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Structure-based mutational analysis of eIF4E in relation to sbm1 resistance to pea seed-borne mosaic virus in pea.

Ashby JA, Stevenson CE, Jarvis GE, Lawson DM, Maule AJ - PLoS ONE (2011)

Bottom Line: The crystallographic asymmetric unit contained eight independent copies of the protein, providing insights into the structurally conserved and flexible regions of eIF4E.The mutants also dissected individual contributions from polymorphisms present in eIF4E(R) and compared the impact of individual residues altered in orthologous resistance alleles from other crop species.The work describes the most extensive structural analysis of eIF4E in relation to potyvirus resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT

Background: Pea encodes eukaryotic translation initiation factor eIF4E (eIF4E(S)), which supports the multiplication of Pea seed-borne mosaic virus (PSbMV). In common with hosts for other potyviruses, some pea lines contain a recessive allele (sbm1) encoding a mutant eIF4E (eIF4E(R)) that fails to interact functionally with the PSbMV avirulence protein, VPg, giving genetic resistance to infection.

Methodology/principal findings: To study structure-function relationships between pea eIF4E and PSbMV VPg, we obtained an X-ray structure for eIF4E(S) bound to m(7)GTP. The crystallographic asymmetric unit contained eight independent copies of the protein, providing insights into the structurally conserved and flexible regions of eIF4E. To assess indirectly the importance of key residues in binding to VPg and/or m(7)GTP, an extensive range of point mutants in eIF4E was tested for their ability to complement PSbMV multiplication in resistant pea tissues and for complementation of protein translation, and hence growth, in an eIF4E-defective yeast strain conditionally dependent upon ectopic expression of eIF4E. The mutants also dissected individual contributions from polymorphisms present in eIF4E(R) and compared the impact of individual residues altered in orthologous resistance alleles from other crop species. The data showed that essential resistance determinants in eIF4E differed for different viruses although the critical region involved (possibly in VPg-binding) was conserved and partially overlapped with the m(7)GTP-binding region. This overlap resulted in coupled inhibition of virus multiplication and translation in the majority of cases, although the existence of a few mutants that uncoupled the two processes supported the view that the specific role of eIF4E in potyvirus infection may not be restricted to translation.

Conclusions/significance: The work describes the most extensive structural analysis of eIF4E in relation to potyvirus resistance. In addition to defining functional domains within the eIF4E structure, we identified eIF4E alleles with the potential to convey novel virus resistance phenotypes.

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Related in: MedlinePlus

The structural organisation of pea eIF4EΔN51.(A) Topology diagram depicting the secondary structure organisation of pea eIF4EΔN51 derived from the PSbMV susceptible pea line JI2009. In contrast to previously reported eIF4E crystal structures from mammals, Schistosoma and wheat, pea eIF4EΔN51 contains a short helical segment (α′) within the β1–β2 loop. (B) Cartoon representation of pea eIF4EΔN51 chain H. The strands comprising the core β-sheet of the cap-binding site are labelled from one to eight and the position of the polymorphisms conferring sbm1 resistance are coloured orange. (C) Surface representation of pea eIF4EΔN51 chain H in complex with m7GTP. Residues interacting with the m7GTP cap analogue are coloured magenta. (D) Residues in pea eIF4EΔN51 chain H making direct polar interactions with m7GTP. An additional van der Waals contact is made by the conserved W180 residue with the methyl group of m7GTP. The γ-phosphate of m7GTP was not visible in the electron density of any of the eight eIF4E molecules within the crystallographic asymmetric unit. (E) Superposition of the Cα backbones of pea eIF4EΔN51 structure (chain H; green) and the wheat eIF4EC113S mutant structure (PDB accession code 2IDV; magenta) giving a root mean square deviation of 0.708 Å over 171 structurally equivalent residues. Significant conformational differences between these orthologues can be observed in the β1–β2 loop and equivalent Cα atoms deviating by at least 5 Å between each structure are represented by dashed lines.
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pone-0015873-g001: The structural organisation of pea eIF4EΔN51.(A) Topology diagram depicting the secondary structure organisation of pea eIF4EΔN51 derived from the PSbMV susceptible pea line JI2009. In contrast to previously reported eIF4E crystal structures from mammals, Schistosoma and wheat, pea eIF4EΔN51 contains a short helical segment (α′) within the β1–β2 loop. (B) Cartoon representation of pea eIF4EΔN51 chain H. The strands comprising the core β-sheet of the cap-binding site are labelled from one to eight and the position of the polymorphisms conferring sbm1 resistance are coloured orange. (C) Surface representation of pea eIF4EΔN51 chain H in complex with m7GTP. Residues interacting with the m7GTP cap analogue are coloured magenta. (D) Residues in pea eIF4EΔN51 chain H making direct polar interactions with m7GTP. An additional van der Waals contact is made by the conserved W180 residue with the methyl group of m7GTP. The γ-phosphate of m7GTP was not visible in the electron density of any of the eight eIF4E molecules within the crystallographic asymmetric unit. (E) Superposition of the Cα backbones of pea eIF4EΔN51 structure (chain H; green) and the wheat eIF4EC113S mutant structure (PDB accession code 2IDV; magenta) giving a root mean square deviation of 0.708 Å over 171 structurally equivalent residues. Significant conformational differences between these orthologues can be observed in the β1–β2 loop and equivalent Cα atoms deviating by at least 5 Å between each structure are represented by dashed lines.

