Selective Interaction of Sugarcane eIF4E with VPgs from Sugarcane Mosaic Pathogens

Abstract

Eukaryotic translation initiation factor 4E (eIF4E) plays a key role in the infection of potyviruses in susceptible plants by interacting with viral genome-linked protein (VPg). Sugarcane (Saccharum spp.) production is threatened by mosaic disease caused by Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), and Sugarcane streak mosaic virus (SCSMV). In this study, two eIF4Es and their isoform eIF(iso)4E and 4E-binding protein coding genes were cloned from sugarcane cultivar ROC22 and designated SceIF4Ea, SceIF4Eb, SceIF(iso)4E, and ScnCBP, respectively. Real-time quantitative PCR analysis showed different expression profiles of these four genes upon SCMV challenge. A subcellular localization assay showed that SceIF4Ea, SceIF4Eb, SceIF(iso)4E, and ScnCBP were distributed in the nucleus and cytoplasm. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that SceIF4Ea/b and SceIF(iso)4E were selectively employed by different sugarcane mosaic pathogens, i.e., SCMV-VPg interacted with SceIF4Ea/b and SceIF(iso)4E, SrMV-VPg interacted with both SceIF4Eb and SceIF(iso)4E, and SCSMV-VPg interacted only with SceIF(iso)4E. Intriguingly, the BiFC assays, but not the Y2H assays, showed that ScnCBP interacted with the VPgs of SCMV, SrMV, and SCSMV. Competitive interaction assays showed that SCMV-VPg, SrMV-VPg, and SCMV-VPg did not compete with each other to interact with SceIF(iso)4E, and SceIF(iso)4E competed with SceIF4Eb to interact with SrMV-VPg but not SCMV-VPg. This study sheds light on the molecular mechanism of sugarcane mosaic pathogen infection of sugarcane plants and benefits sugarcane breeding against the sugarcane mosaic disease.

Keywords: SCMV; SCSMV; SrMV; VPg; eIF4E.

Figure 1

Alignment of the amino acid sequences of the eIF4E, eIF(iso)4E, and nCBP from several plant species. The accession numbers of the selected amino acid sequences were Arabidopsis thaliana: eIF4E (AT4G18040), eIF(iso)4E (AT5G35620), nCBP (AT5G18110); Triticum aestivum: eIF4E (CAA78262), eIF(iso)4E (AAA34296), nCBP (XM.037569593); Zea mays: eIF4E (AF076954), eIF(iso)4E (CD527566), nCBP (EU959765); Oryza sativa: eIF4E (U34597), eIF(iso)4E (U34598), nCBP (CF328046); Glycine max: eIF4E (BI785638), eIF(iso)4E (BG045933), nCBP (BE474869); Saccharum spp. hybrid ROC22: SceIF4Ea (MW547070), SceIF4Eb (MW547071), SceIF(iso)4E (MW547073), ScnCBP (KX757019). Conserved Trp, Phe, and His residues were yellow shaded: H(X5)W(X2)W(X8–12)W-(X9)F(X5)FW(X20)F(X7)W(X10)W(X9–12)W(X34–35)W(X31–33)H. Numbers to the right of the sequences indicate the positions of residues from the N-terminal Met.

