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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

Mapping the biological properties of eIF4E mutants onto the pea crystal structure.(A and B) Results of the PSbMV infection complementation assay mapped onto chain H of the pea eIF4EΔN51 crystal structure. (A) cartoon representation and (B) surface representation of eIF4E colour coded to depict the three classifications of infection complementation: susceptible-like (S; green), partially-susceptible (S*; pink) and resistant-like (R; magenta). (C and D) Results of the yeast translation complementation assay mapped onto chain H of the pea eIF4EΔN51 crystal structure. (A) cartoon representation and (B) surface representation of eIF4E colour coded to depict the classifications of translation complementation: mutations resulting in full (++) and partial growth (+) are coloured green and those resulting in abolished growth (−) are coloured magenta. (E and F) Results of the evolutionary trace analysis mapped onto chain H of the pea eIF4EΔN51 crystal structure. A sequence alignment was generated for the 68 non-redundant plant eIF4E sequences most closely related to pea eIF4E and was used to calculate the relative degree of evolutionary conservation at each amino acid position through an implementation of the Maximum Likelihood method (see Materials and Methods). A colour-coded scale (G) varying from 1 (highly variable) to 9 (fully conserved) was subsequently mapped onto the cartoon (E) and surface (F) representation of pea eIF4E in the amino acid positions included in the mutagenesis screen.
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pone-0015873-g005: Mapping the biological properties of eIF4E mutants onto the pea crystal structure.(A and B) Results of the PSbMV infection complementation assay mapped onto chain H of the pea eIF4EΔN51 crystal structure. (A) cartoon representation and (B) surface representation of eIF4E colour coded to depict the three classifications of infection complementation: susceptible-like (S; green), partially-susceptible (S*; pink) and resistant-like (R; magenta). (C and D) Results of the yeast translation complementation assay mapped onto chain H of the pea eIF4EΔN51 crystal structure. (A) cartoon representation and (B) surface representation of eIF4E colour coded to depict the classifications of translation complementation: mutations resulting in full (++) and partial growth (+) are coloured green and those resulting in abolished growth (−) are coloured magenta. (E and F) Results of the evolutionary trace analysis mapped onto chain H of the pea eIF4EΔN51 crystal structure. A sequence alignment was generated for the 68 non-redundant plant eIF4E sequences most closely related to pea eIF4E and was used to calculate the relative degree of evolutionary conservation at each amino acid position through an implementation of the Maximum Likelihood method (see Materials and Methods). A colour-coded scale (G) varying from 1 (highly variable) to 9 (fully conserved) was subsequently mapped onto the cartoon (E) and surface (F) representation of pea eIF4E in the amino acid positions included in the mutagenesis screen.

Mentions: Five mutants (S70A, K71A, A73D;A74D, V167A, N169K) showed the R/++ phenotype, one mutant (W62L) the R/+ phenotype, and three mutants (A67E, A74D, D109A) the S*/++ phenotype. These mutations identify amino acid positions critical for PSbMV infection. The location of these residues are displayed on the eIF4E molecular model in Figure 5 (Panels A and B in magenta and pink, respectively). They are located in two general regions of eIF4E. In the first group, A67E, S70A and K71A lie on the α′ helix within the β1–β2 loop, and A74D is located proximal to the cap-binding residue W75 within the same loop. W62L lies at the end of β1 and is somewhat isolated from the other major resistance determinants; the closest being D109A (S*/++) whose Cα atom lies relatively distant to that of W62L at 8.3 Å, although both these residues have side chains facing into the cap-binding pocket. The last group of mutations are located close to the top (D109A, β3; N169K, β5) and central (V167A, β5) region of the cap-binding pocket (according to the orientation depicted in Figure 5). Broadly, these data confirm the distribution of determinants for natural and engineered eIF4E-based resistance to potyviruses and support the view that the physical location for binding of VPg overlaps with that for m7GTP. The β5 strand is, however, a novel location for determinants of any potyvirus resistance and may identify an important site for novel sources of resistance. Two alternative explanations are that it represents a host-specific adaptation not yet identified in pea germplasm or that its absence in the wider plant populations studied so far may also indicate that there are pleiotropic costs associated with such mutations.


