionately near the 5' ends of genes, which disrupted native coding sequences. Microhomology at the recombination site between plasmid and chromosome was common. Studies of transformants silenced for 12 different gene targets indicated that neighbors within 500 nt were often cosilenced, regardless of whether hairpin or sense constructs were employed and the direction of transcription of the target. However, this cis spreading of silencing did not occur in all transformants obtained with the same plasmid. Genome-wide studies indicated that unlinked genes with partial complementarity with the silencing-inducing transgene were not usually down-regulated. We learned that hairpin or sense transgenes were not cosilenced with the target in all transformants, which informs how screens for silencing should be performed. We conclude that transformation and gene silencing can be reliable tools for functional genomics in Phytophthora spp. but must be used carefully, especially by testing for the spread of silencing to genes flanking the target.

Phosphagen kinase function in flagellated spores of the oomycete Phytophthora infestans integrates transcriptional regulation, metabolic dynamics and protein retargeting.

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.

Novo&Stitch: accurate reconciliation of genome assemblies via optical maps.

Motivation: De novo genome assembly is a challenging computational problem due to the high repetitive content of eukaryotic genomes and the imperfections of sequencing technologies (i.e. sequencing errors, uneven sequencing coverage and chimeric reads). Several assembly tools are currently available, each of which has strengths and weaknesses in dealing with the trade-off between maximizing contiguity and minimizing assembly errors (e.g. mis-joins). To obtain the best possible assembly, it is common practice to generate multiple assemblies from several assemblers and/or parameter settings and try to identify the highest quality assembly. Unfortunately, often there is no assembly that both maximizes contiguity and minimizes assembly errors, so one has to compromise one for the other. Results: The concept of assembly reconciliation has been proposed as a way to obtain a higher quality assembly by merging or reconciling all the available assemblies. While several reconciliation methods have been introduced in the literature, we have shown in one of our recent papers that none of them can consistently produce assemblies that are better than the assemblies provided in input. Here we introduce Novo&Stitch, a novel method that takes advantage of optical maps to accurately carry out assembly reconciliation (assuming that the assembled contigs are sufficiently long to be reliably aligned to the optical maps, e.g. 50 Kbp or longer). Experimental results demonstrate that Novo&Stitch can double the contiguity (N50) of the input assemblies without introducing mis-joins or reducing genome completeness. Availability and implementation: Novo&Stitch can be obtained from https://github.com/ucrbioinfo/Novo_Stitch.

Gene Expression and Silencing Studies in Phytophthora infestans Reveal Infection-Specific Nutrient Transporters and a Role for the Nitrate Reductase Pathway in Plant Pathogenesis.

To help learn how phytopathogens feed from their hosts, genes for nutrient transporters from the hemibiotrophic potato and tomato pest Phytophthora infestans were annotated. This identified 453 genes from 19 families. Comparisons with a necrotrophic oomycete, Pythium ultimum var. ultimum, and a hemibiotrophic fungus, Magnaporthe oryzae, revealed diversity in the size of some families although a similar fraction of genes encoded transporters. RNA-seq of infected potato tubers, tomato leaves, and several artificial media revealed that 56 and 207 transporters from P. infestans were significantly up- or down-regulated, respectively, during early infection timepoints of leaves or tubers versus media. About 17 were up-regulated >4-fold in both leaves and tubers compared to media and expressed primarily in the biotrophic stage. The transcription pattern of many genes was host-organ specific. For example, the mRNA level of a nitrate transporter (NRT) was about 100-fold higher during mid-infection in leaves, which are nitrate-rich, than in tubers and three types of artificial media, which are nitrate-poor. The NRT gene is physically linked with genes encoding nitrate reductase (NR) and nitrite reductase (NiR), which mobilize nitrate into ammonium and amino acids. All three genes were coregulated. For example, the three genes were expressed primarily at mid-stage infection timepoints in both potato and tomato leaves, but showed little expression in potato tubers. Transformants down-regulated for all three genes were generated by DNA-directed RNAi, with silencing spreading from the NR target to the flanking NRT and NiR genes. The silenced strains were nonpathogenic on leaves but colonized tubers. We propose that the nitrate assimilation genes play roles both in obtaining nitrogen for amino acid biosynthesis and protecting P. infestans from natural or fertilization-induced nitrate and nitrite toxicity.

How Does Phytophthora infestans Evade Control Efforts? Modern Insight Into the Late Blight Disease.

