HMG-like DSP1 is a damage signal to mediate the western flower thrips, Frankliniella occidentalis, immune responses to tomato spotted wilt virus infection.

Tomato spotted wilt virus (TSWV) causes a serious plant disease and is transmitted by specific thrips including the western flower thrips, Frankliniella occidentalis. The persistent and circulative virus transmission suggests an induction of immune defenses in the thrips. We investigated the immune responses of F. occidentalis to TSWV infection. Immunofluorescence assay demonstrated viral infection in the larval midguts at early stage and subsequent propagation to the salivary gland in adults. In the larval midgut, TSWV infection led to the release of DSP1, a damage-associated molecular pattern, from the gut epithelium into the hemolymph. DSP1 up-regulated PLA2 activity, which would lead to biosynthesis of eicosanoids that activate cellular and humoral immune responses. Phenoloxidase (PO) activity was enhanced following induction of PO and its activating protease gene expressions. Antimicrobial peptide genes and dual oxidase, which produces reactive oxygen species, were induced by the viral infection. Expression of four caspase genes increased and TUNEL assay confirmed apoptosis in the larval midgut after the virus infection. These immune responses to viral infection were significantly suppressed by the inhibition of DSP1 release. We infer that TSWV infection induces F. occidentalis immune responses, which are activated by the release of DSP1 from the infection foci within midguts.

Piperazine Derivatives Containing the α-Ketoamide Moiety Discovered as Potential Anti-Tomato Spotted Wilt Virus Agents.

A total of 35 piperazine derivatives were designed and synthesized, and their activities against tomato spotted wilt virus (TSWV) were evaluated systematically. Compounds 34 and 35 with significant anti-TSWV activity were obtained. Their EC50 values were 62.4 and 59.9 µg/mL, prominently better than the control agents ningnanmycin (113.7 µg/mL) and ribavirin (591.1 µg/mL). To explore the mechanism of the interaction between these compounds and the virus, we demonstrated by agrobacterium-mediated, molecular docking, and microscale thermophoresis (MST) experimental methods that compounds 34 and 35 could inhibit the infection of TSWV by binding with the N protein to prevent the assembly of the virus core structure ribonucleoprotein (RNP), and it also meant that the arginine at 94 of the N protein was the key site of interaction between the compounds and the TSWV N target. Therefore, this study demonstrated the potential for forming antiviral agents from piperazine derivatives containing α-ketoamide moieties.

A new NLR gene for resistance to Tomato spotted wilt virus in tomato (Solanum lycopersicum).

KEY MESSAGE: A typical NLR gene, Sl5R-1, which regulates Tomato spotted wilt virus resistance, was fine mapped to a region less than 145 kb in the tomato genome. Tomato spotted wilt is a viral disease caused by Tomato spotted wilt virus (TSWV), which is a devastating disease that affects tomato (Solanum lycopersicum) production worldwide, and the resistance provided by the Sw-5 gene has broken down in some cases. In order to identify additional genes that confer resistance to TSWV, the F2 population was mapped using susceptible (M82) and resistant (H149) tomato lines. After 3 years of mapping, the main quantitative trait locus on chromosome 05 was narrowed to a genomic region of 145 kb and was subsequently identified by the F2 population, with 1971 plants in 2020. This region encompassed 14 candidate genes, and in it was found a gene cluster consisting of three genes (Sl5R-1, Sl5R-2, and Sl5R-3) that code for NBS-LRR proteins. The qRT-PCR and virus-induced gene silencing approach results confirmed that Sl5R-1 is a functional resistance gene for TSWV. Analysis of the Sl5R-1 promoter region revealed that there is a SlTGA9 transcription factor binding site caused by a base deletion in resistant plants, and its expression level was significantly up-regulated in infected resistant plants. Analysis of salicylic acid (SA) and jasmonic acid (JA) levels and the expression of SA- and JA-regulated genes suggest that SlTGA9 interacts or positively regulates Sl5R-1 to affect the SA- and JA-signaling pathways to resist TSWV. These results demonstrate that the identified Sl5R-1 gene regulates TSWV resistance by its own promoter interacting with the transcription factor SlTGA9.

