A System to Explore Tomato Spotted Wilt Virus Gene Function

Tomato spotted wilt virus (TSWV) has one negative sense (L) and two ambisense (M and S) RNAs. The lack of a tractable TSWV reverse genetics system has impeded direct demonstration of gene function and prevented the usual types of pathogen characterization. The TSWV NSm (non-structural) protein has been presumed to be the viral movement protein (MP) based on a few lines of indirect evidence. We determined that a Florida field isolate of TSWV could complement local, and to a lesser extent, systemic movement of a movement-defective Tobacco mosaic virus (TMV) expression vector in Nicotiana benthamiana. To determine if NSm was responsible for this complementation, we cloned the NSm open reading frame (ORF) from this isolate and constructed TMV hybrids expressing the NSm ORF to test whether this tubule forming, putative MP could functionally substitute for the TMV MP protein. Initially, the NSm ORF was expressed from either the MP or coat protein subgenomic (sg) promoter of TMV. Both of these TSWV-TMV hybrids replicated in tobacco suspension cells and directed expression of NSm. Both of these free-RNA hybrids moved cell-to-cell in inoculated leaves of Nicotiana tabacum and moved into upper leaves of N. benthamiana, directly demonstrating that NSm is the TSWV MP. To monitor movement in plants, the ORF for the jellyfish green fluorescent protein (GFP) was inserted behind an additional sg promoter downstream of NSm. These TSWV-TMV hybrids moved and expressed both NSm and GFP in plants. These results demonstrate the utility of a well characterized viral genetic system to dissect gene functions for a virus lacking a reverse genetics system and provide a means to explore the function of TSWV genes in tomato and other economically important crops. INTRODUCTION Tomato spotted wilt virus (TSWV) is the type member of the plant-infecting Tospovirus genus in the family Bunyaviridae, a large group of predominantly vertebrateand insect-infecting RNA viruses. Typical of the tospoviruses, TSWV has a unique genome organization and expression strategy and a distinctive particle morphology among other plant-infecting viruses (Adkins, 2000). The TSWV genome consists of three negativeor ambisense ssRNAs designated S (2.9 kb), M (4.8 kb) and L (8.9 kb), with partially complementary terminal sequences that allow the RNA to adopt a pseudocircular or panhandle conformation. The lack of a tractable TSWV reverse genetics system has impeded direct demonstration of gene function and prevented the usual types of pathogen characterization. The TSWV NSm (non-structural) protein has been presumed to be the viral movement protein (MP) based on several lines of indirect evidence. A wide range of plant species are susceptible to TSWV (Goldbach and Peters, 1994) and the virus also replicates in its insect vector, thrips (Thysanoptera: Thripidae) (Ullman et al., 1993; Wijkamp et al., 1993). Tobacco mosaic virus (TMV) is the type member of the Tobamovirus genus (Lewandowski, 2000) and its discovery in the late 1800’s marks the beginning of the science of virology (Scholthof et al., 1999). The early detection and ease of experimental manipulation of TMV has made it one of the most intensively studied of all viruses (Hull, 2002). TMV has rigid, rod-shaped particles of ~300 nm in length. The TMV genome 85 Proc. 1st IS on Tomato Diseases Eds. M.T. Momol, P. Ji and J.B. Jones Acta Hort 695, ISHS 2005 consists of one positive-sense ssRNA of 6.4 kb. Development of a full-length infectious clone of TMV yielded an extremely useful reverse genetics system (Dawson et al., 1986) and has facilitated studies of viral gene function in protoplasts and plants. Development of TMV-based vectors has enabled the transient expression of heterologous proteins (Dawson et al., 1989; Shivprasad et al., 1999). The overall objective of this research was to determine if an available virus genetic system could be used to demonstrate protein function for another more complex virus lacking such a genetic system. Specifically, we wanted to test whether the TSWV NSm protein was sufficient for cell-to-cell movement of TMV. MATERIALS AND METHODS A Florida field isolate of TSWV was collected from a tomato research plot in Bradenton, FL. The TSWV isolate was characterized by serological and molecular means and tested on a variety of indicator host plants. Following this characterization, Nicotiana benthamiana plants were co-inoculated with this TSWV isolate and a movement-deficient TMV-GFP (jellyfish green fluorescent protein) vector to assay for complementation of movement. The NSm open reading frame (ORF) from this TSWV isolate was cloned by reverse transcription-polymerase chain reaction (RT-PCR) following standard procedures (Sambrook and Russell, 2001) and sequenced. Unique restriction sites were added by a second round of PCR to facilitate insertion into the TMV constructs. Initial hybrids expressed the NSm ORF from either the MP or coat protein (CP) subgenomic (sg) promoter of TMV as mpNSm and cpNSm, respectively (Fig. 1). Protoplasts from a tobacco suspension cell line were inoculated with in vitro RNA transcripts of the TMV hybrids as previously described (Lewandowski and Dawson, 2000). To determine if the TMV hybrids replicated in tobacco protoplasts, total RNA was extracted at ~22 h postinoculation (pi) and analyzed by northern blot hybridization as previously described (Lewandowski and Dawson, 1998). Positive-strand RNA was detected with a digoxigenin-labeled riboprobe complementary to the TMV 3’-non-translated region. NSm expression and tubule formation were visualized using indirectimmunofluorescence. Briefly, tobacco protoplasts transfected with the TMV-TSWV hybrids or TMV, or mock inoculated with water were fixed at 22-24 hpi on Poly-L-lysine coated slides with 95% ethanol, probed sequentially with anti-NSm rabbit polyclonal antiserum and goat anti-rabbit FITC conjugate as previously described (Van Lent et al., 1991). Slides of fixed protoplasts were observed on a fluorescence microscope equipped with filters for visualizing FITC and photographed. The GFP ORF was expressed from derivatives of cpNSm and mpNSm using a second CP sg promoter inserted downstream of the NSm ORF to visualize cell-to-cell movement. Expression of GFP and movement within inoculated leaves was visualized by illumination with a long-wave ultraviolet (UV) lamp. RESULTS AND DISCUSSION The Florida field isolate of TSWV complemented local, and to a lesser extent, systemic movement of the movement-defective TMV-GFP expression vector in N. benthamiana (data not shown). To determine if NSm was sufficient for complementation of movement, the NSm ORF was cloned into TMV constructs lacking the MP and CP ORFs. NSm was expressed from either the TMV MP or CP sg promoter as mpNSm or cpNSm (Fig. 1). Both of these free-RNA hybrids replicated in tobacco suspension cells and produced sg RNAs of the predicted sizes (data not shown), indicating that the mpNSm and cpNSm hybrids were viable. To determine whether NSm could functionally substitute for the 30-kDa TMV MP, in vitro transcripts of mpNSm and cpNSm were used to inoculate expanded leaves of N. tabacum ‘Xanthi nc,’ a local lesion host for TMV, and N. benthamiana, a susceptible host for TMV. Both hybrids formed necrotic local lesions in inoculated leaves of N. tabacum ‘Xanthi nc,’ evidence of cell-to-cell movement (Fig. 2A), demonstrating that NSm is the