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Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire.

Lévesque CA, Brouwer H, Cano L, Hamilton JP, Holt C, Huitema E, Raffaele S, Robideau GP, Thines M, Win J, Zerillo MM, Beakes GW, Boore JL, Busam D, Dumas B, Ferriera S, Fuerstenberg SI, Gachon CM, Gaulin E, Govers F, Grenville-Briggs L, Horner N, Hostetler J, Jiang RH, Johnson J, Krajaejun T, Lin H, Meijer HJ, Moore B, Morris P, Phuntmart V, Puiu D, Shetty J, Stajich JE, Tripathy S, Wawra S, van West P, Whitty BR, Coutinho PM, Henrissat B, Martin F, Thomas PD, Tyler BM, De Vries RP, Kamoun S, Yandell M, Tisserat N, Buell CR - Genome Biol. (2010)

Bottom Line: The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes.A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species.Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agriculture and Agri-Food Canada, 960 Carling Ave, Ottawa, ON, K1A 0C6, Canada.

ABSTRACT

Background: Pythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species.

Results: The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome, including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report of these in a genome outside the metazoans.

Conclusions: Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae.

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Related in: MedlinePlus

An original repertoire of candidate effector proteins in P. ultimum. (a) The number of candidate RXLR effectors estimated by Hidden Markov Model (HMM) searches of predicted proteins with amino-terminal signal peptides. The numbers of false positives were derived from HMM searches of the permutated protein sequences. (b) The number of candidate RXLR effectors discovered by motif searching. The search was performed on the total set of six-frame translated ORFs from the genome sequences that encode proteins with an amino-terminal signal peptide. The motif RXLR and two more degenerate motifs, RXLX or RX[LMIFY][HKR], were required to occur within 100 amino acids of the amino termini. (c) The typical architecture of a YxSL[RK] effector candidate inferred from 91 sequences retrieved from P. ultimum, three Phytophthora genomes and A. euteiches. (d) The YxSL[RK] motif is enriched and positionally constrained in secreted proteins in P. ultimum and Phytophthora spp. The top graph compares the abundance of YxSL[RK]-containing proteins among secreted and non-secreted proteins from four oomycete genomes. The middle and bottom graphs show the frequency of the YxSL[RK] motif among non-secreted and secreted proteins, respectively, according to its position in the protein sequence. (e) Cladogram based on the conserved motifs region of the 91YxSL[KR] proteins, showing boostrap support for the main branches.
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Figure 1: An original repertoire of candidate effector proteins in P. ultimum. (a) The number of candidate RXLR effectors estimated by Hidden Markov Model (HMM) searches of predicted proteins with amino-terminal signal peptides. The numbers of false positives were derived from HMM searches of the permutated protein sequences. (b) The number of candidate RXLR effectors discovered by motif searching. The search was performed on the total set of six-frame translated ORFs from the genome sequences that encode proteins with an amino-terminal signal peptide. The motif RXLR and two more degenerate motifs, RXLX or RX[LMIFY][HKR], were required to occur within 100 amino acids of the amino termini. (c) The typical architecture of a YxSL[RK] effector candidate inferred from 91 sequences retrieved from P. ultimum, three Phytophthora genomes and A. euteiches. (d) The YxSL[RK] motif is enriched and positionally constrained in secreted proteins in P. ultimum and Phytophthora spp. The top graph compares the abundance of YxSL[RK]-containing proteins among secreted and non-secreted proteins from four oomycete genomes. The middle and bottom graphs show the frequency of the YxSL[RK] motif among non-secreted and secreted proteins, respectively, according to its position in the protein sequence. (e) Cladogram based on the conserved motifs region of the 91YxSL[KR] proteins, showing boostrap support for the main branches.