Figure 2

Phylogenetic tree analysis of eIF4E, eIF(iso)4E, and nCBP proteins from different plant species. Protein sequences were retrieved from NCBI and the accession numbers are as follows. Arabidopsis thaliana: eIF4E (AT4G18040), eIF(iso)4E (AT5G35620), nCBP (AT5G18110); Triticum aestivum: eIF4E (CAA78262), eIF(iso)4E (AAA34296), nCBP (XM.037569593); Zea mays: eIF4E (AF076954), eIF(iso)4E (CD527566), nCBP (EU959765); Vitis vinifera: eIF4E (BM437772), eIF(iso)4E (CB918946), nCBP (BM437090); Solanum tuberosum: eIF4E (BI178346), eIF(iso)4E (BG598942), nCBP (CK269484); Saccharum officinarum: eIF4E (CA248584), eIF(iso)4E (CA120131), nCBP (CA241493); Populus deltoides: eIF4E (CX176909), eIF(iso)4E (CV130917), nCBP (CX173803); Oryza sativa: eIF4E (U34597), eIF(iso)4E (U34598), nCBP (CF328046); Medicago truncatula: eIF4E (AJ502732), eIF(iso)4E (AL378243), nCBP (AL381239); Hordeum vulgare: eIF4E (CA008276), eIF(iso)4E (BU987334), nCBP (BQ467551); Glycine max: eIF4E (BI785638), eIF(iso)4E (BG045933), nCBP (BE474869); Brassica napus: eIF4E (CD814531), eIF(iso)4E (CD826731), nCBP (CD842187); Solanum lycopersicum: eIF4E (Solyc03g005870), eIF(iso)4E (Solyc09g090580), nCBP (Solyc10g080660); Manihot esculenta: eIF4E (MANES.17G063100), eIF(iso)4E (MANES.03G160000), nCBP (MANES.09G140300); Saccharum spp. hybrid ROC22: SceIF4Ea (MW547070), SceIF4Eb (MW547071), SceIF(iso)4E (MW547073), ScnCBP (KX757019), which were highlighted by the “leaves” in red. The red circle indicated the dicotyledons. The blue diamond indicated the monocotyledons with the hollow diamond for C4 plants, while the solid core of diamond for C3 plants.

Figure 3

The expression profiles of SceIF4Ea/b, SceIF(iso)4E, and ScnCBP of sugarcane cultivar ROC22 challenged by Sugarcane mosaic virus (SCMV). Leaves of ROC22 plantlets were inoculated with SCMV and sampled at different time points. Mock inoculated plants with 0.01 M phosphate buffer (pH 7.0) were used as the negative controls. The Y axes indicates the relative expression of SCMV-CP (A), SceIF4Ea/b (B), SceIF(iso)4E (C), and ScnCBP (D) at 0, 1, 2, and 5 days post-inoculation. The X axes indicates the time point of material collection. Error bars indicate SD (n = 3), a, b, and c indicate significance at the corresponding time points, t-test, p <0.05. Results were representative of three independent experiments.

Figure 4

Subcellular localization of SceIF4Ea, SceIF4Eb, SceIF(iso)4E, and ScnCBP in Nicotiana benthamiana leaf epidermal cells. Fluorescence of YFP or YFP fusion proteins was detected by 48 h post agroinfiltration. The nucleus was displayed by diamidine phenylindole (DAPI) staining. Bars = 25 μm.

Figure 5

Investigation of protein interaction by yeast two-hybrid assays. The coding sequences of SceIF4Ea, SceIF4Eb, SceIF(iso)4E, and ScnCBP were individually infused into the prey vector pGADT7 and pairwise co-transformed with the vector pGBKT7-SCMV-VPg, pGBKT7-SrMV-VPg, pGBKT7-SCSMV-VPg into the yeast AH109 cells in a 10× dilution series of 10-µL aliquots, which were then plated on a non-selective medium SD/-Leu/-Trp or quadruple dropout medium SD/-Leu/-Trp/-His/-Ade supplemented with X-α-Gal. Yeast cells co-transformed with pGBKT7-53 and pGADT7-T were used as a positive control, pGBKT7-Lam and pGADT7-T were used as a negative control.

Figure 6

Investigation of protein interaction by bimolecular fluorescence complementation assays. Agrobacteria harboring YC/YN fusion proteins were co-infiltrated into Nicotiana benthamiana leaves, respectively. The leaf epidermal cells pairwise co-transformed with SCMV-VPg-YN, SrMV-VPg-YN or SCSMV-VPg-YN and ScELC-YC were used as positive controls, while SCMV-VPg-YN, SrMV-VPg-YN or SCSMV-VPg-YN and YC were used as negative controls. The images were captured at 48 h post agroinfiltration. Bars = 25 μm.

Figure 7

Competitive yeast two-hybrid assays. SCMV-VPg, SrMV-VPg, SCSMV-VPg, eIF4Eb, eIF(iso)4E were used as the competitor to test the interaction of SCMV-VPg, SrMV-VPg, SCSMV-VPg with eIF4Eb, eIF(iso)4E, respectively. The competitor was co-transformed with the paired interacting proteins fused with the activation domain (AD) or DNA-binding domain (BD) into the yeast AH109 cells in a 10× dilution series of 10-µL aliquots, which were then plated on the quadruple dropout medium SD/-Leu/-Trp/-His/-Ade.