Eukaryotic translation initiation factor 4E family member nCBP facilitates the accumulation of TGB-encoding viruses by recognizing the viral coat protein in potato and tobacco

Abstract

Due to their limited coding capacity, plant viruses have to depend on various host factors for successful infection of the host. Loss of function of these host factors will result in recessively inherited resistance, and therefore, these host factors are also described as susceptibility genes or recessive resistance genes. Most of the identified recessive resistance genes are members of the eukaryotic translation initiation factors 4E family (eIF4E) and its isoforms. Recently, an eIF4E-type gene, novel cap-binding protein (nCBP), was reported to be associated with the infection of several viruses encoding triple gene block proteins (TGBps) in Arabidopsis. Here, we, for the first time, report that the knockdown of nCBP in potato (StnCBP) compromises the accumulation of potato virus S (PVS) but not that of potato virus M (PVM) and potato virus X (PVX), which are three potato viruses encoding TGBps. Further assays demonstrated that StnCBP interacts with the coat proteins (CPs) of PVS and PVM but not with that of PVX, and substitution of PVS CP in the PVS infectious clone by PVM CP recovered the virus infection in StnCBP-silenced transgenic plants, suggesting that the recognition of PVS CP is crucial for StnCBP-mediated recessive resistance to PVS. Moreover, the knockdown of nCBP in Nicotiana benthamiana (NbnCBP) by virus-induced gene silencing suppressed PVX accumulation but not PVM, while NbnCBP interacted with the CPs of both PVX and PVM. Our results indicate that the nCBP orthologues in potato and tobacco have conserved function as in Arabidopsis in terms of recessive resistance against TGB-encoding viruses, and the interaction between nCBP and the CP of TGB-encoding virus is necessary but not sufficient to determine the function of nCBP as a susceptibility gene.

Keywords: TGB-encoding virus; coat protein; eukaryotic translation initiation factors 4E; nCBP; recessive resistance; susceptibility genes.

FIGURE 1

Phylogenetic analysis of eIF4E genes in potato, Arabidopsis, tobacco, and tomato (A), and an amino acid sequence alignment of ncbp proteins from various species (B). The prefix of accession numbers with “Soltu.DM” represents proteins from S. tuberosum; the prefix of accession numbers with “AT” represents proteins from A. thaliana; the prefix of accession numbers with “Solyc” represents proteins from S. lycopersicum; the prefix of accession numbers with “Niben101Scf” represents proteins from N. benthamiana. The eIF4E subgroup, eIF(iso)4E subgroup, and nCBP subgroup are distinguished by pink, blue, and yellow regions, respectively. The phylogenetic tree was generated with neighbor-joining tree and 1000 bootstraps. The red underline represents the eIF4E superfamily domain.

FIGURE 2

Morphology and virus resistance identification of the RiStnCBP transgenic lines. (A) Morphology of RiStnCBP transgenic lines under field conditions. (B–D) Virus resistance identification in RiStnCBP transgenic lines against PVS (B), PVM (C), and PVX (D), respectively. The virus accumulation was determined using qRT-PCR (left) and ELISA (right). Error bars indicate ± SD of three replicates (n = 3). Three independent experiments were performed with similar results. p < 0.05*, p < 0.01**, and p < 0.001*** (Student’s t-test).

FIGURE 3

StnCBP interacted with PVS CP in the nucleus and cytoplasm. (A) Interaction between StnCBP and proteins of PVS in Y2H assays. -W-L represents a medium lacking tryptophan and leucine, while -W-L-H-A represents a medium lacking tryptophan, leucine, histidine, and adenine. Paired combinations PGADT7-T/PGBKT7-53 and PGADT7-T/PGBKT7-lam represent positive and negative controls, respectively. (B) Interaction between StnCBP and PVS CP in BiFC assays. Merge means the overlay of YFP and visible light on single confocal planes. Scale bar: 50 μm. (C) Subcellular localization and co-localization of StnCBP and PVS CP. Merge means the overlay of GFP and RFP on single confocal planes. Scale bar: 20 μm. (D) Identification of full-length proteins of StnCBP and PVS CP successfully expressed with GFP in the Western blotting. Total proteins were immunoprecipitated with anti-GFP antibody, and Rubisco was stained with Ponceau S as a loading control.

FIGURE 4

PVS CP played a vital role in the StnCBP-mediated recessive resistance to PVS. (A) Schematic illustration of the construction of PVS infectious clone. PVS-A, PVS-B, and PVS-C represent the fragments from PVS for the assembly. The gray shadow bars indicate the overlapping regions of adjacent fragments, with the number of overlapping nucleotides. (B) Electrophoretic separation of PCR products amplified with detection primers (above) and CP-flanking primers (below) from plants agroinoculated with PVS^CP/S (1–3), PVS^CP/M (4–6), and PVS^CP/X (7–9). M: Trans2K^® Plus DNA Marker. The upper non-inoculated leaves were collected 14 days after inoculation. (C,D) The accumulation of PVS^CP/S (C) and PVS^CP/M (D) infectious clones in the RiStnCBP and control plants (WT). The virus accumulation was determined using qRT-PCR (left) and ELISA (right). Error bars indicate ± SD of three replicates (n = 3). Three independent experiments were performed with similar results. p < 0.01** and p < 0.001*** (Student’s t-test).

FIGURE 5

Antiviral identification of NbnCBP against PVX, and interaction analyses between NbnCBP and proteins of PVX. (A) The silencing efficiency of NbnCBP in N. benthamiana by VIGS. The gene expression was determined using qRT-PCR. Error bars indicate ± SD of five replicates (n = 5). p < 0.001*** (Student’s t-test). (B,C) The determination of PVM and PVX accumulation in the control and NbnCBP-silenced plants by RT-qPCR (B) and ELISA (C). Error bars indicate ± SD of three replicates (n = 3). Three independent experiments were performed with similar results. p < 0.01** (Student’s t-test). (D) Green fluorescence emission in PVX-GFP inoculated leaves of the control, NbnCBP-silenced, and NbnCBP-silenced: complemented plants. The photographs were taken at 8 dpi. (E,F). The determination of PVX-GFP accumulation in inoculated leaves of the control, NbnCBP-silenced and NbnCBP-silenced: complemented plants by RT-qPCR (E) and ELISA (F). Error bars indicate ± SD of three replicates (n = 3). Three independent experiments were performed with similar results. p < 0.01** (Student’s t-test). (G) Interaction analysis between NbnCBP and the proteins of PVX in Y2H. -W-L represents a medium lacking tryptophan and leucine, while -W-L-H-A represents a medium lacking tryptophan, leucine, histidine, and adenine. Paired combinations PGADT7-T/PGBKT7-53 and PGADT7-T/PGBKT7-lam represent positive and negative controls, respectively. (H) Interaction analysis between NbnCBP and PVX CP in BiFC assay. nYFP and cYFP represent their empty vectors. Merge means the overlay of YFP and visible light on single confocal planes. Scale bar: 50 μm.