Mentions: In pea, the sbm1 resistance gene is effective against both BYMV [4] and a range of isolates of PSbMV [5] with lines carrying the dominant SBM1 allele being universally susceptible to PSbMV, unless a second unlinked recessive resistance (sbm2) was present [6]. The sbm1 gene was characterised as a mutant allele of pea eIF4E which differed from its wild type counterpart in five non-conservative amino acid substitutions [7] in the β1, β1–β2 loop, β3, and β5 regions, as defined for the crystal structure of pea eIF4E (Figure 1 A and B). Potyvirus resistance specificities from other plant species are similarly located proximal to these regions (reviewed in [2]). The extent to which these polymorphisms can confer resistance independently and the extent to which knowledge of individual mutations can be useful in selecting novel resistances across different plant species has not been comprehensively investigated. However, naturally occurring single polymorphisms in the pepper pvr24 (V67E; [8]), pepper pvr1 (G107R; [9]), and lettuce mo12 (A70P; [10]) genes, and engineered single amino acid substitutions in the lettuce mo10 gene (W64A, F65A, W77L, R173A, W182A; [11]) do confer resistance to PVY in pepper, TEV in pepper and LMV in lettuce, respectively. In total, previously identified mutations associated with potyvirus resistance, found either alone or in combination [7], [8], [10], [12], [13], [14], [15], [16], are located on the β1, β1–β2 loop, α′, β3, β3–β4 loop, β4, β5, β5–β6 loop and α3-β7 loop secondary structures.


Structure-based mutational analysis of eIF4E in relation to sbm1 resistance to pea seed-borne mosaic virus in pea.

Ashby JA, Stevenson CE, Jarvis GE, Lawson DM, Maule AJ - PLoS ONE (2011)

The structural organisation of pea eIF4EΔN51.(A) Topology diagram depicting the secondary structure organisation of pea eIF4EΔN51 derived from the PSbMV susceptible pea line JI2009. In contrast to previously reported eIF4E crystal structures from mammals, Schistosoma and wheat, pea eIF4EΔN51 contains a short helical segment (α′) within the β1–β2 loop. (B) Cartoon representation of pea eIF4EΔN51 chain H. The strands comprising the core β-sheet of the cap-binding site are labelled from one to eight and the position of the polymorphisms conferring sbm1 resistance are coloured orange. (C) Surface representation of pea eIF4EΔN51 chain H in complex with m7GTP. Residues interacting with the m7GTP cap analogue are coloured magenta. (D) Residues in pea eIF4EΔN51 chain H making direct polar interactions with m7GTP. An additional van der Waals contact is made by the conserved W180 residue with the methyl group of m7GTP. The γ-phosphate of m7GTP was not visible in the electron density of any of the eight eIF4E molecules within the crystallographic asymmetric unit. (E) Superposition of the Cα backbones of pea eIF4EΔN51 structure (chain H; green) and the wheat eIF4EC113S mutant structure (PDB accession code 2IDV; magenta) giving a root mean square deviation of 0.708 Å over 171 structurally equivalent residues. Significant conformational differences between these orthologues can be observed in the β1–β2 loop and equivalent Cα atoms deviating by at least 5 Å between each structure are represented by dashed lines.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3025909&req=5

