A natural substitution of a conserved amino acid in eIF4E confers resistance against multiple potyviruses

Abstract

Eukaryotic translation initiation factor 4E (eIF4E), which plays a pivotal role in initiating translation in eukaryotic organisms, is often hijacked by the viral genome-linked protein to facilitate the infection of potyviruses. In this study, we found that the naturally occurring amino acid substitution D71G in eIF4E is widely present in potyvirus-resistant watermelon accessions and disrupts the interaction between watermelon eIF4E and viral genome-linked protein of papaya ringspot virus-watermelon strain, zucchini yellow mosaic virus or watermelon mosaic virus. Multiple sequence alignment and protein modelling showed that the amino acid residue D⁷¹ located in the cap-binding pocket of eIF4E is strictly conserved in many plant species. The mutation D71G in watermelon eIF4E conferred resistance against papaya ringspot virus-watermelon strain and zucchini yellow mosaic virus, and the equivalent mutation D55G in tobacco eIF4E conferred resistance to potato virus Y. Therefore, our finding provides a potential precise target for breeding plants resistant to multiple potyviruses.

Keywords: Potyvirus; PRSV-W; PVY; ZYMV; eIF4E; recessive resistance.

FIGURE 1

Geographic distribution of potyvirus‐resistant watermelon accessions and variation of eukaryotic translation initiation factor 4E (eIF4E). (a) The eIF4E amino acid substitutions and information of potyvirus‐resistant watermelon accessions. (b) Geographic distribution of watermelon accessions with amino acid substitutions D71G, R114K/D212V or T81P in eIF4E. The red colour indicates the accessions with D71G, the orange colour indicates the accessions with R114K/D212V, and the blue colour indicates the accessions with T81P.

FIGURE 2

The interactions between watermelon eIF4E (CleIF4E) or its mutants and viral genome‐associated protein (VPg) of papaya ringspot virus‐watermelon strain (PRSV‐W) or zucchini yellow mosaic virus (ZYMV) in vitro and in vivo. (a) Yeast two‐hybrid analysis of the interaction between CleIF4E or its mutants and VPg of PRSV or ZYMV. The yeast cells co‐transformed with AD‐CleIF4E or its mutants and BD‐PRSV VPg or BD‐ZYMV VPg were subjected to 10‐fold serial dilutions and plated on the SD/−Trp/−Leu/−His selection medium for 4 days. (b) Bimolecular fluorescence complementation analysis of the interactions between CleIF4E or its mutants and VPg of PRSV‐W or ZYMV in Nicotiana benthamiana leaves. PRSV VPg‐CE or ZYMV VPg‐CE were individually co‐expressed with CleIF4E‐NE or its mutants in N. benthamiana leaves. Confocal imaging was performed at 48 h post‐inoculation (hpi). Scale bars = 20 μm.

FIGURE 3

The location and conservation analysis of amino acid substitutions in eIF4E proteins of watermelon accessions. (a) Single amino acid substitutions in the eIF4E protein of different watermelon accessions. A schematic representation of the eIF4E secondary structure is placed above the alignment. (b) Amino acid substitutions mapped onto the 3D structure of the watermelon eIF4E protein predicted using the crystal structure of melon eIF4E (5ME7) as the template. The two black arrows point to the cap‐binding pocket and dorsal surface. (c) Multiple amino acid sequence alignment and WebLogo analysis of eIF4E of multiple plant species. The height of the letter shows the conservation of the amino acid. The regions highlighted in yellow and green boxes indicate the two regions involved in eIF4E‐mediated resistance against potyviruses in the cap‐binding pocket. The amino acid substitution site D71G is highlighted in red, the amino acid substitution sites R114K and D212V are highlighted in orange, and the amino acid substitution site T81P is highlighted in blue. Other amino acid substitution sites are highlighted in pink.

FIGURE 4

The infection of pCB301‐PRSV‐GFP, pCB301‐PRSV‐CleIF4E^WT and pCB301‐PRSV‐CleIF4E^D71G in PI 244019 plants. (a) Schematic representation of pCB301‐PRSV‐GFP, pCB301‐PRSV‐CleIF4E^WT and pCB301‐PRSV‐CleIF4E^D71G. GFP is marked in green, CleIF4E^WT and CleIF4E^D71G are marked in purple. (b) Phenotypes of PI 244019 plants individually inoculated with pCB301‐PRSV‐GFP, pCB301‐PRSV‐CleIF4E^WT and pCB301‐PRSV‐CleIF4E^D71G at 45 days post‐inoculation (dpi). Plants in the absence of virus infection (Mock) were used as the control. (c) Western blotting analysis of accumulation levels of PRSV coat protein (CP) in the upper leaves of PI 244019 plants individually inoculated with pCB301‐PRSV‐GFP, pCB301‐PRSV‐CleIF4E^WT and pCB301‐PRSV‐CleIF4E^D71G at 45 dpi. The Ponceau S staining of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) shows sample loadings. (d) Endogenous eIF4E expression levels of PI 244019 plants individually inoculated with pCB301‐PRSV‐GFP, pCB301‐PRSV‐CleIF4E^WT and pCB301‐PRSV‐CleIF4E^D71G. Error bars represented the standard deviations of the means from biological repeats. The t test was used to test the significance of the differences. ‘ns’ represents no significant difference (α = 0.05).

