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. 2024 Nov 25;25(11):e70033. doi: 10.1111/mpp.70033

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

Ling‐Xi Zhou 1, Xiao Yin 2, Zhi‐Yong Yan 1, Jun Jiang 1, Yan‐Ping Tian 1, Rui Gao 3, Chao Geng 1,✉, Xiang‐Dong Li 1,2,✉
PMCID: PMC11588673  PMID: 39587435

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: eIF(iso)4E, eIF4E, PRSV‐W, resistance breaking, VPg

Papaya ringspot virus isolates with amino acid substitution of K105Q or E108G in VPg can break the eIF4ED71G‐mediated resistance by re‐hijacking another susceptibility factor eIF(iso)4E in watermelon.

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1. Introduction

Plant viruses threaten crop health and cause huge economic losses worldwide (Varanda et al. 2021). Planting resistant cultivars is the most cost‐effective way to control plant viral diseases. In the past few years, researchers have revealed many defence mechanisms of plants against virus infection and have made many achievements in identifying virus‐resistant genes in plants (Galvez et al. 2014; Kang, Yeam, and Jahn 2005). Some plant species have specific resistance genes against a particular virus to trigger an immune response. Immunity due to resistance genes in these plants is known as dominant resistance (de Ronde, Butterbach, and Kormelink 2014). In contrast, immunity led by the lack of host factors required for virus infection is known as recessive resistance (Palukaitis and Yoon 2020).

Mutation frequently occurs during viral replication and infection (Domingo 1997). RNA viruses are more error‐prone than DNA viruses, due to their short replication cycle and lack of error correction mechanisms of their replicases (Duffy, Shackelton, and Holmes 2008; Domingo 2016). As a result, RNA viruses usually exist in quasispecies and evolve rapidly so that they can expand their host range, escape plant resistance and adapt to the ever‐changing environment (Elena et al. 2011; Mattenberger, Vila‐Nistal, and Geller 2021; Thresh 2006; Kim, Kwon, and Seo 2021).

Eukaryotic translation initiation factor 4E (eIF4E) family proteins play essential roles in mRNA translation and transportation. The eIF4E family members have similar structures and functions (Estevan et al. 2014). However, multiple members of the eIF4E family proteins may play different roles in different plants (Zlobin and Taranov 2023). eIF4E and its isoform eIF(iso)4E are the most widely exploited recessive resistance genes in plants (Liu, Li, and Liu 2017; Kang, Yeam, and Jahn 2005). The interaction between the viral genome‐linked protein (VPg) of potyviruses and the eIF4E family proteins of their host plants is important for virus infection (Coutinho de Oliveira et al. 2019; Miras et al. 2017; Saha and Mäkinen 2020; Tavert‐Roudet et al. 2017). eIF4E family protein variations disrupting the interaction between eIF4E family proteins and VPg confer plant resistance against potyviruses (Ruffel et al. 2002; Grzela et al. 2006; Yeam et al. 2007; Charron et al. 2008).

Some potyviruses accumulate at a low level in eIF4E family gene‐mediated resistant plants (Acosta‐Leal and Xiong 2008; Montarry et al. 2011). In this context, these viruses may use other eIF4E family genes for their infection. Because virus accumulation is very low in resistant plants, this kind of infection does not cause severe damage to the host plants. During the infection in resistant hosts or adaptation to these hosts, virus populations will gradually change, and some mutations may result in the re‐interaction of VPg with the eIF4E family resistant protein or the interaction between VPg and other eIF4E family proteins (Gallois et al. 2010; Perez et al. 2012). The symptomless accumulation of potyviruses in the tissues of resistant plants allows them to evolve, constituting a major source of resistance‐breaking progeny viruses (Quenouille et al. 2013). Synergistic evolution between plants and viruses often leads to the variation of eIF4E family genes and VPg (Charron et al. 2008; Ibiza, Cañizares, and Nuez 2010; Jeong et al. 2012; Moury et al. 2004).

In this study, we found that the α1–α2 loop is the most variable domain of PRSV VPg; the naturally occurring PRSV VPg variants with mutation in the α1–α2 loop could interact with eIF(iso)4E to break the eIF4E‐mediated recessive resistance to PRSV in watermelon (Citrullus lanatus var. citroides) accession PI 244019. We further screened the watermelon eIF(iso)4E mutant that breaks the interaction with the naturally occurring PRSV VPg variants. Above all, our study provides a basis for developing new cultivars resistant to resistance‐breaking PRSV isolates.

2. Results

2.1. PRSV VPg Selectively Interacts With eIF4E in Watermelon

To identify the eIF4E family genes in watermelon, we conducted a BLAST search of the watermelon genome in the CuGenDBv2 database using Arabidopsis thaliana eIF4E family genes. Accordingly, three watermelon genes were found with the GeneIDs of Cla019623, Cla007614 and Cla017165 (Table S2). A phylogenetic tree was constructed using the amino acid sequences of the three watermelon eIF4E family genes and their corresponding homologues from A. thaliana , tobacco, maize and wheat. The phylogenetic analysis revealed that Cla019623, Cla007614 and Cla017165 were clustered with the eIF4E, eIF(iso)4E and nCBP subgroups (Figure 1a). Based upon this phylogenetic analysis, the three eIF4E family genes encoding Cla019623, Cla007614, and Cla017165 were respectively named CleIF4E, CleIF(iso)4E and ClnCBP according to their subgroups (Figure 1b). The amino acid identities between CleIF4E and CleIF(iso)4E, CleIF4E and ClnCBP and CleIF(iso)4E and ClnCBP were 46.61%, 28.63% and 26.21%, respectively (Figure 1c).

