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. 2017 Mar 10:8:338.
doi: 10.3389/fmicb.2017.00338. eCollection 2017.

Translation Initiation Factor eIF4E and eIFiso4E Are Both Required for Peanut stripe virus Infection in Peanut (Arachis hypogaea L.)

Manlin Xu  1 , Hongfeng Xie  2 , Juxiang Wu  2 , Lianhui Xie  3 , Jinguang Yang  4 , Yucheng Chi  2
Affiliations

Translation Initiation Factor eIF4E and eIFiso4E Are Both Required for Peanut stripe virus Infection in Peanut (Arachis hypogaea L.)

Manlin Xu et al. Front Microbiol. .

Abstract

Peanut stripe virus (PStV) belongs to the genus Potyvirus and is the most important viral pathogen of cultivated peanut (Arachis hypogaea L.). The eukaryotic translation initiation factor, eIF4E, and its isoform, eIF(iso)4E, play key roles during virus infection in plants, particularly Potyvirus. In the present study, we cloned the eIF4E and eIF(iso)4E homologs in peanut and named these as PeaeIF4E and PeaeIF(iso)4E, respectively. Quantitative real-time PCR (qRT-PCR) analysis showed that these two genes were expressed during all growth periods and in all peanut organs, but were especially abundant in young leaves and roots. These also had similar expression levels. Yeast two-hybrid analysis showed that PStV multifunctional helper component proteinase (HC-Pro) and viral protein genome-linked (VPg) both interacted with PeaeIF4E and PeaeIF(iso)4E. Bimolecular fluorescence complementation assay showed that there was an interaction between HC-Pro and PeaeIF4E/PeaeIF(iso)4E in the cytoplasm and between VPg and PeaeIF4E/PeaeIF(iso)4E in the nucleus. Silencing either PeaeIF4E or PeaeIF(iso)4E using a virus-induced gene silencing system did not significantly affect PStV accumulation. However, silencing both PeaeIF4E and PeaeIF(iso)4E genes significantly weakened PStV accumulation. The findings of the present study suggest that PeaeIF4E and PeaeIF(iso)4E play important roles in the PStV infection cycle and may potentially contribute to PStV resistance.

Keywords: Peanut stripe virus; gene silencing; peanut; protein–protein interaction; translation initiation factor 4E.

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Figures

FIGURE 1
FIGURE 1
Phylogenetic analysis of eIF4E and eIF(iso)4E sequences of different plant species. The phylogenetic tree was constructed using ClustalW (http://www.ebi.ac.uk/clustalw/). The GenBank accession numbers of the amino acid sequences used are listed in Supplementary Table S2. The two peanut sequences are highlighted in bold and italics.
FIGURE 2
FIGURE 2
mRNA transcript levels (A) of PeaeIF4E and PeaeIF(iso)4E and their subcellular localization (B). Relative mRNA expression levels of peaeIF4E and peaeIF(iso)4E were determined by real-time reverse transcript PCR (RT-PCR). The values represent means of three biological repeats and the value of each biological repeat is the mean of three technical repeats. All values were normalized to the reference gene peanut actin. PeaeIF4E and PeaeIF(iso)4E were fused with green fluorescent protein (GFP) are delivered into protoplasts of Arabidopsis. The GFP fluorescence was observed 12–16 h after transfection. Scale bars = 10 μm.
FIGURE 3
FIGURE 3
Subcellular localization of PStV HC-Pro and PStV VPg. PStV HC-Pro and PStV VPg fused with GFP were transfected into protoplasts of Arabidopsis. The GFP fluorescence was observed 12–16 h after transfection. Scale bars = 10 μm.
FIGURE 4
FIGURE 4
Yeast two-hybrid assay of protein–protein interaction between the PeaeIF4E/PeaeIF(iso)4E from peanut and PStV-HC-pro/PStV-VPg. Yeast co-transformants were grown on selective medium SD/-Leu-Trp-His-Ade plus X-α-Gal and incubated for 4 days at 30°C (A). Frame (B) corresponds to the clones left (A).
FIGURE 5
FIGURE 5
Bimolecular fluorescence complementation (BiFC) assay showing interaction between PeaeIF4E/PeaeIF(iso)4E and PStV HC-Pro/PStV VPg. The full-length open reading frame (ORF) of PeaeIF4E/PeaeIF(iso)4E was cloned into the vector pEarleyGate202-NYFP [eIF4E-NY, eIF(iso)4E-NY] and that of PStV HC-Pro/PStV VPg into pEarleyGate202-CYFP (HC-Pro-CY, VPg-CY). The recombinant plasmids were transfected into protoplasts of Arabidopsis. Fluorescence was observed at 14–16 h post-transfection by confocal laser-scanning microscopy. Scale bars = 10 μm. (A) BiFC analysis of PeaeIF4E/PeaeIF(iso)4E and PStV HC-Pro. (B) A range of negative controls. (C) BiFC analysis of PeaeIF4E/PeaeIF(iso)4E and PStV VPg.
FIGURE 6
FIGURE 6
Real-time PCR analysis for target gene expression in peanut (A), accumulation of PStV RNA (B) and the growth condition and symptoms of peanut after inoculated PStV (C). Virus-induced gene silencing of PeaeIF4E/PeaeIF(iso)4E in representative plants belonging to each of the four different treatments. For qRT-PCR detection of the expression of PeaeIF4E/PeaeIF(iso)4E, three plants from each group were pooled as one sample and the experiments were performed in triplicate (A). Effects of silencing of PeaeIF4E/PeaeIF(iso)4E on PStV infection. The accumulation of PStV RNA in inoculated peanut plants was detected by RT-PCR 15 days after PStV inoculation (B). The growth condition and symptoms of peanut after inoculated PStV (C). Four different treatments peanuts were mechanically infected by PStV. Three plants from each group were pooled as one sample and the experiments were performed in triplicate, 10–14 days later, PStV disease symptom began to appeared. The peanut growth condition after inoculated with PStV, except the different symptoms on peanut leaves, different treatments peanuts growth condition showed no significant differences compared with control. 1, control group; 2, silencing PeaeIF4E; 3, silencing PeaeIF(iso)4E; 4, silencing both PeaeIF4E and PeaeIF(iso)4E.
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