The infamous oomycete Phytophthora infestans has been a persistent threat to potato and tomato production worldwide, causing the diseases known as late blight. This pathogen has proved to be remarkably adept at overcoming control strategies including host-based resistance and fungicides. This review describes the features of P. infestans that make it such a daunting challenge to agriculture. These include a stealthy lifestyle that helps P. infestans evade plant defenses, effectors that suppress host defenses and promote susceptibility, profuse sporulation with a short latent period that enables rapid dissemination, and a genome structure that promotes the adaptive evolution of P. infestans by fostering genetic diversity. Nevertheless, there is reason to be optimistic that accumulated knowledge about the biology of P. infestans and its hosts will lead to improved management of late blight.

Rethinking the evolution of eukaryotic metabolism: novel cellular partitioning of enzymes in stramenopiles links serine biosynthesis to glycolysis in mitochondria.

BACKGROUND: An important feature of eukaryotic evolution is metabolic compartmentalization, in which certain pathways are restricted to the cytosol or specific organelles. Glycolysis in eukaryotes is described as a cytosolic process. The universality of this canon has been challenged by recent genome data that suggest that some glycolytic enzymes made by stramenopiles bear mitochondrial targeting peptides. RESULTS: Mining of oomycete, diatom, and brown algal genomes indicates that stramenopiles encode two forms of enzymes for the second half of glycolysis, one with and the other without mitochondrial targeting peptides. The predicted mitochondrial targeting was confirmed by using fluorescent tags to localize phosphoglycerate kinase, phosphoglycerate mutase, and pyruvate kinase in Phytophthora infestans, the oomycete that causes potato blight. A genome-wide search for other enzymes with atypical mitochondrial locations identified phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase, which form a pathway for generating serine from the glycolytic intermediate 3-phosphoglycerate. Fluorescent tags confirmed the delivery of these serine biosynthetic enzymes to P. infestans mitochondria. A cytosolic form of this serine biosynthetic pathway, which occurs in most eukaryotes, is missing from oomycetes and most other stramenopiles. The glycolysis and serine metabolism pathways of oomycetes appear to be mosaics of enzymes with different ancestries. While some of the noncanonical oomycete mitochondrial enzymes have the closest affinity in phylogenetic analyses with proteins from other stramenopiles, others cluster with bacterial, plant, or animal proteins. The genes encoding the mitochondrial phosphoglycerate kinase and serine-forming enzymes are physically linked on oomycete chromosomes, which suggests a shared origin. CONCLUSIONS: Stramenopile metabolism appears to have been shaped through the acquisition of genes by descent and lateral or endosymbiotic gene transfer, along with the targeting of the proteins to locations that are novel compared to other eukaryotes. Colocalization of the glycolytic and serine biosynthesis enzymes in mitochondria is apparently necessary since they share a common intermediate. The results indicate that descriptions of metabolism in textbooks do not cover the full diversity of eukaryotic biology.

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.

BACKGROUND: The 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. RESULTS: RNA-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 Gß-like protein, which contributed to fitness based on knock-down analysis. CONCLUSIONS: Spore 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 Gß-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.

Lifestyle, gene gain and loss, and transcriptional remodeling cause divergence in the transcriptomes of Phytophthora infestans and Pythium ultimum during potato tuber colonization.

BACKGROUND: How pathogen genomes evolve to support distinct lifestyles is not well-understood. The oomycete Phytophthora infestans, the potato blight agent, is a largely biotrophic pathogen that feeds from living host cells, which become necrotic only late in infection. The related oomycete Pythium ultimum grows saprophytically in soil and as a necrotroph in plants, causing massive tissue destruction. To learn what distinguishes their lifestyles, we compared their gene contents and expression patterns in media and a shared host, potato tuber. RESULTS: Genes related to pathogenesis varied in temporal expression pattern, mRNA level, and family size between the species. A family's aggregate expression during infection was not proportional to size due to transcriptional remodeling and pseudogenization. Ph. infestans had more stage-specific genes, while Py. ultimum tended towards more constitutive expression. Ph. infestans expressed more genes encoding secreted cell wall-degrading enzymes, but other categories such as secreted proteases and ABC transporters had higher transcript levels in Py. ultimum. Species-specific genes were identified including new Pythium genes, perforins, which may disrupt plant membranes. Genome-wide ortholog analyses identified substantial diversified expression, which correlated with sequence divergence. Pseudogenization was associated with gene family expansion, especially in gene clusters. CONCLUSION: This first large-scale analysis of transcriptional divergence within oomycetes revealed major shifts in genome composition and expression, including subfunctionalization within gene families. Biotrophy and necrotrophy seem determined by species-specific genes and the varied expression of shared pathogenicity factors, which may be useful targets for crop protection.