FIGURE 1.

FIGURE 1

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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.

To investigate the interaction between PRSV VPg and watermelon eIF4E family proteins, we performed yeast two‐hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Y2H results showed that PRSV VPg interacted with CleIF4E (Figure 1d). The fluorescence of the yellow fluorescence protein (YFP) was observed in Nicotiana benthamiana leaf cells co‐expressing with PRSV VPg‐CE and CleIF4E‐NE (Figure 1e). In contrast, the leaf cells co‐expressing with PRSV VPg‐CE and CleIF(iso)4E‐NE or ClnCBP‐NE showed no YFP fluorescence (Figure 1e). These results indicated that PRSV VPg interacted with CleIF4E but not with CleIF(iso)4E or ClnCBP.

2.2. The α1–α2 Loop is the Most Variable Domain of PRSV VPg

We predicted the structure of the PRSV VPg based on the structure of VPg from Potato virus Y (PVY) (PDB ID: 6nfw.1) (Coutinho de Oliveira et al. 2019). Results showed that PRSV VPg contained five β‐sheet domains and two consecutive α‐helix domains (Figure 2a,b). Among them, the α1–α2 loop domain (amino acid residues 92–119) may be essential for VPg binding eIF4E (Coutinho de Oliveira et al. 2019, Figure 2a,b). Next, we performed multiple sequence alignment and WebLogo analysis of VPg amino acid sequences of various PRSV watermelon strain (PRSV‐W) isolates (Figure 2c and Table S3). The results showed that the amino acid identities of the β1–β5 sheet domains of PRSV VPg were 100%, 100%, 97.5%, 100% and 95.7%, respectively, while the amino acid identities of the α1 and α2 helix domains were 95.3% and 93.0%, respectively (Figure 2d). These results indicated that the α1–α2 loop was variable in PRSV VPg.

FIGURE 2.

FIGURE 2

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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.

The eIF4E family resistance alleles confer resistance to potyviruses by breaking their interaction with VPg. Some isolates of many potyviruses, such as barley yellow mosaic virus (BaYMV), bean common mosaic virus (BCMV), bean yellow mosaic virus (BYMV), cucumber vein yellowing virus (CVYV), pea seed‐borne mosaic virus (PSbMV), PVY, tobacco etch virus (TEV), plum pox virus (PPV) and turnip mosaic virus (TuMV), can break the recessive resistance mediated by eIF4E family genes by accumulating amino acid substitutions in VPg (Ayme et al. 2006, 2007; Li et al. 2016; Desbiez et al. 2022; Perez et al. 2012; Moury et al. 2004; Janzac et al. 2014; Gallois et al. 2010; Borgstrøm and Johansen 2001; Bruun‐Rasmussen et al. 2007; Charron et al. 2008; Martinez‐Turino et al. 2021). Therefore, we conducted an amino acid sequence alignment analysis of VPg in different isolates of the potyviruses mentioned above (Figure S1a) and analysed the amino acid identities of the β1, β2, β3, β4, β5, α1 and α2 domains (Figure S1b). The results showed that the α1 and α2 helix domains of the central region were the most variable regions of VPg in most potyviruses (Figure S1a,b). The analysis of the altered amino acid sites in the VPg of potyvirus isolates that have been reported to overcome eIF4E family resistance allele‐mediated resistance showed that most of the altered amino acid sites were located in the α1–α2 loop of VPg, and a few of the altered amino acid sites were sporadically distributed around the α1–α2 loop (Figure S1c) (Ayme et al. 2006, 2007; Li et al. 2016; Desbiez et al. 2022; Perez et al. 2012; Moury et al. 2004; Janzac et al. 2014; Gallois et al. 2010; Borgstrøm and Johansen 2001; Bruun‐Rasmussen et al. 2007; Charron et al. 2008; Martinez‐Turino et al. 2021), suggesting that the α1–α2 loop domain of VPg is a critical region in recognition of eIF4E family proteins and is relatively easy to mutate under the selective pressure of recessive resistance mediated by the eIF4E family genes.

2.3. The Naturally Occurring Amino Substitution of K105Q or E108G in the α1–α2 Loop Enables PRSV VPg to Interact With eIF(iso)4E

We then analysed single‐nucleotide polymorphisms in the VPg of different PRSV‐W isolates. We identified 10 naturally occurring nonsynonymous nucleotide substitutions in the PRSV VPg α1–α2 loop that individually led to the amino acid changes of M94 to I, V102 to I, N104 to S, K105 to Q, E108 to D, E108 to G, S111 to N, R114 to K, S116 to T and S116 to A (Figure 3a).

