Antiviral RNA silencing suppression activity of Tomato spotted wilt virus NSs protein.

In addition to regulating gene expression, RNA silencing is an essential antiviral defense system in plants. Triggered by double-stranded RNA, silencing results in degradation or translational repression of target transcripts. Viruses are inducers and targets of RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressors that interfere with this process, such as the Tomato spotted wilt virus (TSWV) NSs protein. The mechanism by which NSs suppresses RNA silencing and its role in viral infection and movement remain to be determined. We cloned NSs from the Hawaii isolate of TSWV and using two independent assays show for the first time that this protein restored pathogenicity and supported the formation of local infection foci by suppressor-deficient Turnip mosaic virus and Turnip crinkle virus. Demonstrating the suppression of RNA silencing directed against heterologous viruses establishes the foundation to determine the means used by NSs to block this antiviral process.

The receptor-like kinase SlSOBIR1 is differentially modulated by virus infection but its overexpression in tobacco has no significant impact on virus accumulation.

KEY MESSAGE: The role of the tomato receptor-like kinase SlSOBIR1 in antiviral defense was investigated. SlSOBIR1 was transcriptionally modulated by unrelated viruses but its ectopic expression had no effect on virus accumulation. Leucine-rich repeat receptor-like kinases (LRR-RLK) constitute a diverse group of proteins allowing the cell to recognize and respond to the extracellular environment. In the present study we focused on a gene encoding a tomato LRR-RLK (named SlSOBIR1) involved in the host defense against fungal pathogens. Curiously, SlSOBIR1 has been previously reported to be down-regulated by Pepper yellow mosaic virus (PepYMV) infection. Here, we show that SlSOBIR1 is responsive to wounding and differentially modulated by unrelated virus infection, i.e., down-regulated by PepYMV and up-regulated by Tomato chlorotic spot virus (TCSV). Despite these divergent expression profiles, SlSOBIR1 overexpression in transgenic tobacco plants had no evident effect on TCSV and PepYMV accumulation. On the other hand, overexpression of SlSOBIR1 significantly increased the expression of selected defense genes (PR-1a and PR-6) and exacerbated superoxide production in wounded leaves. Our data indicate that the observed modulation of SlSOBIR1 expression is probably triggered by secondary effects of the virus infection process and suggest that SlSOBIR1 is not directly involved in antiviral signaling response.

The Tomato spotted wilt virus cell-to-cell movement protein (NSM ) triggers a hypersensitive response in Sw-5-containing resistant tomato lines and in Nicotiana benthamiana transformed with the functional Sw-5b resistance gene copy.

Although the Sw-5 gene cluster has been cloned, and Sw-5b has been identified as the functional gene copy that confers resistance to Tomato spotted wilt virus (TSWV), its avirulence (Avr) determinant has not been identified to date. Nicotiana tabacum 'SR1' plants transformed with a copy of the Sw-5b gene are immune without producing a clear visual response on challenge with TSWV, whereas it is shown here that N. benthamiana transformed with Sw-5b gives a rapid and conspicuous hypersensitive response (HR). Using these plants, from all structural and non-structural TSWV proteins tested, the TSWV cell-to-cell movement protein (NSM ) was confirmed as the Avr determinant using a Potato virus X (PVX) replicon or a non-replicative pEAQ-HT expression vector system. HR was induced in Sw-5b-transgenic N. benthamiana as well as in resistant near-isogenic tomato lines after agroinfiltration with a functional cell-to-cell movement protein (NSM ) from a resistance-inducing (RI) TSWV strain (BR-01), but not with NSM from a Sw-5 resistance-breaking (RB) strain (GRAU). This is the first biological demonstration that Sw-5-mediated resistance is triggered by the TSWV NSM cell-to-cell movement protein.

Elimination of TSWV from impatiens hawkerii Bull. and regeneration of virus-free plant

The possibility for obtaining virus free plants from Impatiens hawkerii Bull. shoots infected with Tomato spotted wilt virus (TSWV) through meristem-tip culture was examined. TSWV presence in I. hawkerii plants was detected by DAS-ELISA and RT-PCR and identification of the virus was confirmed by sequencing one of the chosen isolate (GenBank Accesion CQ132190). Meristem-tip explants (0.3-1.5 mm) from virus-infected shoots are cultured on MS media supplemented with different concentrations of the cytokinins, CPPU or TDZ (0.01-1.0 uM), respectively. Using this system, a large number of in vitro shoots could be produced from a single explant. Also, cytokinins showed a stimulatory effect on the length, fresh and dry weights of the newly formed shoots. Plant pigments content in I. hawkerii shoots increased significantly in the presence of cytokinins. Rooting of shoots was spontaneous on the same media. Rooted plantlets were transferred to soil where 97 percent successfully acclimatized. By DAS-ELISA and RT-PCR, 80 percent of the in vitro plantlets were shown to be a virus-free. Considering these, the present protocol seems to be an efficient method for in vitro generation of virus-free I. hawkerii plantlets by meristem tip cultures.

The N protein of Tomato spotted wilt virus (TSWV) is associated with the induction of programmed cell death (PCD) in Capsicum chinense plants, a hypersensitive host to TSWV infection.

In sweet pepper, the Tsw gene, originally described in Capsicum chinense, has been widely used as an efficient gene for inducing a hypersensitivity response (HR) derived Tomato spotted wilt virus (TSWV) resistance. Since previously reported studies suggested that the TSWV-S RNA mutation(s) are associated with the breakdown of Tsw mediated TSWV resistance in peppers, the TSWV genes N (structural nucleocapsid protein) and NS(S) (non-structural silencing suppressor protein) were cloned into a Potato virus X (PVX)-based expression vector, and inoculated into the TSWV-resistant C. chinense genotype, PI 159236, to identify the Tsw-HR viral elicitor. Typical HR-like chlorotic and necrotic lesions followed by leaf abscission were observed only in C. chinense plants inoculated with the PVX-N construct. Cytopathological analyses of these plants identified fragmented genomic DNA, indicative of programmed cell death (PCD), in mesophyll cell nuclei surrounding PVX-N-induced necrotic lesions. The other constructs induced only PVX-like symptoms without HR-like lesions and there were no microscopic signs of PCD. The mechanism of TSWV N-gene HR induction is apparently species specific as the N gene of a related tospovirus, Tomato chlorotic spot virus, was not a HR elicitor and did not cause PCD in infected cells.

Expression of a viral polymerase-bound host factor turns human cell lines permissive to a plant- and insect-infecting virus.

Tospoviruses are the only plant-infecting members of the Bunyaviridae family of ambisense ssRNA viruses. Tomato spotted wilt tospovirus (TSWV), the type-member, also causes mild infection on its main insect vector, Frankliniella occidentalis. Herein, we identified an F. occidentalis putative transcription factor (FoTF) that binds to the TSWV RNA-dependent RNA polymerase and to viral RNA. Using in vitro RNA synthesis assays, we show that addition of purified FoTF improves viral replication, but not transcription. Expression of FoTF deletion mutants, unable to bind the RNA-dependent RNA polymerase or viral RNA, blocks TSWV replication in F. occidentalis cells. Finally, expression of FoTF wild-type turns human cell lines permissive to TSWV replication. These data indicate that FoTF is a host factor required for TSWV replication in vitro and in vivo, provide an experimental system that could be used to compare molecular defense mechanisms in plant, insect, and human cells against the same pathogen (TSWV), and could lead to a better understanding of evolutionary processes of ambisense RNA viruses.