Functional analysis of the nonstructural protein NSs of tomato zonate spot virus.

Tomato zonate spot virus (TZSV), a member of the genus orthotospovirus, causes severe damage to vegetables and ornamental crops in southwest China. The NSs protein is an RNA silencing suppressor in various orthotospovirus like TZSV, but its mechanism and role in virus infection are poorly understood. Here, we observed that an NSs-GFP fusion protein was transiently expressed on the plasma membrane and Golgi bodies in Nicotiana benthamiana plants. The TZSV NSs gene was silenced and infiltrated into N. benthamiana and N. tabacum cv. K326. RT-qPCR and Indirect enzyme-linked immunosorbent assay (ID-ELISA) showed that the transcription and the protein expression of the NSs gene were inhibited by more than 90.00%, and the symptoms on silenced plants were alleviated. We also found that the expression of the Zingipain-2-like gene significantly decreased when the NSs gene was silenced, resulting in co-localization of the NSs-GFP and the Zingipain-2-like-mCherry fusion protein. The findings of this study provide new insights into the mechanism of silencing suppression by NSs, as well as its effect on systemic virus infection, and also support the theory of disease resistance breeding and control and prevention of TZSV in the field.

Characterization of the Roles of SGT1/RAR1, EDS1/NDR1, NPR1, and NRC/ADR1/NRG1 in Sw-5b-Mediated Resistance to Tomato Spotted Wilt Virus.

The tomato Sw-5b gene confers resistance to tomato spotted wilt virus (TSWV) and encodes a nucleotide-binding leucine-rich repeat (NLR) protein with an N-terminal Solanaceae-specific domain (SD). Although our understanding of how Sw-5b recognizes the viral NSm elicitor has increased significantly, the process by which Sw-5b activates downstream defense signaling remains to be elucidated. In this study, we used a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system to investigate the roles of the SGT1/RAR1, EDS1/NDR1, NPR1, and NRC/ADR1/NRG1 genes in the Sw-5b-mediated signaling pathway. We found that chaperone SGT1 was required for Sw-5b function, but co-chaperone RAR1 was not. Sw-5b-mediated immune signaling was independent of both EDS1 and NDR1. Silencing NPR1, which is a central component in SA signaling, did not result in TSWV systemic infection in Sw-5b-transgenic N. benthamiana plants. Helper NLR NRCs (NLRs required for cell death) were required for Sw-5b-mediated systemic resistance to TSWV infection. Suppression of NRC2/3/4 compromised the Sw-5b resistance. However, the helper NLRs ADR1 and NRG1 may not participate in the Sw-5b signaling pathway. Silencing ADR1, NRG1, or both genes did not affect Sw-5b-mediated resistance to TSWV. Our findings provide new insight into the requirement for conserved key components in Sw-5b-mediated signaling pathways.

Progression of Watermelon Bud Necrosis Virus Infection in Its Vector, Thrips palmi.

Thrips are important pests of agricultural, horticultural, and forest crops worldwide. In addition to direct damages caused by feeding, several thrips species can transmit diverse tospoviruses. The present understanding of thrips-tospovirus relationships is largely based on studies of tomato spotted wilt virus (TSWV) and Western flower thrips (Frankliniella occidentalis). Little is known about other predominant tospoviruses and their thrips vectors. In this study, we report the progression of watermelon bud necrosis virus (WBNV) infection in its vector, melon thrips (Thrips palmi). Virus infection was visualized in different life stages of thrips using WBNV-nucleocapsid protein antibodies detected with FITC-conjugated secondary antibodies. The anterior midgut was the first to be infected with WBNV in the first instar larvae. The midgut of T. palmi was connected to the principal salivary glands (PSG) via ligaments and the tubular salivary glands (TSG). The infection progressed to the PSG primarily through the connecting ligaments during early larval instars. The TSG may also have an ancillary role in disseminating WBNV from the midgut to PSG in older instars of T. palmi. Infection of WBNV was also spread to the Malpighian tubules, hindgut, and posterior portion of the foregut during the adult stage. Maximum virus-specific fluorescence in the anterior midgut and PSG indicated the primary sites for WBNV replication. These findings will help to better understand the thrips-tospovirus molecular relationships and identify novel potential targets for their management. To our knowledge, this is the first report of the WBNV dissemination path in its vector, T. palmi.

