1.11.65
GENOMIC AND BIOLOGICAL PROPERTIES OF TOMATO MOSAIC VIRUS (TOMV)

N BHARATHAN1, S CHERIAN2 and V MUNIYAPPA2

1Department of Biology, Indiana University of Pennsylvania, Indiana, PA 15701, USA; 2Department of Plant Pathology, University of Agricultural Sciences, Bangalore 560065, India

Background and objectives
Tomato mosaic virus (ToMV) causing severe systemic mosaic and necrosis was isolated from tomatoes (Lycopersicon esculentum) grown in the central USA, and identified as a member of the tobamovirus group by host range, electron microscopy, serology and nucleic acid hybridization. The US strain of ToMV hybridized strongly to ToMV RNA from the Indian isolate. Tomato mosaic virus symptoms are known to be caused by several strains of tobacco mosaic virus. The virus is a predominant virus infecting several agricultural crops worldwide, as well as forest ecosystems. Typical symptoms in tomato include light and dark green mosaic leaf mottle with distortion of younger leaves. Fruits from such plants generally show reduced yields in fresh market tomatoes by 10-50% [1]. Although there is no known invertebrate vector, the virus spreads rapidly under field conditions through cultural operations. ToMV is a stable soil- and waterborne virus. Widespread transmission of ToMV is reported in red spruce in the USA through fog and clouds [2]. In this paper we report the isolation and nucleic acid properties of two isolates of ToMV and expression of the tomato mosaic viral genome in a cell-free system. Polyproteins and viral-specific proteins were analysed by Western blot analysis using polyclonal antibody to ToMV.

Results and conclusions
Like other tobamoviruses, both the isolates when mechanically inoculated to Nicotiana clevelandii plants produced typical light and dark green mosaic symptoms. However, necrotic local lesions developed in Gomphrena globosa and Nicotiana rustica. Out of 80 plant species inoculated, 18 host species were infected. ToMV produced necrotic local lesions on 11 of them; chlorotic local lesion on one host; and six of them developed systemic symptoms. ELISA readily detected the Indian strain of ToMV in the seeds from several commercially grown tomato varieties. The UV absorption spectrum of the purified virus showed maximum and minimum absorption at 261 and 250 nm. Electron microscopy of the purified preparations revealed rod-shaped particles mostly measuring 16x300 nm. SDS-PAGE of disrupted viral coat protein revealed the presence of a single polypeptide of 17,800 Da. RNA preparations from purified virus particles migrated as a single band in denaturing glyoxal gels, The approximate size of the single-stranded (ss) RNA was estimated to be 6240 bp. In contrast, double-stranded (ds) RNA analysis from infected tobacco plants revealed nucleic acids twice the size of the genomic viral RNA. The in vitro translation of ToMV RNA in the rabbit reticulocyte lysate (RRL) system (Amersham) stimulated incorporation of RRP-conjugated streptavidin into proteins 30-50-fold higher than endogenous controls. The major translation products, as determined through SDS-PAGE, ranged in size from 18,000 to 14,000 Da. The translation products were analysed by immunoprecipitation, and this confirmed the translation of the 17,800-Da coat protein. The translation products were detected due to their ability to react with streptavidin-HRP, which detected the biotinylated lysine present in the translation products. The translation products did not reveal the presence of large non-structural proteins. Protein bands representing both 17,800- and 14,400-Da proteins reacted strongly with polyclonal antibodies specific to ToMV in Western blot analysis. Dot-spot nucleic acid hybridization studies using US isolate RNA as the probe revealed strong sequence homology between the two isolates of ToMV. Work is currently being done to optimize non-radioactive detection of ToMV RNA in infected tomato seeds and leaves using the principle of enhanced chemiluminescence (ECL).

References
1. Ahoonmanesh A, Shalla TA, 1981. Plant Disease 65, 56-58.
2. Castello JD, Lakshman DK, Tavantzis SM et al., 1995. Phytopathology 85, 1409-1412.