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 $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 approximately 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 approximately 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.

A Cas12a-based gene editing system for Phytophthora infestans reveals monoallelic expression of an elicitor.

Phytophthora infestans is a destructive pathogen of potato and a model for investigations of oomycete biology. The successful application of a CRISPR gene editing system to P. infestans is so far unreported. We discovered that it is difficult to express CRISPR/Cas9 but not a catalytically inactive form in transformants, suggesting that the active nuclease is toxic. We were able to achieve editing with CRISPR/Cas12a using vectors in which the nuclease and its guide RNA were expressed from a single transcript. Using the elicitor gene Inf1 as a target, we observed editing of one or both alleles in up to 13% of transformants. Editing was more efficient when guide RNA processing relied on the Cas12a direct repeat instead of ribozyme sequences. INF1 protein was not made when both alleles were edited in the same transformant, but surprisingly also when only one allele was altered. We discovered that the isolate used for editing, 1306, exhibited monoallelic expression of Inf1 due to insertion of a copia-like element in the promoter of one allele. The element exhibits features of active retrotransposons, including a target site duplication, long terminal repeats, and an intact polyprotein reading frame. Editing occurred more often on the transcribed allele, presumably due to differences in chromatin structure. The Cas12a system not only provides a tool for modifying genes in P. infestans, but also for other members of the genus by expanding the number of editable sites. Our work also highlights a natural mechanism that remodels oomycete genomes.

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

BACKGROUND: Oomycetes 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. RESULTS: Bioinformatic 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. CONCLUSION: The 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.

An RNA symbiont enhances heat tolerance and secondary homothallism in the oomycete Phytophthora infestans.

Some strains of Phytophthora infestans, the potato late blight pathogen, harbour a small extrachromosomal RNA called PiERE1. A previous study reported that this RNA symbiont does not noticeably affect its host. Here it is revealed that PiERE1 exerts subtle effects on P. infestans, which result in greater thermotolerance during growth and an increase in secondary homothallism, i.e. oospore formation in the absence of the opposite mating type. The interaction can be considered mutualistic since these traits may increase the fitness of P. infestans in nature. Assays of biomarkers for cellular stress revealed that an Hsp70 chaperone was upregulated by PiERE1. A genome-wide search for more members of the Hsp70 family identified ten belonging to the DnaK subfamily, one in the Hsp110/SSE subfamily, and pseudogenes. Four DnaK subfamily genes encoding predicted cytoplasmic or endoplasmic reticulum proteins were upregulated in strains harbouring PiERE1. This may explain the greater thermotolerance conferred by the RNA element, and suggests that Hsp70 may be a useful biomarker for testing organisms for the cellular effects of symbiotic elements.

Invertases in Phytophthora infestans Localize to Haustoria and Are Programmed for Infection-Specific Expression.

The oomycete Phytophthora infestans, the causal agent of potato and tomato blight, expresses two extracellular invertases. Unlike typical fungal invertases, the P. infestans genes are not sucrose induced or glucose repressed but instead appear to be under developmental control. Transcript levels of both genes were very low in mycelia harvested from artificial medium but high in preinfection stages (sporangia, zoospores, and germinated cysts), high during biotrophic growth in leaves and tubers, and low during necrotrophy. Genome-wide analyses of metabolic enzymes and effectors indicated that this expression profile was fairly unusual, matched only by a few other enzymes, such as carbonic anhydrases and a few RXLR effectors. Genes for other metabolic enzymes were typically downregulated in the preinfection stages. Overall metabolic gene expression during the necrotrophic stage of infection clustered with artificial medium, while the biotrophic phase formed a separate cluster. Confocal microscopy of transformants expressing green fluorescent protein (GFP) fusions indicated that invertase protein resided primarily in haustoria during infection. This localization was not attributable to haustorium-specific promoter activity. Instead, the N-terminal regions of proteins containing signal peptides were sufficient to deliver proteins to haustoria. Invertase expression during leaf infection was linked to a decline in apoplastic sucrose, consistent with a role of the enzymes in plant pathogenesis. This was also suggested by the discovery that invertase genes occur across multiple orders of oomycetes but not in most animal pathogens or a mycoparasite.IMPORTANCE Oomycetes cause hundreds of diseases in economically and environmentally significant plants. How these microbes acquire host nutrients is not well understood. Many oomycetes insert specialized hyphae called haustoria into plant cells, but unlike their fungal counterparts, a role in nutrition has remained unproven. The discovery that Phytophthora invertases localize to haustoria provides the first strong evidence that these structures participate in feeding. Since regions of proteins containing signal peptides targeted proteins to the haustorium-plant interface, haustoria appear to be the primary machinery for secreting proteins during biotrophic pathogenesis. Although oomycete invertases were acquired laterally from fungi, their expression patterns have adapted to the Phytophthora lifestyle by abandoning substrate-level regulation in favor of developmental control, allowing the enzymes to be produced in anticipation of plant colonization. This study highlights how a widely distributed hydrolytic enzyme has evolved new behaviors in oomycetes.