FIGURE 3.

FIGURE 3

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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 VPgK105Q or VPgE108G and watermelon eIF4E family proteins in vitro. The yeast cells co‐transformed with BD‐VPgK105Q, BD‐VPgE108G or BD‐VPgWT 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 VPgK105Q or VPgE108G and watermelon eIF4E family proteins in Nicotiana benthamiana leaves. VPgK105Q‐CE, VPgE108G‐CE or VPgWT‐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.

Next, we explored the interactions between PRSV VPg with naturally occurring mutations in the α1–α2 loop and the eIF4E family proteins of watermelon. Because amino acid properties are essential for protein interactions, we analysed the properties of the amino acids that were altered. The results showed that the change from K105 to Q led to amino acid change from basic amino acid to neutral amino acid. The change from E108 to G led to amino acid change from acidic amino acid to neutral amino acid. We then performed Y2H and BiFC assays to test the interactions between eIF4E family proteins and VPgK105Q or VPgE108G. Y2H results showed that robust colony growth was observed in yeast transformed with BD‐VPgWT and AD‐CleIF4E compared with yeast transformed with BD‐VPgWT and AD‐CleIF(iso)4E or AD‐ClnCBP on the SD/−Trp/−Leu/−His selection medium (Figure 3b), while yeast transformed with BD‐VPgK105Q or BD‐VPgE108G and AD‐CleIF(iso)4E grew on the SD/−Trp/−Leu/−His selection medium (Figure 3b). The western blotting analysis showed that the expression levels of different PRSV VPg variants were similar (Figure S4b). To further verify the interaction between VPgK105Q or VPgE108G and eIF4E family proteins, we separately co‐expressed VPgK105Q‐CE, VPgE108G‐CE or VPgWT‐CE with CleIF4E‐NE, CleIF(iso)4E‐NE or ClnCBP‐NE in N. benthamiana leaves. Confocal microscopy results revealed YFP fluorescence in the leaf cells co‐expressing with VPgK105Q‐CE or VPgE108G‐CE and CleIF(iso)4E‐NE, or VPgK105Q‐CE or VPgWT‐CE and CleIF4E‐NE (Figure 3c). Above all, the substitutions of K105Q and E108G in the α1–α2 loop enable PRSV VPg to interact with watermelon eIF(iso)4E.

2.4. The Naturally Occurring Amino Substitution of K105Q or E108G in the VPg α1–α2 Loop Breaks the Resistance to PRSV in PI 244019 Plants

Our previous results have shown that the naturally occurring substitution of D71G in the eIF4E of watermelon accession PI 244019 is critical for the resistance to PRSV‐W (Zhou et al. 2024). To identify whether PRSV with VPgK105Q or VPgE108G use eIF(iso)4E for their infection in watermelon, we obtained mutants using the parental wild‐type PRSV infectious clone and separately infiltrated these mutants into the cotyledons of PI 244019 and Fufeng plants. At 30 days post‐inoculation (dpi), PRSVK105Q, PRSVE108G and PRSVWT all induced distortion symptoms in the upper leaves of Fufeng plants and induced strong green fluorescence under UV illumination (Figure 4a). Western blotting analysis showed that the coat protein (CP) accumulation level of PRSVK105Q, PRSVE108G and PRSVWT was similar in Fufeng plants (Figure 4b). We sequenced the inoculated viruses, and the results showed that no additional mutations were found beyond VPg (Figure S5). In contrast, PRSVK105Q and PRSVE108G induced stronger green fluorescence than PRSVWT in PI 244019 plants under UV illumination (Figure 4a). Western blotting analysis showed that the CP accumulation level of PRSVK105Q and PRSVE108G was higher than PRSVWT in watermelon accession PI 244019 plants (Figure 4b). Thus, the naturally occurring amino substitution of K105Q or E108G in the VPg α1–α2 loop could break the resistance to PRSV in watermelon accession PI 244019 carrying the eIF4E resistance allele.

FIGURE 4.

FIGURE 4

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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 PRSVK105Q, PRSVE108G and PRSVWT 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 PRSVK105Q, PRSVE108G and PRSVWT. The Ponceau S staining of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) shows sample loadings.

In addition, we verified the interaction between VPgK105Q or VPgE108G and CleIF4ED71G using Y2H and BiFC analysis. The results showed that neither VPgK105Q nor VPgE108G interacted with CleIF4ED71G (Figure S2a,b). The western blotting analysis showed that the expression levels of CleIF4EWT and CleIF4ED71G were similar (Figure S4a). In summary, the resistance breaking of watermelon accession PI 244019 induced by PRSVK105Q and PRSVE108G was due to the PRSV VPg interaction with eIF(iso)4E rather than the re‐interaction with the resistance allele CleIF4ED71G.