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)

Mapping the biological properties of eIF4E mutants onto the pea crystal structure.(A and B) Results of the PSbMV infection complementation assay mapped onto chain H of the pea eIF4EΔN51 crystal structure. (A) cartoon representation and (B) surface representation of eIF4E colour coded to depict the three classifications of infection complementation: susceptible-like (S; green), partially-susceptible (S*; pink) and resistant-like (R; magenta). (C and D) Results of the yeast translation complementation assay mapped onto chain H of the pea eIF4EΔN51 crystal structure. (A) cartoon representation and (B) surface representation of eIF4E colour coded to depict the classifications of translation complementation: mutations resulting in full (++) and partial growth (+) are coloured green and those resulting in abolished growth (−) are coloured magenta. (E and F) Results of the evolutionary trace analysis mapped onto chain H of the pea eIF4EΔN51 crystal structure. A sequence alignment was generated for the 68 non-redundant plant eIF4E sequences most closely related to pea eIF4E and was used to calculate the relative degree of evolutionary conservation at each amino acid position through an implementation of the Maximum Likelihood method (see Materials and Methods). A colour-coded scale (G) varying from 1 (highly variable) to 9 (fully conserved) was subsequently mapped onto the cartoon (E) and surface (F) representation of pea eIF4E in the amino acid positions included in the mutagenesis screen.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0015873-g005: Mapping the biological properties of eIF4E mutants onto the pea crystal structure.(A and B) Results of the PSbMV infection complementation assay mapped onto chain H of the pea eIF4EΔN51 crystal structure. (A) cartoon representation and (B) surface representation of eIF4E colour coded to depict the three classifications of infection complementation: susceptible-like (S; green), partially-susceptible (S*; pink) and resistant-like (R; magenta). (C and D) Results of the yeast translation complementation assay mapped onto chain H of the pea eIF4EΔN51 crystal structure. (A) cartoon representation and (B) surface representation of eIF4E colour coded to depict the classifications of translation complementation: mutations resulting in full (++) and partial growth (+) are coloured green and those resulting in abolished growth (−) are coloured magenta. (E and F) Results of the evolutionary trace analysis mapped onto chain H of the pea eIF4EΔN51 crystal structure. A sequence alignment was generated for the 68 non-redundant plant eIF4E sequences most closely related to pea eIF4E and was used to calculate the relative degree of evolutionary conservation at each amino acid position through an implementation of the Maximum Likelihood method (see Materials and Methods). A colour-coded scale (G) varying from 1 (highly variable) to 9 (fully conserved) was subsequently mapped onto the cartoon (E) and surface (F) representation of pea eIF4E in the amino acid positions included in the mutagenesis screen.
Mentions: Five mutants (S70A, K71A, A73D;A74D, V167A, N169K) showed the R/++ phenotype, one mutant (W62L) the R/+ phenotype, and three mutants (A67E, A74D, D109A) the S*/++ phenotype. These mutations identify amino acid positions critical for PSbMV infection. The location of these residues are displayed on the eIF4E molecular model in Figure 5 (Panels A and B in magenta and pink, respectively). They are located in two general regions of eIF4E. In the first group, A67E, S70A and K71A lie on the α′ helix within the β1–β2 loop, and A74D is located proximal to the cap-binding residue W75 within the same loop. W62L lies at the end of β1 and is somewhat isolated from the other major resistance determinants; the closest being D109A (S*/++) whose Cα atom lies relatively distant to that of W62L at 8.3 Å, although both these residues have side chains facing into the cap-binding pocket. The last group of mutations are located close to the top (D109A, β3; N169K, β5) and central (V167A, β5) region of the cap-binding pocket (according to the orientation depicted in Figure 5). Broadly, these data confirm the distribution of determinants for natural and engineered eIF4E-based resistance to potyviruses and support the view that the physical location for binding of VPg overlaps with that for m7GTP. The β5 strand is, however, a novel location for determinants of any potyvirus resistance and may identify an important site for novel sources of resistance. Two alternative explanations are that it represents a host-specific adaptation not yet identified in pea germplasm or that its absence in the wider plant populations studied so far may also indicate that there are pleiotropic costs associated with such mutations.

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