Variability in eukaryotic initiation factor iso4E in Brassica rapa influences interactions with the viral protein linked to the genome of Turnip mosaic virus

Abstract

Plant potyviruses require eukaryotic translation initiation factors (eIFs) such as eIF4E and eIF(iso)4E to replicate and spread. When Turnip mosaic virus (TuMV) infects a host plant, its viral protein linked to the genome (VPg) needs to interact with eIF4E or eIF(iso)4E to initiate translation. TuMV utilizes BraA.eIF4E.a, BraA.eIF4E.c, BraA.eIF(iso)4E.a, and BraA.eIF(iso)4E.c of Brassica rapa to initiate translation in Arabidopsis thaliana. In this study, the BraA.eIF4E.a, BraA.eIF4E.c, BraA.eIF(iso)4E.a, and BraA.eIF(iso)4E.c genes were cloned and sequenced from eight B. rapa lines, namely, two BraA.eIF4E.a alleles, four BraA.eIF4E.c alleles, four BraA.eIF(iso)4E.a alleles, and two BraA.eIF(iso)4E.c alleles. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) analyses indicated that TuMV VPg could not interact with eIF4E, but only with eIF(iso)4E of B. rapa. In addition, the VPgs of the different TuMV isolates interacted with various eIF(iso)4E copies in B. rapa. In particular, TuMV-UK1/CDN1 VPg only interacted with BraA.eIF(iso)4E.c, not with BraA.eIF(iso)4E.a. Some single nucleotide polymorphisms (SNPs) were identified that may have affected the interaction between eIF(iso)4E and VPg such as the SNP T₁₀₆C in BraA.eIF(iso)4E.c and the SNP A₁₅₄C in VPg. Furthermore, a three-dimensional structural model of the BraA.eIF(iso)4E.c-1 protein was constructed to identify the specific conformation of the variable amino acids from BraA.eIF(iso)4E.c. The 36^th amino acid in BraA.eIF(iso)4E.c is highly conserved and may play an important role in establishing protein structural stability. The findings of the present study may lay the foundation for future investigations on the co-evolution of TuMV and eIF(iso)4E.

Figure 1

TuMV C4, TuMV UK1, and TuMV CDN1 VPgs do not interact with BraA.eIF4E.a or BraA.eIF4E.c. (A) The interaction was confirmed by yeast two-hybrid (Y2H) assays. Negative control: the empty vectors pGADT7 and pGBKT7 (data not shown); positive controls: the murine p53 and SV40 large T antigen from the Matchmaker GAL4 two-hybrid system 3; TuMV-VPg and Arabidopsis eIF(iso)4E (lsp); assay controls: each partner and empty vector (data not shown). (B) The interactions were confirmed by bimolecular fluorescence complementation (BiFC). P: positive controls (the combination of bZIP63YN and bZIP63YC); N: negative controls (YNE-empty and YCE-empty vectors); the assay controls: each partner and empty vectors (data not shown).

Figure 2

TuMV CDN1 VPgs interacts with BraA.eIF(iso)4E.c, but not with BraA.eIF(iso)4E.a. (A) The results are from the Y2H. Negative control: the empty vectors pGADT7 and pGBKT7 (data not shown); positive controls: the murine p53 and SV40 large T antigen from the Matchmaker GAL4 two-hybrid system 3; TuMV-VPg and Arabidopsis eIF(iso)4E (lsp); assay controls: each partner and empty vector (data not shown). (B) Verification of the results using BiFC. P: positive controls (the combination of bZIP63YN and bZIP63YC); N: negative controls (YNE-empty and YCE-empty vectors); each partner and empty vector were used as controls (data not shown).

Figure 3

Specific SNPs affect the interaction between eIF(iso)4E and TuMV VPg. Variations between BraA.eIF(iso)4E.c-1 and BraA.eIF(iso)4E.c-2 could affect the interaction as confirmed by Y2H (A) and by BiFC. (B,C) Multiple sequence alignment of TuMV C4 VPg TuMV UK1 VPg and TuMV CDN1 VPg. Five amino acid substitutions were identified between TuMV C4 and CDN1, whereas four amino acid substitutions were detected between TuMV C4 and UK1. Variations between TuMV-C4 VPg and TuMV-CDN1 VPg could affect the interaction as indicated by Y2H (D) and by BiFC. (E) Y2H: negative control, the empty vectors pGADT7 and pGBKT7 (data not shown); positive controls, the murine p53 and SV40 large T antigen from the Matchmaker GAL4 two-hybrid system 3; TuMV-VPg and Arabidopsis eIF(iso)4E (lsp); assay controls: each partner and empty vector (data not shown). BiFC: P-positive controls (the combination of bZIP63YN and bZIP63YC); N-negative controls (YNE-empty and YCE-empty vectors); the assay controls were each partner and empty vectors (data not shown).

Figure 4

Analysis of BraA.eIF(iso)4E.c-1 protein domain tertiary structure. (A) The three-dimensional structural model was built based on the wheat eIF(iso)4E protein. (B) Four amino acid variations were detected between the BraA.eIF(iso)4E.c-1 and BraA.eIF(iso)4E.c-2 proteins. (C) The location of the three different amino acid variations is depicted in the structural model of the entire protein. (D) The 36^th amino acid changed from phenylalanine to leucine. (E) The 52^th amino acids changed from valine to alanine. (F) The 80^th changed from isoleucine to threonine. (G) The 150^th changed from proline to glutamine.