Metabolic Novelties in the Oomycete Phytopathogen, Phytophthora infestans

Phytophthora infestans, the causal agent of potato late blight, is a significant threat to global food security. Little is known about the metabolism of this oomycete, including how it adapts to different growth and nutrient conditions, while such aspects are one key to understanding host-pathogen interactions. To identify nutrient transporter and metabolic genes involved in pathogenicity, genome and RNA-seq data were mined. One of the major findings was the identification of nitrate assimilation genes that were strongly upregulated at mid time-point tomato leaf tissues compared to late time-point. Mutants silenced for these genes were then generated, which showed a reduction in virulence. This confirmed involvement of the nitrate assimilation genes in pathogenicity. Even though P. infestans can not grow using nitrate as the sole nitrogen source, enzyme assays and isotopic labeling studies indicated that its nitrate assimilation pathway is functional. Enzyme assays revealed few biochemical differences in the activity of nitrate reductase from P. infestans and the enzyme from Phytophthora mirabilis and Pythium ultimum, which are two oomycetes that can grow on nitrate. Isotopic labelling studies indicated that P. infestans and P. mirabilis assimilated little nitrate into amino acids in young cultures, but incorporated substantial nitrate in older cultures. In contrast, Py. ultimum utilized nitrate at both early and late growth stages. Studies of other metabolic pathways revealed the presence of mitochondrial glycolytic pay-off phase in addition to the canonical cytosolic one, and a mitochondrial serine biosynthesis pathway, which is cytoplasmic in other species; both pathways are linked through the intermediate, 3-phosphoglycerate. These novel mitochondrial genes are restricted to stramenopiles. Such novel enzymes could be used as targets for the chemical control of oomycetes. I purpose that manipulation of metabolism is a promising approach for control of P. infestans, one of the most devastating phytopathogens

Metabolic Novelties in the Oomycete Phytopathogen, Phytophthora infestans

Phytophthora infestans, the causal agent of potato late blight, is a significant threat to global food security. Little is known about the metabolism of this oomycete, including how it adapts to different growth and nutrient conditions, while such aspects are one key to understanding host-pathogen interactions. To identify nutrient transporter and metabolic genes involved in pathogenicity, genome and RNA-seq data were mined. One of the major findings was the identification of nitrate assimilation genes that were strongly upregulated at mid time-point tomato leaf tissues compared to late time-point. Mutants silenced for these genes were then generated, which showed a reduction in virulence. This confirmed involvement of the nitrate assimilation genes in pathogenicity. Even though P. infestans can not grow using nitrate as the sole nitrogen source, enzyme assays and isotopic labeling studies indicated that its nitrate assimilation pathway is functional. Enzyme assays revealed few biochemical differences in the activity of nitrate reductase from P. infestans and the enzyme from Phytophthora mirabilis and Pythium ultimum, which are two oomycetes that can grow on nitrate. Isotopic labelling studies indicated that P. infestans and P. mirabilis assimilated little nitrate into amino acids in young cultures, but incorporated substantial nitrate in older cultures. In contrast, Py. ultimum utilized nitrate at both early and late growth stages. Studies of other metabolic pathways revealed the presence of mitochondrial glycolytic pay-off phase in addition to the canonical cytosolic one, and a mitochondrial serine biosynthesis pathway, which is cytoplasmic in other species; both pathways are linked through the intermediate, 3-phosphoglycerate. These novel mitochondrial genes are restricted to stramenopiles. Such novel enzymes could be used as targets for the chemical control of oomycetes. I purpose that manipulation of metabolism is a promising approach for control of P. infestans, one of the most devastating phytopathogens

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

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

Abstract 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

Additional file 2: of Rethinking the evolution of eukaryotic metabolism: novel cellular partitioning of enzymes in stramenopiles links serine biosynthesis to glycolysis in mitochondria

Species represented in Fig. 2 and accession numbers of the analyzed protein sequences. (XLSX 43 kb

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

Additional file 3: of Rethinking the evolution of eukaryotic metabolism: novel cellular partitioning of enzymes in stramenopiles links serine biosynthesis to glycolysis in mitochondria

Accession numbers of protein sequences used in phylogenetic analyses of payoff phase glycolytic enzymes and serine biosynthesis enzymes. (DOCX 177 kb

Additional file 1: Table S1. of Rethinking the evolution of eukaryotic metabolism: novel cellular partitioning of enzymes in stramenopiles links serine biosynthesis to glycolysis in mitochondria

Mitochondrial targeting scores of enzymes. (XLS 66 kb

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

<div><p>To help learn how phytopathogens feed from their hosts, genes for nutrient transporters from the hemibiotrophic potato and tomato pest <i>Phytophthora infestans</i> were annotated. This identified 453 genes from 19 families. Comparisons with a necrotrophic oomycete, <i>Pythium ultimum</i> var. <i>ultimum</i>, and a hemibiotrophic fungus, <i>Magnaporthe oryzae</i>, 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 <i>P</i>. <i>infestans</i> 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 <i>P</i>. <i>infestans</i> from natural or fertilization-induced nitrate and nitrite toxicity.</p></div