Crystal structure of tomato spotted wilt virus GN reveals a dimer complex formation and evolutionary link to animal-infecting viruses.

Tospoviridae is a family of enveloped RNA plant viruses that infect many field crops, inflicting a heavy global economic burden. These tripartite, single-stranded, negative-sense RNA viruses are transmitted from plant to plant by thrips as the insect vector. The medium (M) segment of the viral genome encodes two envelope glycoproteins, GN and GC, which together form the envelope spikes. GC is considered the virus fusogen, while the accompanying GN protein serves as an attachment protein that binds to a yet unknown receptor, mediating the virus acquisition by the thrips carrier. Here we present the crystal structure of glycoprotein N (GN) from the tomato spotted wilt virus (TSWV), a representative member of the Tospoviridae family. The structure suggests that GN is organized as dimers on TSWV's outer shell. Our structural data also suggest that this dimerization is required for maintaining GN structural integrity. Although the structure of the TSWV GN is different from other bunyavirus GN proteins, they all share similar domain connectivity that resembles glycoproteins from unrelated animal-infecting viruses, suggesting a common ancestor for these accompanying proteins.

A convenient in vivo cap donor delivery system to investigate the cap snatching of plant bunyaviruses.

Like their animal-infecting counterparts, plant bunyaviruses use capped RNA leaders cleaved from host cellular mRNAs to prime viral genome transcription in a process called cap-snatching, but in vivo systems to investigate the details of this process are lacking for them. Here, we report that Rice stripe tenuivirus (RSV) and Tomato spotted wilt tospovirus (TSWV) cleave capped RNA leaders from mRNAs transiently expressed by agroinfiltration, which makes it possible to artificially deliver defined cap donors to the two plant bunyaviruses with unprecedented convenience. With this system, some ideas regarding how plant bunyaviruses select and use capped RNA leaders can be tested easily. We were also able to obtain clear evidence that the capped RNA leaders selected by TSWV are generally longer than those by RSV. TSWV frequently uses the prime-and-realign mechanism in transcription primed by capped RNA leaders shorter than a certain length, like that has been demonstrated recently for RSV.

Discovery of Novel Thrips Vector Proteins That Bind to the Viral Attachment Protein of the Plant Bunyavirus Tomato Spotted Wilt Virus.