2.5. The Amino Acid Substitution of the DNQS Motif in the Watermelon eIF(iso)4E Cap‐Binding Pocket Breaks the Interaction Between eIF(iso)4E and VPgK105Q or VPgE108G

We then analysed the single‐nucleotide polymorphisms in the eIF(iso)4E coding sequences of multiple watermelon accessions in the watermelon genome database CuGenDBv2. The results showed that there were seven nonsynonymous nucleotide mutations in the eIF(iso)4E coding sequence: G46 to A, A52 to C, G64 to C, G142 to A, G162 to C, T253 to A and G525 to C, which respectively resulted in amino acid changes V16 to M, T18 to P, D22 to H, A48 to T, L54 to P, F85 to I and E175 to D (Figure S3a). Among them, amino acid substitutions of A48T and L54P were located in the cap‐binding pocket of eIF(iso)4E (Figure S3b), and A48T was present in the PRSV‐resistant watermelon accession PI 595203 (Ling et al. 2009).

Next, we investigated whether the amino acid mutations A48T and L54P that are naturally present in the cap‐binding pocket of watermelon eIF(iso)4E could break the interaction between eIF(iso)4E and naturally occurring mutants VPgK105Q or VPgE108G. We then verified the interactions of CleIF(iso)4EWT, CleIF(iso)4EA48T or CleIF(iso)4EL54P with VPgK105Q or VPgE108G using the Y2H assay. The Y2H result showed that yeast cells co‐transformed with CleIF(iso)4EWT, AD‐CleIF(iso)4EA48T or AD‐CleIF(iso)4EL54P and BD‐VPgK105Q or BD‐VPgE108G grew vigorously on the selective medium SD/−Trp/−Leu/−His (Figure S3c). The interactions between VPgK105Q or VPgE108G and CleIF(iso)4EA48T or CleIF(iso)4EL54P were further verified using the BiFC assay. The results showed that YFP fluorescence could be observed in N. benthamiana cells co‐expressing with VPgK105Q‐CE or VPgE108G‐CE and CleIF(iso)4EWT‐NE, CleIF(iso)4EA48T‐NE or CleIF(iso)4EL54P‐NE (Figure S3d). This result suggested that the naturally occurring amino acid mutations in the watermelon eIF(iso)4E cap‐binding pocket could not break the interaction with VPgK105Q or VPgE108G.

We then performed alanine scanning to screen the critical amino acids affecting the interactions between CleIF(iso)4E and VPgK105Q or VPgE108G, in which the mutation of the conserved DNQS motif in the CleIF(iso)4E cap‐binding pocket could break the interactions with VPgK105Q or VPgE108G (Figure 5a,b). Y2H was used to verify the interactions between CleIF(iso)4EGAAA and VPgK105Q or VPgE108G. The results showed that yeast cells co‐transformed with AD‐CleIF(iso)4EWT and BD‐VPgK105Q or BD‐VPgE108G could grow vigorously on the selective medium SD/−Trp/−Leu/−His, while yeast cells co‐transformed with AD‐CleIF(iso)4EGAAA and BD‐VPgK105Q or BD‐VPgE108G could not (Figure 5c). The western blotting analysis showed that the expression levels of different CleIF(iso)4E variants were similar (Figure S4a). The interaction between CleIF(iso)4EGAAA with VPgK105Q or VPgE108G was further tested using BiFC. The results of the BiFC assay showed that YFP fluorescence could be observed in N. benthamiana cells co‐expressing CleIF(iso)4EWT‐NE and VPgK105Q‐CE or VPgE108G‐CE. In contrast, YFP fluorescence was not observed in N. benthamiana cells co‐expressing CleIF(iso)4EGAAA‐NE and VPgK105Q‐CE or VPgE108G‐CE (Figure 5d). Multiple amino acid sequence alignment and WebLogo analysis indicated that the DNQS motif was conserved in cucurbit plants (Figure 5a). In summary, the DNQS motif of CleIF(iso)4E can be used as a target for breeding watermelon accessions resistant to the PRSV mutants that break the resistance mediated by eIF4E such as PRSVK105Q and PRSVE108G.

FIGURE 5.

FIGURE 5

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Analysis of the interaction between the eIF(iso)4E cap‐binding pocket mutant and VPgK105Q or VPgE108G. (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 VPgK105Q or VPgE108G and CleIF(iso)4EGAAA in vitro. The yeast cells co‐transformed with BD‐VPgK105Q, BD‐VPgE108G or BD and AD‐CleIF(iso)4EGAAA or AD‐CleIF(iso)4EWT 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 VPgK105Q or VPgE108G and CleIF(iso)4EGAAA in Nicotiana benthamiana leaves. VPgK105Q‐CE, VPgE108G‐CE or GUS‐CE were co‐expressed with CleIF(iso)4EGAAA‐NE or CleIF(iso)4EWT‐NE in N. benthamiana. Confocal imaging was performed at 48 h post‐inoculation. Scale bars = 20 μm.