pone-0015873-g001: The structural organisation of pea eIF4EΔN51.(A) Topology diagram depicting the secondary structure organisation of pea eIF4EΔN51 derived from the PSbMV susceptible pea line JI2009. In contrast to previously reported eIF4E crystal structures from mammals, Schistosoma and wheat, pea eIF4EΔN51 contains a short helical segment (α′) within the β1–β2 loop. (B) Cartoon representation of pea eIF4EΔN51 chain H. The strands comprising the core β-sheet of the cap-binding site are labelled from one to eight and the position of the polymorphisms conferring sbm1 resistance are coloured orange. (C) Surface representation of pea eIF4EΔN51 chain H in complex with m7GTP. Residues interacting with the m7GTP cap analogue are coloured magenta. (D) Residues in pea eIF4EΔN51 chain H making direct polar interactions with m7GTP. An additional van der Waals contact is made by the conserved W180 residue with the methyl group of m7GTP. The γ-phosphate of m7GTP was not visible in the electron density of any of the eight eIF4E molecules within the crystallographic asymmetric unit. (E) Superposition of the Cα backbones of pea eIF4EΔN51 structure (chain H; green) and the wheat eIF4EC113S mutant structure (PDB accession code 2IDV; magenta) giving a root mean square deviation of 0.708 Å over 171 structurally equivalent residues. Significant conformational differences between these orthologues can be observed in the β1–β2 loop and equivalent Cα atoms deviating by at least 5 Å between each structure are represented by dashed lines.
Mentions: In pea, the sbm1 resistance gene is effective against both BYMV [4] and a range of isolates of PSbMV [5] with lines carrying the dominant SBM1 allele being universally susceptible to PSbMV, unless a second unlinked recessive resistance (sbm2) was present [6]. The sbm1 gene was characterised as a mutant allele of pea eIF4E which differed from its wild type counterpart in five non-conservative amino acid substitutions [7] in the β1, β1–β2 loop, β3, and β5 regions, as defined for the crystal structure of pea eIF4E (Figure 1 A and B). Potyvirus resistance specificities from other plant species are similarly located proximal to these regions (reviewed in [2]). The extent to which these polymorphisms can confer resistance independently and the extent to which knowledge of individual mutations can be useful in selecting novel resistances across different plant species has not been comprehensively investigated. However, naturally occurring single polymorphisms in the pepper pvr24 (V67E; [8]), pepper pvr1 (G107R; [9]), and lettuce mo12 (A70P; [10]) genes, and engineered single amino acid substitutions in the lettuce mo10 gene (W64A, F65A, W77L, R173A, W182A; [11]) do confer resistance to PVY in pepper, TEV in pepper and LMV in lettuce, respectively. In total, previously identified mutations associated with potyvirus resistance, found either alone or in combination [7], [8], [10], [12], [13], [14], [15], [16], are located on the β1, β1–β2 loop, α′, β3, β3–β4 loop, β4, β5, β5–β6 loop and α3-β7 loop secondary structures.

Bottom Line: The crystallographic asymmetric unit contained eight independent copies of the protein, providing insights into the structurally conserved and flexible regions of eIF4E.The mutants also dissected individual contributions from polymorphisms present in eIF4E(R) and compared the impact of individual residues altered in orthologous resistance alleles from other crop species.The work describes the most extensive structural analysis of eIF4E in relation to potyvirus resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT

Background: Pea encodes eukaryotic translation initiation factor eIF4E (eIF4E(S)), which supports the multiplication of Pea seed-borne mosaic virus (PSbMV). In common with hosts for other potyviruses, some pea lines contain a recessive allele (sbm1) encoding a mutant eIF4E (eIF4E(R)) that fails to interact functionally with the PSbMV avirulence protein, VPg, giving genetic resistance to infection.

Methodology/principal findings: To study structure-function relationships between pea eIF4E and PSbMV VPg, we obtained an X-ray structure for eIF4E(S) bound to m(7)GTP. The crystallographic asymmetric unit contained eight independent copies of the protein, providing insights into the structurally conserved and flexible regions of eIF4E. To assess indirectly the importance of key residues in binding to VPg and/or m(7)GTP, an extensive range of point mutants in eIF4E was tested for their ability to complement PSbMV multiplication in resistant pea tissues and for complementation of protein translation, and hence growth, in an eIF4E-defective yeast strain conditionally dependent upon ectopic expression of eIF4E. The mutants also dissected individual contributions from polymorphisms present in eIF4E(R) and compared the impact of individual residues altered in orthologous resistance alleles from other crop species. The data showed that essential resistance determinants in eIF4E differed for different viruses although the critical region involved (possibly in VPg-binding) was conserved and partially overlapped with the m(7)GTP-binding region. This overlap resulted in coupled inhibition of virus multiplication and translation in the majority of cases, although the existence of a few mutants that uncoupled the two processes supported the view that the specific role of eIF4E in potyvirus infection may not be restricted to translation.

Conclusions/significance: The work describes the most extensive structural analysis of eIF4E in relation to potyvirus resistance. In addition to defining functional domains within the eIF4E structure, we identified eIF4E alleles with the potential to convey novel virus resistance phenotypes.

Show MeSH
Related in: MedlinePlus