De novo pyrimidine biosynthesis in the oomycete plant pathogen Phytophthora infestans

García-Bayona, Leonor; Garavito, Manuel F.; Lozano, Gabriel L.; Vasquez, Juan J.; Myers, Kevin; Fry, William E.; Bernal, Adriana; Zimmermann, Bárbara H.; Restrepo, Silvia

doi:10.1016/j.gene.2013.12.009

Cited by 21 publications

(30 citation statements)

References 75 publications

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“…phylogenetic analysis of UPs in oomycete. The expression patterns for oomycete enzymes in the pyrimidine biosynthesis and salvage pathways were first described for the potato pathogen P. infestans 7 . BLAST analysis of the P. capsici orthologue PcUP1 against other oomycetes revealed orthologues in all the other Phytophthora species as well as Pythium species.…”

Section: Resultsmentioning

confidence: 99%

Structural and catalytic analysis of two diverse uridine phosphorylases in Phytophthora capsici

Yang

Huang

et al. 2020

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Uridine phosphorylase (Up) is a key enzyme of pyrimidine salvage pathways that enables the recycling of endogenous or exogenous-supplied pyrimidines and plays an important intracellular metabolic role. Here, we biochemically and structurally characterized two evolutionarily divergent uridine phosphorylases, PcUP1 and PcUP2 from the oomycete pathogen Phytophthora capsici. our analysis of other oomycete genomes revealed that both uridine phosphorylases are present in Phytophthora and Pythium genomes, but only UP2 is seen in Saprolegnia spp. which are basal members of the oomycetes. Moreover, uridine phosphorylases are not found in obligate oomycete pathogens such as Hyaloperonospora arabidopsidis and Albugo spp. PcUP1 and PcUP2 are upregulated 300 and 500 fold respectively, within 90 min after infection of pepper leaves. The crystal structures of PcUP1 in ligand-free and in complex with uracil/ribose-1-phosphate, 2′-deoxyuridine/phosphate and thymidine/ phosphate were analyzed. crystal structure of this uridine phosphorylase showed strict conservation of key residues in the binding pocket. Structure analysis of PcUP1 with bound ligands, and site-directed mutagenesis of key residues provide additional support for the "push-pull" model of catalysis. our study highlights the importance of pyrimidine salvage during the earliest stages of infection. Phytophthora capsici, one of the most destructive plant pathogens of Phytophthora spp., causes economic losses to tomatoes, peppers, cucurbits, lima, and snap bean crops 1. The most effective control strategies of Phytophthora require a combination of different methods 1. However, chemical control of Phytophthora, is becoming less effective because of growing resistance to existing chemicals, and because oomycetes lack the targets for most of the broad-spectrum agrochemicals deployed against phytopathogenic fungi. Since metabolism and nutrient acquisition play an important role in disease establishment, infection, and development of plant pathogens 2 a complete, detailed analysis of metabolic strategies used by foliar oomycete pathogens is warranted to identify new strategies for control of these pathogens 3. The biosynthesis of purine and pyrimidine nucleotides which are critical substrates for growth and replication, can proceed via de novo synthesis from amino acids, or by salvage pathways that enable the recycling of bases and nucleosides formed during the degradation of RNA and DNA 4. While the enzymatic strategies for synthesis of purine and pyrimidine are strongly conserved across the domains of life, different salvage pathways are seen in mammals, plants, and fungi 5,6 Earlier work indicated that during the initial biotrophic phase of the foliar infection by the potato blight pathogen P. infestans, biosynthesis of pyrimidines was strongly upregulated, indicating that invasive growth was not dependent on plant nucleotides, while the salvage pathway appeared to become more important during the necrotrophic stage of infection 7. The nucleotide salvage pathway has also b...

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Section: Resultsmentioning

confidence: 99%

Structural and catalytic analysis of two diverse uridine phosphorylases in Phytophthora capsici

Yang

Huang

et al. 2020

Sci Rep

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“…For example, endogenous production of crucial, central metabolites should be enhanced during the biotrophic phase of hemibiotropic plant pathogens (Fernandez et al, 2013 ), while metabolities involved in nutrient scavenging should be enhanced during the necrotrophic phase. A good example of a metabolite that is changing during infection and could be used as an objective function is pyrimidine (García-Bayona et al, 2014 ). Given this context, metabolites that are used as precursors for the biosynthesis of amino acids and nucleotides, among others, should be the objective functions of the CSMs related to biotrophic phases, and metabolites corresponding to salvage pathways should be included in necrotrophic phases.…”