3. Discussion

Plant viruses hijack host factors for their infection. Potyviruses use VPg to translate in a cap‐independent manner, in which VPg is covalently linked to the 5′ end of the viral RNA and interacts with eIF4E family proteins by serving as an m7G cap analogue of the mRNAs (Khan et al. 2008; Choudhary and Suresh 2020). VPg has long been believed to be a disordered protein. However, recent studies showed that the central region (α1–α2 loop) of VPg has a globular structure and is critical for its recognition with eIF4E family proteins (Walter et al. 2020; Coutinho de Oliveira et al. 2019; Lebedeva et al. 2021). The most common reason for potyvirus breaking resistance mediated by eIF4E family genes is the mutation in their VPg. In this study, we found that most of the variable amino acids in the VPg of potyvirus isolates overcoming eIF4E family resistance allele‐mediated resistance were clustered in the α1–α2 loop, forming the surface of VPg bound to eIF4E (Figures 2c,d and S1c) (Coutinho de Oliveira et al. 2019). However, the molecular mechanism for breaking resistance is unclear.

Plants can acquire resistance by accumulating mutations in host factors necessary for virus infection (Schmitt‐Keichinger 2019). Most recessive resistance to potyviruses has been related to eIF4E or its isoforms (Zlobin and Taranov 2023). Our previous study has shown that amino acid substitution of D71G in eIF4E conferred resistance to multiple potyviruses (Zhou et al. 2024). The mutation sites of eIF4E or its isoforms are highly clustered in or around the cap‐binding pocket (Charron et al. 2008; Robaglia and Caranta 2006), leading to changes in the charge or hydrophobicity of the corresponding protein regions (Nicaise et al. 2003; Gao et al. 2004; Charron et al. 2008; Poulicard et al. 2016). These mutations usually do not affect the original function of the eIF4E family proteins (Charron et al. 2008; Moury et al. 2014; Zhou et al. 2024). Potyviruses may break the eIF4E family protein‐mediated resistance by re‐interacting with the eIF4E family resistance allele or interacting with and using other eIF4E family proteins to complete its infection process. Mutants of TEV VPg break pvr1‐mediated resistance to TEV in peppers by re‐interacting with the eIF4E resistance allele pvr1 (Perez et al. 2012). Mutations within the VPg enable TuMV to recruit both eIF(iso)4E and eIF4E1 in A. thaliana plants (Gallois et al. 2010; Bastet et al. 2018). In this study, we found that VPg with the natural amino acid substitution of K105Q or E108G in the α1–α2 loop could interact with eIF(iso)4E, which enables PRSV to break the resistance of watermelon accession PI 244019 (Figures 3b,c and S2a,b). PVY isolates carrying equivalent amino acid mutations of K105E or V108I in VPg can re‐hijack eIF(iso)4E and infect eIF4E‐knockdown tobacco plants (Takakura et al. 2018). These results suggested that the amino acids at positions 105 and 108 of potyviral VPg may be critical for breaking eIF4E‐mediated resistance. Re‐hijacking versus target switch has been reported to be a pathway for potyviruses to break resistance (Gallois, Moury, and German‐Retana 2018). Our results in this study further showed that it may be a general process of resistance breaking of potyviruses.

CRSPR‐based gene editing technology is highly desirable for crop improvement. Base editing and prime editing are two newly developed precision gene editing systems, which have also been gradually used in cucurbit germplasm improvement in recent years (Tian et al. 2018; Wang et al. 2023; Li et al. 2023; Shirazi Parsa et al. 2023). In this study, we found that the DNQS motif in eIF(iso)4E was critical for the interaction with VPgK105Q or VPgE108G and the mutation of the DNQS motif to GAAA broke the interactions with VPgK105Q or VPgE108G (Figure 5), suggesting that the DNQS motif of eIF(iso)4E can be used as a potential antiviral target. It is worth checking whether the mutation of DNQS affects the function of eIF(iso)4E in translation initiation. However, the transformation efficiency of watermelon is very low; so far, we have not obtained the relevant results. Overexpression of eIF(iso)4E W95L and W95L/K150E confers resistance to multiple TuMV strains in susceptible Chinese cabbage cultivars (Kim et al. 2014). In the future, the DNQS motif of eIF(iso)4E can be edited to produce watermelon accessions resistant to the resistance‐breaking PRSV isolates.

Combining the results of this paper and our previous study, we come to the following conclusions (Figure 6). PRSV VPg hijacks eIF4E, which enables PRSV to infect most watermelon cultivars. In some watermelon accessions, the amino acid at position 71 is G rather than D in eIF4E (eIF4ED71G), which cannot interact with PRSV VPg. Watermelon accessions such as PI 244019, which has amino acid G at position 71 of eIF4E, are resistant to PRSV and zucchini yellow mosaic virus (ZYMV) (Zhou et al. 2024). Several natural PRSV isolates with Q105 or G108 in VPg (VPgK105Q or VPgE108G) can break the resistance mediated by eIF4EG71 by re‐hijacking susceptibility factor eIF(iso)4E. However, eIF(iso)4E with the mutation of the DNQS motif to GAAA cannot interact with VPg with VPgK105Q or VPgE108G. Motif GAAA could be used to produce watermelon cultivars resistant to the resistance‐breaking PRSV isolates via precise gene editing. Therefore, our results provide a precise target for breeding watermelon plants with broad and durable resistance to potyviruses.

FIGURE 6.

FIGURE 6

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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.

4. Experimental Procedures

4.1. Plant Growth

The watermelon and N. benthamiana plants used in this study were grown at 22°C ± 2°C, on a 16 h light/8 h dark cycle, with 60% relative humidity.

