The Naturally Occurring Amino Acid Substitution in the VPg α1-α2 Loop Breaks eIF4E-Mediated Resistance to PRSV by Enabling VPg to Re-Hijack Another eIF4E Isoform eIF(iso)4E in Watermelon

Abstract

Plant resistance, which acts as a selective pressure that affects viral population fitness, leads to the emergence of resistance-breaking virus strains. Most recessive resistance to potyviruses is related to the mutation of eukaryotic translation initiation factor 4E (eIF4E) or its isoforms that break their interactions with the viral genome-linked protein (VPg). In this study, we found that the VPg α1-α2 loop, which is essential for binding eIF4E, is the most variable domain of papaya ringspot virus (PRSV) VPg. PRSV VPg with the naturally occurring amino acid substitution of K105Q or E108G in the α1-α2 loop fails to interact with watermelon (Citrullus lanatus) eIF4E but interacts with watermelon eIF(iso)4E instead. Moreover, PRSV carrying these mutations can break the eIF4E-mediated resistance to PRSV in watermelon accession PI 244019. We further revealed that watermelon eIF(iso)4E with the amino acid substitutions of DNQS to GAAA in the cap-binding pocket could not interact with PRSV VPg with natural amino acid substitution of K105Q or E108G. Therefore, our finding provides a precise target for engineering watermelon germplasm resistant to resistance-breaking PRSV isolates.

Keywords: PRSV‐W; VPg; eIF(iso)4E; eIF4E; resistance breaking.

FIGURE 1

The interaction between papaya ringspot virus (PRSV) viral genome‐associated protein (VPg) and eukaryotic translation initiation factor 4E (eIF4E) family proteins. (a) Phylogenetic relationships of eIF4E family genes from watermelon, Arabidopsis thaliana , tobacco, maize and wheat. Phylogenetic analysis of eIF4E family genes from watermelon, A. thaliana , tobacco, maize and wheat was conducted using MEGA7 using the neighbour‐joining (NJ) method. A total of 1000 bootstrap replicates were used. Percentages of replicate trees (when ≥ 50%) in which the associated taxa clustered together are shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Genes marked with red triangles indicate eIF4E family genes in watermelon. (b) Descriptions of watermelon eIF4E family genes based upon phylogenetic relationships. Cla019623, Cla007614 and Cla017165 were, respectively, named CleIF4E, CleIF(iso)4E and ClnCBP. (c) The amino acid identity of watermelon eIF4E family proteins. The amino acid identities between CleIF4E and CleIF(iso)4E, CleIF4E and ClnCBP and CleIF(iso)4E and ClnCBP are highlighted in orange, blue and green, respectively. (d) Yeast two‐hybrid (Y2H) analysis of the interaction between PRSV VPg and eIF4E family proteins. The yeast cells co‐transformed with BD‐PRSV VPg or BD and AD‐CleIF4E, AD‐CleIF(iso)4E or AD‐ClnCBP were subjected to 10‐fold serial dilutions and plated on the SD/−Trp/−Leu/−His selection medium for 4 days. (e) Bimolecular fluorescence complementation (BiFC) analysis of the interactions between PRSV VPg and eIF4E family proteins in Nicotiana benthamiana leaves. PRSV VPg‐CE or β‐glucuronidase (GUS)‐CE was individually co‐expressed with CleIF4E‐NE, CleIF(iso)4E‐NE or ClnCBP‐NE in N. benthamiana leaves. Confocal imaging was performed at 48 h post‐inoculation. Scale bars = 20 μm.

FIGURE 2

The amino acid identity analysis of VPg amino acid sequences of multiple papaya ringspot virus watermelon strain (PRSV‐W) isolates. (a) Schematic representation of the eIF4E secondary structure. The β1–β5 sheets, the α1 helix and the α2 helix are highlighted in grey, yellow and blue, respectively. (b) Amino acid substitutions mapped onto the 3D structure of the PRSV VPg protein predicted using the Potato virus Y (PVY) VPg protein structure as the template. The β1–β5 sheets are highlighted in grey, and the α1 and α2 helices are highlighted in yellow and blue, respectively. (c) Multiple amino acid sequence alignment and WebLogo analysis of VPg amino acid sequences of various PRSV‐W isolates. The height of the letter shows the conservation of the amino acid. The regions highlighted in yellow and blue boxes indicate the α1 and α2 helices, respectively. (d) Multiple amino acid sequence identity analysis of VPg from multiple PRSV‐W isolates using a heatmap.

