Mutational analysis of interaction between coat protein and helper component-proteinase of Soybean mosaic virus involved in aphid transmission

Abstract

Soybean mosaic virus (SMV), a member of the genus Potyvirus, is transmitted by aphids in a non-persistent manner. It has been well documented that the helper component-proteinase (HC-Pro) plays a role as a 'bridge' between virion particles and aphid stylets in the aphid transmission of potyviruses. Several motifs, including the KITC and PTK motifs on HC-Pro and the DAG motif on the coat protein (CP), have been found to be involved in aphid transmission. Previously, we have shown strong interaction between SMV CP and HC-Pro in a yeast two-hybrid system (YTHS). In this report, we further analysed this CP-HC-Pro interaction based on YTHS and an in vivo binding assay to identify crucial amino acid residues for this interaction. Through this genetic approach, we identified two additional amino acid residues (H256 on CP and R455 on HC-Pro), as well as G12 on the DAG motif, crucial for the CP-HC-Pro interaction. We introduced mutations into the identified residues using an SMV infectious clone and showed that these mutations affected the efficiency of aphid transmission of SMV. We also investigated the involvement of the PTK and DAG motifs in the CP-HC-Pro interaction and aphid transmission of SMV. Our results support the concept that physical interaction between CP and HC-Pro is important for potyviral aphid transmission. Based on the combination of our current results with previous findings, the possibility that aphid transmission may be regulated by more complex molecular interactions than the simple involvement of HC-Pro as a bridge is discussed.

Figure 1

Map of interaction between Soybean mosaic virus (SMV) coat protein (CP) and helper component‐proteinase (HC‐Pro) in a yeast two‐hybrid system (YTHS). Full‐length CP and its truncations were fused downstream of the GAL4 activation domain (GAL4‐AD) of pACT2. Full‐length HC‐Pro and its truncations were fused downstream of the GAL4 DNA‐binding domain (GAL4‐BD) of pAS2‐1. Yeast cells co‐transformed with pACT2 and pAS2‐1 fusion derivatives were selected on synthetic dropout (SD) agar medium lacking leucine, tryptophan, histidine and adenine (–LWHA), and their α‐galactosidase activities were assayed on SD/–LW and SD/–LWHA agar plates containing X‐α‐Gal. The number of ‘+’ symbols indicates the comparative number of colonies formed on SD medium. (a) Interactions between HC‐Pro and CP truncations. (b) Interactions between CP and HC‐Pro truncations.

Figure 2

Identification of amino acid residues crucial for the coat protein–helper component‐proteinase (CP–HC‐Pro) interaction in a yeast two‐hybrid system (YTHS). Schematic representations of Soybean mosaic virus (SMV) CP (a) and HC‐Pro (b), showing the amino acid positions of the site‐directed mutations tested in this study. Alignment of the C‐terminal amino acid sequences of potyviral CPs (c) and HC‐Pros (d). Sequences were aligned by ClustalX2. Arrows indicate the amino acid positions on SMV CP and HC‐Pro. Arrowhead indicates the location of the HC‐Pro self‐cleavage site. Potyviral sequence data were obtained from the National Center for Biotechnology Information (NCBI) database: TEV, Tobacco etch virus (NC001555); TVMV, Tobacco vein mottling virus (NC001768); PVA, Potato virus A (NC004039); PPV, Plum pox virus (NC001445); LMV, Lettuce mosaic virus (NC003605); PVY, Potato virus Y (NC001616); SMV, Soybean mosaic virus (FJ807700); ZYMV, Zucchini yellow mosaic virus (NC003224); PMV, Peanut mottle virus (NC002600).

Figure 3

Detection of in planta interactions between coat protein (CP) and helper component‐proteinase (HC‐Pro) by bimolecular fluorescence complementation (BiFC) assay. (a) Schematic representation of the constructs used for BiFC assay. The basal BiFC vector contains, in sequential order, a left border of T‐DNA (LB), a double 35S promoter (35S), cloning sites, six histidine residues, either nYFP or cYFP sequences, a 35S terminator (T35S) and a right border of T‐DNA (RB). CP and HC‐Pro cistrons and their mutants were in‐frame inserted into the basal BiFC vectors utilizing StuI and SpeI sites. The constructed clones were transferred into Nicotiana benthamiana leaves by agroinfiltration in pairwise combinations as follows: none (healthy; b), nYFP + cYFP (c), CP‐nYFP + cYFP (d), nYFP + HC‐cYFP (e), CP‐nYFP + CP‐cYFP (f), CP‐nYFP + HC‐cYFP (g), CP_G12E‐nYFP + HC‐cYFP (h), CP_H246A‐nYFP + HC‐cYFP (i), CP_R249A‐nYFP + HC‐cYFP (j), CP_D250A‐nYFP + HC‐cYFP (k), CP_H256A‐nYFP + HC‐cYFP (l), CP‐nYFP + HC_T310A‐cYFP (m), CP‐nYFP + HC_E450A‐cYFP (n), CP‐nYFP + HC_K452A‐cYFP (o) and CP‐nYFP + HC_R455A‐cYFP (p). The reconstructed yellow fluorescent protein (YFP) signals were observed in the epidermal cells using epifluorescence microscopy at 3 days post‐infiltration (dpi). Co‐expression of (g), (i), (j), (k), (m), (n) and (o) induced fluorescence signals. Co‐expression of (l) induced weak but detectable fluorescence. YFP signals from the co‐expression of (f) were used as a positive control. Images for fluorescence emitted by YFP (left) and for the transmitted light mode (right) are shown.

Figure 4

Schematic representation of Soybean mosaic virus (SMV) constructs used for the introduction of helper component‐proteinase (HC‐Pro) mutations. (a) SMV genome organization. HC‐Pro is processed by P1 and by HC‐Pro by itself in an in cis manner. Amino acid sequences of the peptide cleavage sites recognized by either P1 or HC‐Pro are underlined, and arrowheads indicate the location of the cleaved peptide bond. (b) Construction of an available insertion cassette between P1 and P3. A cloning site (XbaI) and the NIa‐Pro cleavage site (ESVSLQ/S) were inserted into the polyprotein open reading frame (ORF) between the P1 and P3 cistrons. The amino acid sequences of the peptide cleavage sites recognized by either P1 or NIa‐Pro are underlined, and arrowheads indicate the location of the cleaved peptide bond. (b) Schematic representation of HC‐Pro processing from the reconstructed SMV genome. HC‐Pro, which is inserted into pSMVdHC utilizing the XbaI site, is processed by P1 in cis and by NIa‐Pro in an in cis manner, and is predicted to have two additional amino acids (SR) at its N‐terminus and eight amino acids (SRESVSLQ) at its C‐terminus.