4.2. RNA Extraction, Reverse Transcription and Plasmid Construction

BLAST search of the Citrullus lanatus (97103) v1 genome was performed via the Cucurbit Genomics Database (http://www.cucurbitgenomics.org/) using eIF4E family genes of A. thaliana as the template. Total RNA was extracted from the leaves of Fufeng plants using TRIzol reagent (TransGen Biotech) following the manufacturer's instructions. After gDNA wiper (Vazyme) treatment to eliminate the genomic DNA, total RNA was reverse transcribed using 3′ end‐specific primers and SMART M‐MLV reverse transcriptase (Takara) (Table S1). The coding sequence (CDS) of the eIF4E family genes was PCR‐amplified using Phanta Max Super‐Fidelity DNA polymerase (Vazyme) from reverse transcription products from Fufeng plants. The amplification products were separately cloned into pGADT7 and pCam35S‐NE (a vector containing the coding sequence of the N terminal YFP). The VPg genes of PRSV were separately PCR‐amplified from the pCB301‐PRSV‐GFP plasmids and were separately cloned into pGBKT7 and pCam35S‐CE (a vector containing the coding sequence of the C terminal YFP). Site‐directed mutagenesis was performed through a PCR‐based method using specific primers (Liu and Naismith 2008, Table S1). The resultant product was sequenced by Sangon Biotech Company.

4.3. Agrobacterium‐Mediated Inoculation

Competent cells of Agrobacterium tumefaciens GV3101 transformed with PRSV infectious clones were diluted to OD600 = 0.5 and infiltrated individually into the cotyledon of watermelon leaves. Agrobacterium cultures were adjusted to OD600 = 1.0 and infiltrated individually into N. benthamiana leaves for BiFC.

4.4. Protein Extractions and Western Blotting

Total plant proteins were extracted and treated as described previously (Geng et al. 2015; Zhou et al. 2024). Protein samples were separated in 12% SDS‐polyacrylamide gels through electrophoresis and then blotted onto nitrocellulose membranes. The virus protein was detected by polyclonal antibodies specific to PRSV CP. A horseradish peroxidase‐conjugated goat anti‐rabbit IgG (Sigma‐Aldrich) was used as the secondary antibody. The detection signal was visualised using a chemiluminescent imaging SH‐Compact 523 (Shenhua). Quantification of PRSV CP accumulation levels was estimated using ImageJ software.

4.5. Y2H Analysis

The Y2H assay was performed according to the manufacturer's protocol (Clontech). The Y2H gold yeast cells transformed with constructs of interest grew on an SD selection medium lacking Leu and Trp for 3 days. Then, the Y2H gold yeast cells were transferred to the highly stringent selection medium lacking Leu, Trp and His for 4 days. Total yeast proteins were extracted using a yeast protein extraction kit (Solarbio). The expression levels of different variants were detected through the western blotting assay using the HA‐specific antibody (Abways Technology) or Myc‐specific antibody (abcam).

4.6. BiFC Analysis

The BiFC assay was performed as described previously (Geng et al. 2015). The YFP interaction signal was detected using a Zeiss LSM800 confocal microscope at 48 hpi. The excitation wavelength for YFP was set at 488 nm and the emission was captured at 560–585 nm. The captured images were further processed using ZEN blue v. 3.2 software.

4.7. Gene Data Analysis and Protein Modelling

The amino acid sequences of eIF(iso)4E from other plant species and VPg from multiple potyviruses were retrieved from the NCBI (https://www.ncbi.nlm.nih.gov/). The nucleotide polymorphisms of eIF(iso)4E in different watermelon accessions were analysed using the ‘Genotype’ module in CuGenDBv2 (http://cucurbitgenomics.org/v2). Phylogenetic analysis was performed using MEGA 7 software. Nucleotide and amino acid sequences were analysed using DNAMAN and Bioedit software. The conservation analysis of eIF(iso)4E and VPg was performed using the WebLogo website (https://weblogo.berkeley.edu/logo.cgi). The heatmap was performed using GraphPad software.

The 3D view of the PRSV VPg protein structure and CleIF(iso)4E protein structure was predicted using SWISS‐MODEL, as described previously (Zhou et al. 2024). The protein models were analysed using PyMOL software.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

FIGURE S1. Multiple amino acid sequence identity analysis of VPg from multiple potyviruses. (a) Multiple amino acid sequence identity of VPg from different isolates of multiple potyviruses. (b) Heatmaps of the multiple amino acid sequence identity of VPg from different isolates of multiple potyviruses. (c) Amino acid mutation sites of VPg in potyviruses that break the eIF4E family gene‐mediated recessive resistance. The regions highlighted in grey boxes individually indicated the β1, β2, β3, β4 and β5 sheets. The regions highlighted in yellow and blue boxes respectively indicated the α1 and α2 helices. All of the amino acid substitution sites were highlighted in red boxes.