Mentions: Many plant pathogens, especially biotrophic and hemi-biotrophic ones, produce effector proteins that either enter into host cells or are predicted to do so [27,58,59]. The genomes of Ph. sojae, Ph. ramorum and Ph. infestans encode large numbers (370 to 550) of potential effector proteins that contain an amino-terminal cell-entry domain with the motifs RXLR and dEER [28,29], which mediate entry of these proteins into host cells in the absence of pathogen-encoded machinery [60,61]. RXLR-dEER effectors are thought, and in a few cases shown, to suppress host defense responses, but a subset of these effectors can be recognized by plant immune receptors resulting in programmed cell death and disease resistance. To search for RXLR effectors in the genome of P. ultimum, we translated all six frames of the genome sequence to identify all possible small proteins, exclusive of splicing. Among these, a total of 7,128 translations were found to contain an amino-terminal signal peptide based on SignalP prediction. We then used the RXLR-dEER Hidden Markov Model (HMM) [29] to search the translations for candidate effectors and, as a control, the same set of translations following permutation of their sequences downstream of the signal peptide (Figure 1a). Only 35 sequences with significant scores were found in the non-permuted set while an average of 5 were found in 100 different permuted sets. In comparison to the Ph. ramorum secretome, 300 hits were found without permutation. Examination of the 35 significant sequences revealed that most were members of a secreted proteinase family [62] in which the RXLR motif was part of a conserved subtilisin-like serine protease domain of 300 amino acids in length, and thus unlikely to be acting as a cell entry motif. A string search was then performed for the RXLR motif within the amino terminus of each translation, 30 to 150 residues from the signal peptide. In this case, the number of hits was not significantly different between the real sequences and the permuted sequences. The same result was obtained with the strings RXLX and RX[LMFY][HKR] (Figure 1b). HMMs have been defined to identify carboxy-terminal motifs conserved in about 60% of RXLR-dEER effectors [29,63]. Searching the secretome and the permutated secretome with this HMM also identified no significant numbers of candidate effectors (data not shown). Blast searches with the most conserved Phytophthora effectors likewise produced no hits.


Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire.

Lévesque CA, Brouwer H, Cano L, Hamilton JP, Holt C, Huitema E, Raffaele S, Robideau GP, Thines M, Win J, Zerillo MM, Beakes GW, Boore JL, Busam D, Dumas B, Ferriera S, Fuerstenberg SI, Gachon CM, Gaulin E, Govers F, Grenville-Briggs L, Horner N, Hostetler J, Jiang RH, Johnson J, Krajaejun T, Lin H, Meijer HJ, Moore B, Morris P, Phuntmart V, Puiu D, Shetty J, Stajich JE, Tripathy S, Wawra S, van West P, Whitty BR, Coutinho PM, Henrissat B, Martin F, Thomas PD, Tyler BM, De Vries RP, Kamoun S, Yandell M, Tisserat N, Buell CR - Genome Biol. (2010)

An original repertoire of candidate effector proteins in P. ultimum. (a) The number of candidate RXLR effectors estimated by Hidden Markov Model (HMM) searches of predicted proteins with amino-terminal signal peptides. The numbers of false positives were derived from HMM searches of the permutated protein sequences. (b) The number of candidate RXLR effectors discovered by motif searching. The search was performed on the total set of six-frame translated ORFs from the genome sequences that encode proteins with an amino-terminal signal peptide. The motif RXLR and two more degenerate motifs, RXLX or RX[LMIFY][HKR], were required to occur within 100 amino acids of the amino termini. (c) The typical architecture of a YxSL[RK] effector candidate inferred from 91 sequences retrieved from P. ultimum, three Phytophthora genomes and A. euteiches. (d) The YxSL[RK] motif is enriched and positionally constrained in secreted proteins in P. ultimum and Phytophthora spp. The top graph compares the abundance of YxSL[RK]-containing proteins among secreted and non-secreted proteins from four oomycete genomes. The middle and bottom graphs show the frequency of the YxSL[RK] motif among non-secreted and secreted proteins, respectively, according to its position in the protein sequence. (e) Cladogram based on the conserved motifs region of the 91YxSL[KR] proteins, showing boostrap support for the main branches.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2926784&req=5