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

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.

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

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.

Architecture of the sporulation-specific Cdc14 promoter from the oomycete Phytophthora infestans.

The Cdc14 gene of Phytophthora infestans is transcribed specifically during sporulation, with no mRNA detectable in vegetative hyphae, and is required for sporangium development. To unravel the mechanisms regulating its transcription, mutated Cdc14 promoters plus chimeras of selected Cdc14 sequences and a minimal promoter were tested in stable transformants. This revealed that a tandem repeat of three copies of the motif CTYAAC, located between 67 and 90 nucleotides (nt) upstream of the major transcription start site, is sufficient to determine sporulation-specific expression. All three repeats need to be present for activity, suggesting that they bind a transcription factor through a cooperative mechanism. Electrophoretic mobility shift assays indicated that the CTYAAC repeats are specifically bound by a protein in nuclear extracts. Evidence was also obtained for a second region within the promoter that activates Cdc14 transcription during sporulation which does not involve those repeats. The CTYAAC motif also affects the specificity of transcription initiation. Wild-type Cdc14 is transcribed from a major start site and minor site(s) located about 100 nt upstream of the major site. However, stepwise mutations through the CTYAAC triad caused a graded shift to the upstream sites, as did mutating bases surrounding the major start site; transcripts initiated from the upstream site remained sporulation specific. Replacing the Cdc14 initiation region with the Inr-like region of the constitutive Piexo1 gene had no apparent effect on the pattern of transcription. Therefore, this study reports the first motif determining sporulation-induced transcription in oomycetes and helps define oomycete core promoters.

Illuminating Phytophthora Biology with Fluorescent Protein Tags.

Phytophthora species cause diseases that threaten agricultural, ornamental, and forest plants worldwide. Explorations of the biology of these pathogens have been aided by the availability of genome sequences, but much work remains to decipher the roles of their proteins. Insight into protein function can be obtained by visualizing them within cells, which has been facilitated by recent improvements in fluorescent protein and microscope technologies. Here, we describe strategies to permit investigators to generate strains of Phytophthora that express fluorescently tagged proteins and study their localization during growth in artificial media and during plant infection.

Transcriptomic and proteomic analysis reveals wall-associated and glucan-degrading proteins with potential roles in Phytophthora infestans sexual spore development.

Sexual reproduction remains an understudied feature of oomycete biology. To expand our knowledge of this process, we used RNA-seq and quantitative proteomics to examine matings in Phytophthora infestans. Exhibiting significant changes in mRNA abundance in three matings between different A1 and A2 strains compared to nonmating controls were 1170 genes, most being mating-induced. Rising by >10-fold in at least one cross were 455 genes, and 182 in all three crosses. Most genes had elevated expression in a self-fertile strain. Many mating-induced genes were associated with cell wall biosynthesis, which may relate to forming the thick-walled sexual spore (oospore). Several gene families were induced during mating including one encoding histidine, serine, and tyrosine-rich putative wall proteins, and another encoding prolyl hydroxylases which may strengthen the extracellular matrix. The sizes of these families vary >10-fold between Phytophthora species and one exhibits concerted evolution, highlighting two features of genome dynamics within the genus. Proteomic analyses of mature oospores and nonmating hyphae using isobaric tags for quantification identified 835 shared proteins, with 5% showing >2-fold changes in abundance between the tissues. Enriched in oospores were ß-glucanases potentially involved in digesting the oospore wall during germination. Despite being dormant, oospores contained a mostly normal complement of proteins required for core cellular functions. The RNA-seq data generated here and in prior studies were used to identify new housekeeping controls for gene expression studies that are more stable than existing normalization standards. We also observed >2-fold variation in the fraction of polyA+ RNA between life stages, which should be considered when quantifying transcripts and may also be relevant to understanding translational control during development.