The plant-pathogenic virus tomato spotted wilt virus (TSWV) encodes a structural glycoprotein (GN) that, like with other bunyavirus/vector interactions, serves a role in viral attachment and possibly in entry into arthropod vector host cells. It is well documented that Frankliniella occidentalis is one of nine competent thrips vectors of TSWV transmission to plant hosts. However, the insect molecules that interact with viral proteins, such as GN, during infection and dissemination in thrips vector tissues are unknown. The goals of this project were to identify TSWV-interacting proteins (TIPs) that interact directly with TSWV GN and to localize the expression of these proteins in relation to virus in thrips tissues of principal importance along the route of dissemination. We report here the identification of six TIPs from first-instar larvae (L1), the most acquisition-efficient developmental stage of the thrips vector. Sequence analyses of these TIPs revealed homology to proteins associated with the infection cycle of other vector-borne viruses. Immunolocalization of the TIPs in L1 revealed robust expression in the midgut and salivary glands of F. occidentalis, the tissues most important during virus infection, replication, and plant inoculation. The TIPs and GN interactions were validated using protein-protein interaction assays. Two of the thrips proteins, endocuticle structural glycoprotein and cyclophilin, were found to be consistent interactors with GN These newly discovered thrips protein-GN interactions are important for a better understanding of the transmission mechanism of persistent propagative plant viruses by their vectors, as well as for developing new strategies of insect pest management and virus resistance in plants.IMPORTANCE Thrips-transmitted viruses cause devastating losses to numerous food crops worldwide. For negative-sense RNA viruses that infect plants, the arthropod serves as a host as well by supporting virus replication in specific tissues and organs of the vector. The goal of this work was to identify thrips proteins that bind directly to the viral attachment protein and thus may play a role in the infection cycle in the insect. Using the model plant bunyavirus tomato spotted wilt virus (TSWV), and the most efficient thrips vector, we identified and validated six TSWV-interacting proteins from Frankliniella occidentalis first-instar larvae. Two proteins, an endocuticle structural glycoprotein and cyclophilin, were able to interact directly with the TSWV attachment protein, GN, in insect cells. The TSWV GN-interacting proteins provide new targets for disrupting the viral disease cycle in the arthropod vector and could be putative determinants of vector competence.

Nondestructive Raman Spectroscopy as a Tool for Early Detection and Discrimination of the Infection of Tomato Plants by Two Economically Important Viruses.

Global population forecasts dictate a rapid adoption of multifaceted approaches to fulfill increasing food requirements, ameliorate food dietary value and security using sustainable and economically feasible agricultural processes. Plant pathogens induce up to 25% losses in vegetable crops and their early detection would contribute to limit their spread and economic impact. As an alternative to time-consuming, destructive, and expensive diagnostic procedures, such as immunological assays and nucleic acid-based techniques, Raman spectroscopy (RS) is a nondestructive rapid technique that generates a chemical fingerprinting of a sample, at low operating costs. Here, we assessed the suitability of RS combined to chemometric analysis to monitor the infection of an important vegetable crop plant, tomato, by two dangerous and peculiarly different viral pathogens, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato spotted wilt virus (TSWV). Experimentally inoculated plants were monitored over 28 days for symptom occurrence and subjected to RS analysis, alongside with measuring the virus amount by quantitative real-time PCR. RS allowed to discriminate mock inoculated (healthy) from virus-infected specimens, reaching an accuracy of >70% after only 14 days after inoculation for TYLCSV and >85% only after 8 days for TSWV, demonstrating its suitability for early detection of virus infection. Importantly, RS also highlighted spectral differences induced by the two viruses, providing specific information on the infecting agent.

Distinct Mechanism for the Formation of the Ribonucleoprotein Complex of Tomato Spotted Wilt Virus.

The Tomato spotted wilt virus (TSWV) belongs to the Tospovirus genus of the Bunyaviridae family and represents the sole plant-infecting group within bunyavirus. TSWV encodes a nucleocapsid protein (N) which encapsidates the RNA genome to form a ribonucleoprotein complex (RNP). In addition, the N has multiple roles during the infection of plant cells. Here, we report the crystal structure of the full-length TSWV N. The N features a body domain consisting of an N-lobe and a C-lobe. These lobes clamp a positively charged groove which may constitute the RNA binding site. Furthermore, the body domains are flanked by N- and C-terminal arms which mediate homotypic interactions to the neighboring subunits, resulting in a ring-shaped N trimer. Interestingly, the C terminus of one protomer forms an additional interaction with the protomer of an adjacent trimer in the crystal, which may constitute a higher-order oligomerization contact. In this way, this study provides insights into the structure and trimeric assembly of TSWV N, which help to explain previous functional findings, but also suggests distinct N interactions within a higher-order RNP.IMPORTANCE TSWV is one of the most devastating plant pathogens that cause severe diseases in numerous agronomic and ornamental crops worldwide. TSWV is also the prototypic member of the Tospovirus genus, which is the sole group of plant-infecting viruses in the bunyavirus family. This study determined the structure of full-length TSWV N in an oligomeric state. The structural observations explain previously identified biological properties of TSWV N. Most importantly, the additional homotypic interaction between the C terminus of one protomer with another protomer indicates that there is a distinct mechanism of RNP formation in the bunyavirus family, thereby enhancing the current knowledge of negative-sense single-stranded RNA virus-encoded N. TSWV N is the last remaining representative N with an unknown structure in the bunyavirus family. Combined with previous studies, the structure of TSWV N helps to build a complete picture of the bunyavirus-encoded N family and reveals a close evolutionary relationship between orthobunyavirus, phlebovirus, hantavirus, and tospovirus.