Section: Discussionmentioning

confidence: 99%

A Genome-Scale Metabolic Reconstruction of Phytophthora infestans With the Integration of Transcriptional Data Reveals the Key Metabolic Patterns Involved in the Interaction of Its Host

et al. 2018

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Phytophthora infestans, the causal agent of late blight disease, affects potatoes and tomatoes worldwide. This plant pathogen has a hemibiotrophic lifestyle, having an initial biotrophic infection phase during which the pathogen spreads within the host tissue, followed by a necrotrophic phase in which host cell death is induced. Although increasing information is available on the molecular mechanisms, underlying the distinct phases of the hemibiotrophic lifestyle, studies that consider the entire metabolic processes in the pathogen while undergoing the biotrophic, transition to necrotrophic, and necrotrophic phases have not been conducted. In this study, the genome-scale metabolic reconstruction of P. infestans was achieved. Subsequently, transcriptional data (microarrays, RNA-seq) was integrated into the metabolic reconstruction to obtain context-specific (metabolic) models (CSMs) of the infection process, using constraint-based reconstruction and analysis. The goal was to identify specific metabolic markers for distinct stages of the pathogen's life cycle. Results indicate that the overall metabolism show significant changes during infection. The most significant changes in metabolism were observed at the latest time points of infection. Metabolic activity associated with purine, pyrimidine, fatty acid, fructose and mannose, arginine, glycine, serine, and threonine amino acids appeared to be the most important metabolisms of the pathogen during the course of the infection, showing high number of reactions associated with them and expression switches at important stages of the life cycle. This study provides a framework for future throughput studies of the metabolic changes during the hemibiotrophic life cycle of this important plant pathogen.

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“…Solanum tuberosum cDNA was prepared from commercially available potato plants (Sabanera variety). Total RNA was extracted from frozen mycelia or from 300 mg of plant leaves and cDNA was synthesized (Garcia-Bayona et al, 2014). Subsequently, a fraction of the cDNA reactions (2 μL) was used for PCR amplification.…”

Section: Methodsmentioning

confidence: 99%

Phytophthora infestans Dihydroorotate Dehydrogenase Is a Potential Target for Chemical Control – A Comparison With the Enzyme From Solanum tuberosum

et al. 2019

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The oomycete Phytophthora infestans is the causal agent of tomato and potato late blight, a disease that causes tremendous economic losses in the production of solanaceous crops. The similarities between oomycetes and the apicomplexa led us to hypothesize that dihydroorotate dehydrogenase (DHODH), the enzyme catalyzing the fourth step in pyrimidine biosynthetic pathway, and a validated drug target in treatment of malaria, could be a potential target for controlling P. infestans growth. In eukaryotes, class 2 DHODHs are mitochondrially associated ubiquinone-linked enzymes that catalyze the fourth, and only redox step of de novo pyrimidine biosynthesis. We characterized the enzymes from both the pathogen and a host, Solanum tuberosum . Plant DHODHs are known to be class 2 enzymes. Sequence analysis suggested that the pathogen enzyme (PiDHODHs) also belongs to this class. We confirmed the mitochondrial localization of GFP-PiDHODH showing colocalization with mCherry-labeled ATPase in a transgenic pathogen. N-terminally truncated versions of the two DHODHs were overproduced in E. coli , purified, and kinetically characterized. StDHODH exhibited a apparent specific activity of 41 ± 1 μmol min -1 mg -1 , a k cat app of 30 ± 1 s -1 , and a K m app of 20 ± 1 μM for L -dihydroorotate, and a K m app = 30 ± 3 μM for decylubiquinone (Qd). PiDHODH exhibited an apparent specific activity of 104 ± 1 μmol min -1 mg -1 , a k cat app of 75 ± 1 s -1 , and a K m app of 57 ± 3 μM for L -dihydroorotate, and a K m app of 15 ± 1 μM for Qd. The two enzymes exhibited different activities with different quinones and napthoquinone derivatives, and different sensitivities to compounds known to cause inhibition of DHODHs from other organisms. The IC 50 for A77 1726, a nanomolar inhibitor of human DHODH, was 2.9 ± 0.6 mM for StDHODH, and 79 ± 1 μM for PiDHODH. In vivo , 0.5 mM A77 1726 decreased mycelial growth by approximately 50%, after 92 h. Collectively, our findings suggest that the Pi DHODH could be a target for selective inhibitors and we provide a biochemical background for the development of compounds that could be helpful for the control of the pathogen, opening the way to protein crystallization.

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De novo pyrimidine biosynthesis in the oomycete plant pathogen Phytophthora infestans

Cited by 21 publications

References 75 publications

Structural and catalytic analysis of two diverse uridine phosphorylases in Phytophthora capsici

Structural and catalytic analysis of two diverse uridine phosphorylases in Phytophthora capsici

A Genome-Scale Metabolic Reconstruction of Phytophthora infestans With the Integration of Transcriptional Data Reveals the Key Metabolic Patterns Involved in the Interaction of Its Host

Phytophthora infestans Dihydroorotate Dehydrogenase Is a Potential Target for Chemical Control – A Comparison With the Enzyme From Solanum tuberosum

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