FIGURE 5

The infection of pCB301‐ZYMV‐GFP, pCB301‐ZYMV‐CleIF4E^WT and pCB301‐ZYMVV‐CleIF4E^D71G in PI 244019 plants. (a) Schematic representation of pCB301‐ZYMV‐GFP, pCB301‐ZYMV‐CleIF4E^WT and pCB301‐ZYMV‐CleIF4E^D71G. GFP is marked in green, CleIF4E^WT and CleIF4E^D71G is marked in purple. (b) Phenotype of PI 244019 plants individually inoculated with pCB301‐ZYMV‐GFP, pCB301‐ZYMV‐CleIF4E^WT and pCB301‐ZYMV‐CleIF4E^D71G at 35 days post‐inoculation (dpi). Plants in the absence of virus infection (Mock) were used as the control. (c) Western blotting analysis of accumulation levels of ZYMV coat protein (CP) in the upper leaves of PI 244019 plants individually inoculated with pCB301‐ZYMV‐GFP, pCB301‐ZYMV‐CleIF4E^WT and pCB301‐ZYMV‐CleIF4E^D71G at 35 dpi. The Ponceau S staining of RuBisCO shows sample loadings. (d) Endogenous eIF4E expression level in PI 244019 plants individually inoculated with pCB301‐ZYMV‐GFP, pCB301‐ZYMV‐CleIF4E^WT and pCB301‐ZYMV‐CleIF4E^D71G. Error bars represent the standard deviations of the means from three biological repeats. The t test was used to test the significance of the difference. ‘ns’ represented no significant difference (α = 0.05).

FIGURE 6

Tobacco eIF4E carrying D55G mutation in cap‐binding pocket conferred resistance to PVY. (a) The location of D⁵⁵ in the structure of tobacco eIF4E. Two regions involved in resistance against potyviruses in the cap‐binding pocket are individually indicated in yellow and green. The amino acid site D⁵⁵ is highlighted in red. The two black arrows point to the cap‐binding pocket and dorsal surface. (b) Determination of the interaction between NteIF4E^WT or NteIF4E^D55G and PVY VPg through the yeast two‐hybrid system. The yeast cells co‐transformed with AD‐NteIF4E^WT or AD‐NteIF4E^D55G and BD‐PVY VPg were subjected to 10‐fold serial dilutions and plated on the SD/−Trp/−Leu/−His selection medium for 4 days. Yeast cells co‐transformed with AD and BD‐PVY VPg, AD‐NteIF4E^WT or NteIF4E^D55G and BD were used as negative controls. (c) Bimolecular fluorescence complementation analysis of the interaction between NteIF4E^WT or NteIF4E^D55G and PVY VPg in Nicotiana benthamiana leaves. NteIF4E^WT‐NE or NteIF4E^D55G‐NE were co‐expressed with PVY VPg‐CE in N. benthamiana. Confocal imaging was performed at 48 h post‐inoculation (hpi). Scale bars = 20 μm. (d) Schematic representation of pCB301‐PVY‐NteIF4E^WT and pCB301‐PVY‐NteIF4E^D55G. NteIF4E ^WT and NteIF4E ^D55G are marked in blue. (e) Phenotypes of Nicotiana tabacum 'Virgin A Mutant' (VAM) plants individually inoculated with pCB301‐PVY‐NteIF4E^WT and pCB301‐PVY‐NteIF4E^D55G at 15 days post‐inoculation (dpi). Plants in the absence of virus infection (Mock) were used as the control. (f) Western blot analysis of accumulation levels of coat protein (CP) in the upper leaves of VAM individually inoculated with PVY‐NteIF4E^WT and PVY‐NteIF4E^D55G at 15 dpi. The Ponceau S staining of RuBisCO shows sample loadings. (g) Endogenous eIF4E expression level of PI 244019 individually inoculated with pCB301‐PVY‐GFP, pCB301‐PVY‐NteIF4E^WT and pCB301‐PVY‐NteIF4E^D55G. Error bars represent the standard deviation of the means from biological repeats. The t test was used to test the significance of the differences. ‘ns’ represented no significant difference (α = 0.05).