FIGURE 3

The interaction between papaya ringspot virus (PRSV) VPg with the naturally occurring mutation in the α1–α2 loop and watermelon eIF4E family proteins. (a) Single amino acid substitutions in the VPg α1–α2 loop of different PRSV‐W isolates. The regions highlighted in yellow and blue boxes indicate the α1 and α2 helices, respectively. All of the amino acid substitution sites are highlighted in red boxes. The mutated amino acid substitution sites K105Q and E108G are highlighted in yellow, and the mutated amino acid substitution sites M94I, V102I, N104S, E108D, S111N, R114K, S116T and S116A are highlighted in white. (b) Yeast two‐hybrid analysis of the interaction between the naturally occurring mutant VPg^K105Q or VPg^E108G and watermelon eIF4E family proteins in vitro. The yeast cells co‐transformed with BD‐VPg^K105Q, BD‐VPg^E108G or BD‐VPg^WT and AD‐CleIF4E, AD‐CleIF(iso)4E, AD‐ClnCBP or AD were subjected to 10‐fold serial dilutions and plated on the SD/−Trp/−Leu/−His selection medium for 4 days. (c) Bimolecular fluorescence complementation analysis of the interaction between the naturally occurring mutants VPg^K105Q or VPg^E108G and watermelon eIF4E family proteins in Nicotiana benthamiana leaves. VPg^K105Q‐CE, VPg^E108G‐CE or VPg^WT‐CE were co‐expressed with CleIF4E‐NE, CleIF(iso)4E‐NE, ClnCBP‐NE or GUS‐NE in N. benthamiana. Confocal imaging was performed at 48 h post‐inoculation. Scale bars = 20 μm.

FIGURE 4

The infection of papaya ringspot virus (PRSV) with naturally occurring mutation in the VPg α1–α2 loop in PI 244019 and Fufeng plants. (a) Phenotypes of PI 244019 and Fufeng plants individually inoculated with PRSV^K105Q, PRSV^E108G and PRSV^WT under daylight and UV illumination. (b) Western blotting analysis of the PRSV coat protein (CP) accumulation level in the upper leaves of PI 244019 and Fufeng plants separately inoculated with PRSV^K105Q, PRSV^E108G and PRSV^WT. The Ponceau S staining of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) shows sample loadings.

FIGURE 5

Analysis of the interaction between the eIF(iso)4E cap‐binding pocket mutant and VPg^K105Q or VPg^E108G. (a) Multiple amino acid sequence alignment and WebLogo analysis of eIF(iso)4E of multiple cucurbit 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 eIF(iso)4E‐mediated resistance against potyviruses in the cap‐binding pocket. The DNQS motif is highlighted in the red box. (b) DNQS motif mapped onto the 3D structure of the watermelon eIF(iso)4E protein predicted using the structure of cucumber eIF(iso)4E (PDB ID: B0F832.1.A) as the template. The black arrow points to the cap‐binding pocket. The DNQS motif is highlighted in red. (c) Yeast two‐hybrid analysis of the interaction between VPg^K105Q or VPg^E108G and CleIF(iso)4E^GAAA in vitro. The yeast cells co‐transformed with BD‐VPg^K105Q, BD‐VPg^E108G or BD and AD‐CleIF(iso)4E^GAAA or AD‐CleIF(iso)4E^WT were subjected to 10‐fold serial dilutions and plated on the selection medium SD/−Trp/−Leu/−His for 4 days. (d) Bimolecular fluorescence complementation analysis of the interaction between VPg^K105Q or VPg^E108G and CleIF(iso)4E^GAAA in Nicotiana benthamiana leaves. VPg^K105Q‐CE, VPg^E108G‐CE or GUS‐CE were co‐expressed with CleIF(iso)4E^GAAA‐NE or CleIF(iso)4E^WT‐NE in N. benthamiana. Confocal imaging was performed at 48 h post‐inoculation. Scale bars = 20 μm.

FIGURE 6

A working model for engineering watermelon plants resistant to papaya ringspot virus (PRSV). PRSV can infect the watermelon plant by interacting with and using eIF4E in the susceptible watermelon plant (CleIF4E). Natural amino acid substitution D71G in the eIF4E of some watermelon accessions such as PI 244019 confer resistance to PRSV by disrupting the interaction between VPg and CleIF4E. PRSV isolates with natural amino acid substitution of K105Q or E108G in VPg break the resistance by interacting with CleIF(iso)4E. The CleIF(iso)4E mutant with the mutation of the DNQS motif to GAAA cannot interact with VPg mutants with amino acid substitution of K105Q or E108G and therefore mediate resistance to the resistance‐breaking PRSV isolates.