Figure 1: An original repertoire of candidate effector proteins in P. ultimum. (a) The number of candidate RXLR effectors estimated by Hidden Markov Model (HMM) searches of predicted proteins with amino-terminal signal peptides. The numbers of false positives were derived from HMM searches of the permutated protein sequences. (b) The number of candidate RXLR effectors discovered by motif searching. The search was performed on the total set of six-frame translated ORFs from the genome sequences that encode proteins with an amino-terminal signal peptide. The motif RXLR and two more degenerate motifs, RXLX or RX[LMIFY][HKR], were required to occur within 100 amino acids of the amino termini. (c) The typical architecture of a YxSL[RK] effector candidate inferred from 91 sequences retrieved from P. ultimum, three Phytophthora genomes and A. euteiches. (d) The YxSL[RK] motif is enriched and positionally constrained in secreted proteins in P. ultimum and Phytophthora spp. The top graph compares the abundance of YxSL[RK]-containing proteins among secreted and non-secreted proteins from four oomycete genomes. The middle and bottom graphs show the frequency of the YxSL[RK] motif among non-secreted and secreted proteins, respectively, according to its position in the protein sequence. (e) Cladogram based on the conserved motifs region of the 91YxSL[KR] proteins, showing boostrap support for the main branches.
Mentions: Many plant pathogens, especially biotrophic and hemi-biotrophic ones, produce effector proteins that either enter into host cells or are predicted to do so [27,58,59]. The genomes of Ph. sojae, Ph. ramorum and Ph. infestans encode large numbers (370 to 550) of potential effector proteins that contain an amino-terminal cell-entry domain with the motifs RXLR and dEER [28,29], which mediate entry of these proteins into host cells in the absence of pathogen-encoded machinery [60,61]. RXLR-dEER effectors are thought, and in a few cases shown, to suppress host defense responses, but a subset of these effectors can be recognized by plant immune receptors resulting in programmed cell death and disease resistance. To search for RXLR effectors in the genome of P. ultimum, we translated all six frames of the genome sequence to identify all possible small proteins, exclusive of splicing. Among these, a total of 7,128 translations were found to contain an amino-terminal signal peptide based on SignalP prediction. We then used the RXLR-dEER Hidden Markov Model (HMM) [29] to search the translations for candidate effectors and, as a control, the same set of translations following permutation of their sequences downstream of the signal peptide (Figure 1a). Only 35 sequences with significant scores were found in the non-permuted set while an average of 5 were found in 100 different permuted sets. In comparison to the Ph. ramorum secretome, 300 hits were found without permutation. Examination of the 35 significant sequences revealed that most were members of a secreted proteinase family [62] in which the RXLR motif was part of a conserved subtilisin-like serine protease domain of 300 amino acids in length, and thus unlikely to be acting as a cell entry motif. A string search was then performed for the RXLR motif within the amino terminus of each translation, 30 to 150 residues from the signal peptide. In this case, the number of hits was not significantly different between the real sequences and the permuted sequences. The same result was obtained with the strings RXLX and RX[LMFY][HKR] (Figure 1b). HMMs have been defined to identify carboxy-terminal motifs conserved in about 60% of RXLR-dEER effectors [29,63]. Searching the secretome and the permutated secretome with this HMM also identified no significant numbers of candidate effectors (data not shown). Blast searches with the most conserved Phytophthora effectors likewise produced no hits.

Bottom Line: The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes.A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species.Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agriculture and Agri-Food Canada, 960 Carling Ave, Ottawa, ON, K1A 0C6, Canada.

ABSTRACT

Background: Pythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species.

Results: The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome, including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report of these in a genome outside the metazoans.

Conclusions: Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae.

Show MeSH
Related in: MedlinePlus