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

<p>Abstract</p> <p>Background</p> <p>Oomycetes are a large group of economically and ecologically important species. Its most notorious member is <it>Phytophthora infestans</it>, the cause of the devastating potato late blight disease. The life cycle of <it>P. infestans </it>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 <it>P. infestans </it>and several relatives. The study also provides new insight into kinase evolution since oomycetes are taxonomically distant from organisms with well-characterized kinomes.</p> <p>Results</p> <p>Bioinformatic searches of the genomes of <it>P. infestans</it>, <it>P. ramorum</it>, and <it>P. sojae </it>reveal they have similar kinomes, which for <it>P. infestans </it>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 <it>Hyaloperonospora arabidopsidis</it>, an oomycete with a simpler life cycle than <it>P. infestans</it>, 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.</p> <p>Conclusion</p> <p>The large sizes of the <it>Phytophthora </it>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.</p

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

BackgroundHow 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.ResultsGenes 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.ConclusionThis 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

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.

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 &gt;4200 genes were up-regulated. More than 1350 of these were induced &gt;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

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

BackgroundAn 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.ResultsMining 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.ConclusionsStramenopile 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

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

Novo&amp;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&amp;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&amp;Stitch can double the contiguity (N50) of the input assemblies without introducing mis-joins or reducing genome completeness. Availability and implementation:Novo&amp;Stitch can be obtained from https://github.com/ucrbioinfo/Novo_Stitch

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

MotivationDe 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.ResultsThe 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&amp;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&amp;Stitch can double the contiguity (N50) of the input assemblies without introducing mis-joins or reducing genome completeness.Availability and implementationNovo&amp;Stitch can be obtained from https://github.com/ucrbioinfo/Novo_Stitch