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. 2013;8(3):e57229.
doi: 10.1371/journal.pone.0057229. Epub 2013 Mar 7.

An isoform of eukaryotic initiation factor 4E from Chrysanthemum morifolium interacts with Chrysanthemum virus B coat protein

Aiping Song  1 , Wanghuai Lou, Jiafu Jiang, Sumei Chen, Zuxia Sun, Zhiyong Guan, Weimin Fang, Nianjun Teng, Fadi Chen
Affiliations

An isoform of eukaryotic initiation factor 4E from Chrysanthemum morifolium interacts with Chrysanthemum virus B coat protein

Aiping Song et al. PLoS One. 2013.

Abstract

Background: Eukaryotic translation initiation factor 4E (eIF4E) plays an important role in plant virus infection as well as the regulation of gene translation.

Methodology/principal findings: Here, we describe the isolation of a cDNA encoding CmeIF(iso)4E (GenBank accession no. JQ904592), an isoform of eIF4E from chrysanthemum, using RACE PCR. We used the CmeIF(iso)4E cDNA for expression profiling and to analyze the interaction between CmeIF(iso)4E and the Chrysanthemum virus B coat protein (CVBCP). Multiple sequence alignment and phylogenetic tree analysis showed that the sequence similarity of CmeIF(iso)4E with other reported plant eIF(iso)4E sequences varied between 69.12% and 89.18%, indicating that CmeIF(iso)4E belongs to the eIF(iso)4E subfamily of the eIF4E family. CmeIF(iso)4E was present in all chrysanthemum organs, but was particularly abundant in the roots and flowers. Confocal microscopy showed that a transiently transfected CmeIF(iso)4E-GFP fusion protein distributed throughout the whole cell in onion epidermis cells. A yeast two hybrid assay showed CVBCP interacted with CmeIF(iso)4E but not with CmeIF4E. BiFC assay further demonstrated the interaction between CmeIF(iso)4E and CVBCP. Luminescence assay showed that CVBCP increased the RLU of Luc-CVB, suggesting CVBCP might participate in the translation of viral proteins.

Conclusions/significance: These results inferred that CmeIF(iso)4E as the cap-binding subunit eIF(iso)4F may be involved in Chrysanthemum Virus B infection in chrysanthemum through its interaction with CVBCP in spatial.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Phylogenetic relationship between CmeIF(iso)4E and other eIF4E superfamily proteins.
The phylogenetic tree file was produced using ClustalW (http://www.ebi.ac.uk/clustalW/). The GenBank accession numbers of the amino acid sequences used are: CmeIF_iso_4E (JQ904591, Chrysanthemum morifolium), LseIF_iso_4E (AAP86603.1, Lactuca sativa), SleIF_iso_4E (NP_001234772.1, Solanum lycopersicum), PseIF_iso_4E (BAK53449.1, Pisum sativum), NteIF_iso_4E (AAU06579.1, Nicotiana tabacum), PveIF_iso_4E (ABU54807.1, Phaseolus vulgaris), CpeIF_iso_4E (ACM18197.1, Carica papaya), CseIF_iso_4E (ABY56102.1, Cucumis sativus), CmeIF4E (JQ904591, Chrysanthemum morifolium), AteIF4E (NP_193538.1, Arabidopsis thaliana), CaeIF4E (AAN74644.1, Cayenne pepper), CpeIF4E (ACN38307.1, Carica papaya), HveIF4E (CAR92170.2, Hordeum vulgare), LseIF4E (AAP86602.1, Lactuca sativa), NteIF4E (DK22107.1, Nicotiana tabacum), SleIF4E (AAV88610.1, Solanum lycopersicum), PseIF4E (ABG35119.1, Pisum sativum) and ZmeIF4E (ACG34414.1, Zea mays).
Figure 2
Figure 2. CmeIF(iso)4E expression in chrysanthemum as demonstrated by qRT-PCR.
Figure 3
Figure 3. Subcellular localization of the CmeIF(iso)4E protein in onion epidermis cells.
The upper row shows the 35S::GFP signal alone as a positive control; the middle row displays the signal from 35S::CmeIF(iso)4E-GFP; the lower row shows 35S::CmeIF(iso)4E-GFP following treatment with 0.8 M sucrose to induce plasmolysis. The left panel shows bright field images; the middle panel shows green fluorescence signals detected at 488 nm; the right panel shows the merged GFP signals and bright field images. Bars = 50 µm
Figure 4
Figure 4. Interaction between CmeIF(iso)4E and CVBCP in vitro as demonstrated by Y2H assay.
Yeast two-hybrid screen demonstrating the interaction between CVBCP and CmeIF(iso)4E. The left panel shows the growth of yeast cells containing both plasmids on SD-Leu-Trp medium (SD-2); the middle panel shows the selection of yeast colonies on SD/-Leu/-Trp/-Ade/-His medium (SD-4); the right panel shows the selection of yeast colonies on SD-4 containing α- X-Gal.
Figure 5
Figure 5. BiFC view of the interaction between CmeIF(iso)4E and CVBCP in transiently transfected onion cells.
Confocal microscopy images showing yellow fluorescence in onion cells transfected with nEYFP-CmeIF(iso)4E and cEYFP-CVBCP. No fluorescence was observed in negative control onion cells co-transfected with nEYFP-CmeIF(iso)4E + cEYFP, nEYFP + cEYFP-CVBCP, or nEYFP + cEYFP. The corresponding differential interference contrast (DIC) images are shown at the top. Bars = 50 µm.
Figure 6
Figure 6. Relative luciferase activities in Arabidopsis mesophyll protoplasts after transfection with CVBCP and Luc-CVB constructs.
Mock was a negative control without the constructs. Data are the mean of three independent experiments. RLU, relative light units.
See this image and copyright information in PMC

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This study is supported by the Program for New Century Excellent Talents in University of Chinese Ministry of Education (Grant No. NCET-10-0492), Sci-Tech Support Plan of Jiangsu Province (BE2011325), 863 Project (2011AA100208), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Fundamental Research Funds for the Central Universities (KYZ201147) and Youth Science and Technology Innovation Fund (KJ2011009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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