MPP-25-e70033-s003.docx (335.7KB, docx)

FIGURE S2. Analysis of the interaction between the PRSV VPg α1–α2 loop naturally occurring mutants and CleIF4ED71G. (a) Yeast two‐hybrid analysis of the interaction between the PRSV VPg α1–α2 loop naturally occurring mutants and CleIF4ED71G in vitro. The yeast cells co‐transformed with BD‐VPgWT, BD‐VPgK105Q or BD‐VPgE108G and AD‐CleIF4ED71G or AD‐CleIF4EWT 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 interaction between the naturally occurring mutants VPgK105Q or VPgE108G and CleIF4ED71G in Nicotiana benthamiana leaves. VPgWT‐CE, VPgK105Q‐CE or VPgE108G‐CE was co‐expressed with CleIF4EWT‐NE or CleIF4EWT‐NE in N. benthamiana leaves. Confocal imaging was performed at 48 h post‐inoculation. Scale bars = 20 μm.

MPP-25-e70033-s007.docx (78.7KB, docx)

FIGURE S3. Analysis of the interaction between VPgK105Q or VPgE108G and CleIF(iso)4E with the naturally occurring mutants in the cap‐binding pocket. (a) Single amino acid substitutions in the CleIF(iso)4E protein of different watermelon accessions. The regions highlighted in yellow and green boxes indicated the two regions involved in eIF(iso)4E‐mediated resistance against potyviruses in the cap‐binding pocket. The naturally occurring mutant sites of the CleIF(iso)4E cap‐binding pocket were highlighted in blue boxes. (b) The naturally occurring mutant sites of the CleIF(iso)4E cap‐binding pocket mapped onto the 3D structure of the watermelon eIF(iso)4E protein predicted using the structure of cucumber eIF(iso)4E as the template. The black arrow pointed to the cap‐binding pocket. The naturally occurring mutant sites of the CleIF(iso)4E cap‐binding pocket were highlighted in blue. (c) Yeast two‐hybrid analysis of the interaction between VPgK105Q or VPgE108G and CleIF(iso)4E with the naturally occurring mutants in the cap‐binding pocket in vitro. The yeast cells co‐transformed with BD‐VPgK105Q or BD‐VPgE108G and AD‐CleIF(iso)4EA48T, AD‐CleIF(iso)4EL54P, AD‐CleIF(iso)4EWT or empty vector AD were subjected to 10‐fold serial dilutions and plated on the SD/−Trp/−Leu/−His selection medium for 4 days. (d) Bimolecular fluorescence complementation analysis of the interaction between VPgK105Q or VPgE108G and CleIF(iso)4E with the naturally occurring mutants in the cap‐binding pocket in Nicotiana benthamiana leaves. VPgK105Q‐CE or VPgE108G‐CE was co‐expressed with CleIF(iso)4EA48T‐NE, CleIF(iso)4EL54P‐NE, CleIF(iso)4EWT‐NE or β‐glucuronidase (GUS)‐NE in N. benthamiana leaves. Confocal imaging was performed at 48 h post‐inoculation. Scale bars = 20 μm.

MPP-25-e70033-s002.docx (153.9KB, docx)

FIGURE S4. Western blotting analysis of expression levels of different variants of PRSV VPg, CleIF4E or CleIF(iso)4E. (a) Western blotting analysis of the expression level of different variants of CleIF4E or CleIF(iso)4E in yeast two‐hybrid (Y2H) assays. (b) Western blotting analysis of the expression level of different variants of PRSV VPg in Y2H assays.

MPP-25-e70033-s008.docx (67.4KB, docx)

FIGURE S5. Full‐length sequencing results of inoculated viruses.

MPP-25-e70033-s005.pdf (24.4MB, pdf)

TABLE S1. Primers used in this work.

MPP-25-e70033-s004.docx (17.2KB, docx)

TABLE S2. Sequences of watermelon eIF4E family genes.

MPP-25-e70033-s006.docx (15.7KB, docx)

TABLE S3. List of GenBank numbers of multiple PRSV‐W isolates and amino acid sequences of eIF4E family proteins of Arabidopsis thaliana , tobacco, maize, sugarcane and watermelon.

MPP-25-e70033-s001.docx (20.4KB, docx)

Acknowledgements

This study was funded by the National Natural Science Foundation of China (NSFC; 31801704 and 32472523), Innovation Project of Shandong Academy of Agricultural Sciences (CXGC2024B11 and CXGC2024A05), China Postdoctoral Science Foundation (2021M691973 and 2022T150389), the Youth Innovation Team Plan for Shandong High Education Institution (2022KJ241) and the Taishan Scholar Project (TS2022‐028). The authors appreciate the U.S. National Plant Germplasm System for providing the seeds of PI 244019.

Funding: This study was funded by the National Natural Science Foundation of China (grants 31801704 and 32472523), Innovation Project of Shandong Academy of Agricultural Sciences (grants CXGC2024B11 and CXGC2024A05), China Postdoctoral Science Foundation (grants 2021M691973 and 2022T150389), the Youth Innovation Team Plan for Shandong High Education Institution (grant 2022KJ241) and the Taishan Scholar Project (grant TS2022‐028).

Contributor Information

Chao Geng, Email: chaogeng@sdau.edu.cn.

