Production of serogroup-specific antibodies using the plant viral vector expressed non-structural NSs protein of watermelon silver mottle virus
Yeh S-D1, Chen T-C1, Huang C-W1, Kuo Y-W1, Liu F-L1, YuanC-H1, Hsu H-T2
1Department of Plant Pathology, National Chung Hsing University, Taichung, 40227, Taiwan
2Floral and Nursery Plants Research Unit, U. S. Department of Agriculture, Agricultural Research Service, Beltsville, Maryland 20705, U. S. A.
Correspondence: sdyeh@nchu.edu.tw
Using the Zucchini yellow mosaic virus (ZYMV) vector, the S RNA encoded nonstructural (NSs) protein of Watermelon silver mottle virus (WSMoV) was successfully expressed in squash. The expressed NSs protein with a histidine tag was first isolated by the Ni2+-NTA affinity column, and further purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for production of a rabbit antiserum and mouse monoclonal antibodies (MAbs). The rabbit antiserum specifically reacted with the crude antigen in the extract of WSMoV-infected Nicotiana benthamiana, and cross reacted with that of the high temperature-recovered gloxinia isolate (HT-1) of Capsicum chlorosis virus (CaCV). It is interesting to note that the ascitic fluids, produced from 3 stabilized hybridoma lines 231E6D12, 238C2G10 or 239F1C11, strongly reacted with extracts from plant tissues infected with WSMoV, CCSV or CaCV, all belonging to the WSMoV serogroup. Various deletions of the NSs open reading frame (ORF) were expressed by the ZYMV vector for epitope mapping. The results indicated that all three MAbs target at the amino acids 89จC125 of the expressed WSMoV NSs protein. The serological analysis coupled with the sequence alignment revealed that the MAbs-targeted region of the NSs protein is highly conserved among four available sequences of WSMoV, CaCV, CCSV, and Peanut bud necrosis virus (PBNV) in the WSMoV serogroup. Therefore we conclude that the produced NSs-MAbs react with the common epitope of the NSs proteins and are useful for the detection of different tospovirus members in the WSMoV serogroup.
