1.13.5S
IS THE ACQUISITION OF TOMATO SPOTTED WILT TOSPOVIRUS BY THRIPS A RECEPTOR MEDIATED EVENT?

JL SHERWOOD1, TL GERMAN2 and DE ULLMAN3

1Department of Plant Pathology, University of Georgia, Athens, GA 30602, USA; 2Department of Plant Pathology, University of Wisconsin, Madison, WI 53706, USA; 3Department of Entomology, University of California, Davis, CA 95616, USA

Background and objectives
All the viruses in the family Bunyaviridae are bound by a membrane that contains two glycoproteins (GP1 and GP2). Although the functions of the GPs are not well characterized for most viruses in this family, the GPs of the La Crosse bunyavirus have been shown to serve as viral attachment proteins that interact with cellular receptors to mediate entry to the insect midgut and subsequently into other insect organs. Tomato spotted wilt virus (TSWV), the type member of the genus Tospovirus , is the only genus in the family Bunyaviridae with members that infect plants. TSWV is transmitted by several species of thrips, of which the most ubiquitous is the western flower thrips (WFT), Frankliniella occidentalis (Thysanoptera: Thripidae). The ingestion of TSWV by thrips during feeding on virus-infected plants only leads to virus acquisition in larval WFT. While it has been known for sometime that TSWV replicates in the thrips vector, the mechanism by which the virus initially transcends the midgut barrier has remained an enigma.

That the GPs play an important role in virus acquisition by larval thrips is indicated by the finding that repeated mechanical transmission of TSWV results in envelope deficient isolates that are not thrips transmissible. Observations by electron microscopy show TSWV GPs bound to the brush border plasmalemma in larval WFT, but not in adult WFT [2]. Based on these observations we have suggested that TSWV fuses at the cell membrane, a mechanism of cell entry being consistent with that of other membrane bound viruses such as human immunodeficiency virus and certain baculoviruses, rather than entering via coated vesicles and uncoating via endosomes [3]. Gel overlay assays are routinely used to tentatively identify the corresponding cellular ligand that interacts with the viral attachment protein. While this approach has shown acceptable utility with viruses infecting vertebrate cells, the technique has had limited use in identifying potential cellular receptors for viruses that are transmitted to plants by insect vectors. Overlay assays have provided some evidence for a possible proteinaceous component of a cellular receptor site for TSWV [1] and current immunolabelling experiments also support the hypothesis that acquisition of TSWV by thrips is receptor mediated.

Results and conclusions
That the TSWV GPs serve as viral attachment proteins that interact with one or more cellular receptors in the thrips midgut is supported by several pieces of evidence. In gel overlay assays the TSWV GPs were found to selectively bind a 50 kDa protein in WFT, but not to proteins from the greenhouse thrips, a species that does not transmit TSWV. The intensity of the band observed in the overlay assays was found to be greater in extracts from larval thrips than in adult thrips, an observation that is consistent with the biological findings that larvae acquire the virus and adults are refractory to acquisition. Anti-idiotype antibodies prepared to the monoclonal antibodies against the viral GPs, and thus mimic the TSWV GPs, also labelled a 50 kDa protein in the thrips midgut in Western blots. Immunolabelling of the midgut membranes from WFT first instars with the anti-idiotype antibodies further supports the hypothesis that the TSWV GPs specifically interact with a component of the midgut epithelial. While receptor abundance might be critical to virus acquisition, modification of the gut environment during subsequent development from the larval stage may also influence acquisition. Work is currently under way to characterize the interaction between the TSWV GPs and the tentative viral receptor, to isolate the putative receptor, and determine the parameters that influence vector competency.

References
1. Bandla MD, Campbell LR, UlIman DE, Sherwood JL, 1998. Phytopathology 88, 98-104.
2. UlIman DE, German TL, Sherwood JL, Westcot DM, 1995. In Thrips Biology and Management. Plenum Press, New York, pp. 135-152.
3. White JM, 1990. Annual Review of Physiology 52, 675-97.