Xiang‐Dong Li, Email: xdongli@sdau.edu.cn.

Data Availability Statement

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supporting Information. Additional data related to this paper may be requested from the authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

FIGURE S1. Multiple amino acid sequence identity analysis of VPg from multiple potyviruses. (a) Multiple amino acid sequence identity of VPg from different isolates of multiple potyviruses. (b) Heatmaps of the multiple amino acid sequence identity of VPg from different isolates of multiple potyviruses. (c) Amino acid mutation sites of VPg in potyviruses that break the eIF4E family gene‐mediated recessive resistance. The regions highlighted in grey boxes individually indicated the β1, β2, β3, β4 and β5 sheets. The regions highlighted in yellow and blue boxes respectively indicated the α1 and α2 helices. All of the amino acid substitution sites were highlighted in red boxes.

MPP-25-e70033-s003.docx (335.7KB, docx)

FIGURE S2. Analysis of the interaction between the PRSV VPg α1–α2 loop naturally occurring mutants and CleIF4ED71G. (a) Yeast two‐hybrid analysis of the interaction between the PRSV VPg α1–α2 loop naturally occurring mutants and CleIF4ED71G in vitro. The yeast cells co‐transformed with BD‐VPgWT, BD‐VPgK105Q or BD‐VPgE108G and AD‐CleIF4ED71G or AD‐CleIF4EWT 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 interaction between the naturally occurring mutants VPgK105Q or VPgE108G and CleIF4ED71G in Nicotiana benthamiana leaves. VPgWT‐CE, VPgK105Q‐CE or VPgE108G‐CE was co‐expressed with CleIF4EWT‐NE or CleIF4EWT‐NE in N. benthamiana leaves. Confocal imaging was performed at 48 h post‐inoculation. Scale bars = 20 μm.

MPP-25-e70033-s007.docx (78.7KB, docx)

FIGURE S3. Analysis of the interaction between VPgK105Q or VPgE108G and CleIF(iso)4E with the naturally occurring mutants in the cap‐binding pocket. (a) Single amino acid substitutions in the CleIF(iso)4E protein of different watermelon accessions. The regions highlighted in yellow and green boxes indicated the two regions involved in eIF(iso)4E‐mediated resistance against potyviruses in the cap‐binding pocket. The naturally occurring mutant sites of the CleIF(iso)4E cap‐binding pocket were highlighted in blue boxes. (b) The naturally occurring mutant sites of the CleIF(iso)4E cap‐binding pocket mapped onto the 3D structure of the watermelon eIF(iso)4E protein predicted using the structure of cucumber eIF(iso)4E as the template. The black arrow pointed to the cap‐binding pocket. The naturally occurring mutant sites of the CleIF(iso)4E cap‐binding pocket were highlighted in blue. (c) Yeast two‐hybrid analysis of the interaction between VPgK105Q or VPgE108G and CleIF(iso)4E with the naturally occurring mutants in the cap‐binding pocket in vitro. The yeast cells co‐transformed with BD‐VPgK105Q or BD‐VPgE108G and AD‐CleIF(iso)4EA48T, AD‐CleIF(iso)4EL54P, AD‐CleIF(iso)4EWT or empty vector AD were subjected to 10‐fold serial dilutions and plated on the SD/−Trp/−Leu/−His selection medium for 4 days. (d) Bimolecular fluorescence complementation analysis of the interaction between VPgK105Q or VPgE108G and CleIF(iso)4E with the naturally occurring mutants in the cap‐binding pocket in Nicotiana benthamiana leaves. VPgK105Q‐CE or VPgE108G‐CE was co‐expressed with CleIF(iso)4EA48T‐NE, CleIF(iso)4EL54P‐NE, CleIF(iso)4EWT‐NE or β‐glucuronidase (GUS)‐NE in N. benthamiana leaves. Confocal imaging was performed at 48 h post‐inoculation. Scale bars = 20 μm.

MPP-25-e70033-s002.docx (153.9KB, docx)

FIGURE S4. Western blotting analysis of expression levels of different variants of PRSV VPg, CleIF4E or CleIF(iso)4E. (a) Western blotting analysis of the expression level of different variants of CleIF4E or CleIF(iso)4E in yeast two‐hybrid (Y2H) assays. (b) Western blotting analysis of the expression level of different variants of PRSV VPg in Y2H assays.

MPP-25-e70033-s008.docx (67.4KB, docx)

FIGURE S5. Full‐length sequencing results of inoculated viruses.

MPP-25-e70033-s005.pdf (24.4MB, pdf)

TABLE S1. Primers used in this work.

MPP-25-e70033-s004.docx (17.2KB, docx)

TABLE S2. Sequences of watermelon eIF4E family genes.

MPP-25-e70033-s006.docx (15.7KB, docx)

TABLE S3. List of GenBank numbers of multiple PRSV‐W isolates and amino acid sequences of eIF4E family proteins of Arabidopsis thaliana , tobacco, maize, sugarcane and watermelon.

MPP-25-e70033-s001.docx (20.4KB, docx)

Data Availability Statement

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supporting Information. Additional data related to this paper may be requested from the authors.

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