Induction of cell death by tospoviral protein NSs and the motif critical for cell death does not control RNA silencing suppression activity.

Groundnut bud necrosis virus induces necrotic symptoms in different hosts. Previous studies showed reactive oxygen species-mediated programmed cell death (PCD) resulted in necrotic symptoms. Transgenic expression of viral protein NSs mimics viral symptoms. Here, we showed a role for NSs in influencing oxidative burst in the cell, by analyzing H2O2 accumulation, activities of antioxidant enzymes and expression levels of vacuolar processing enzymes, H2O2-responsive microRNA 319a.2 plus its possible target metacaspase-8. The role of NSs in PCD, was shown using two NSs mutants: one in the Trp/GH3 motif (a homologue of pro-apototic domain) (NSsS189R) and the other in a non-Trp/GH3 motif (NSsL172R). Tobacco rattle virus (TRV) expressing NSsS189R enhanced the PCD response, but not TRV-NSsL172R, while RNA silencing suppression activity was lost in TRV-NSsL172R, but not in TRV-NSsS189R. Therefore, we propose dual roles of NSs in RNA silencing suppression and induction of cell death, controlled by different motifs.

Salicylic Acid Is Involved in the Basal Resistance of Tomato Plants to Citrus Exocortis Viroid and Tomato Spotted Wilt Virus.

Tomato plants expressing the NahG transgene, which prevents accumulation of endogenous salicylic acid (SA), were used to study the importance of the SA signalling pathway in basal defence against Citrus Exocortis Viroid (CEVd) or Tomato Spotted Wilt Virus (TSWV). The lack of SA accumulation in the CEVd- or TSWV-infected NahG tomato plants led to an early and dramatic disease phenotype, as compared to that observed in the corresponding parental Money Maker. Addition of acibenzolar-S-methyl, a benzothiadiazole (BTH), which activates the systemic acquired resistance pathway downstream of SA signalling, improves resistance of NahG tomato plants to CEVd and TSWV. CEVd and TSWV inoculation induced the accumulation of the hydroxycinnamic amides p-coumaroyltyramine, feruloyltyramine, caffeoylputrescine, and feruloylputrescine, and the defence related proteins PR1 and P23 in NahG plants earlier and with more intensity than in Money Maker plants, indicating that SA is not essential for the induction of these plant defence metabolites and proteins. In addition, NahG plants produced very high levels of ethylene upon CEVd or TSWV infection when compared with infected Money Maker plants, indicating that the absence of SA produced additional effects on other metabolic pathways. This is the first report to show that SA is an important component of basal resistance of tomato plants to both CEVd and TSWV, indicating that SA-dependent defence mechanisms play a key role in limiting the severity of symptoms in CEVd- and TSWV-infected NahG tomato plants.

Comparison of small RNA profiles in Nicotiana benthamiana and Solanum lycopersicum infected by polygonum ringspot tospovirus reveals host-specific responses to viral infection.

Viral small RNAs (vsRNAs) are one of the key elements involved in RNA silencing-based defense against viruses in plants. We analyzed the vsRNA profiles in Nicotiana benthamiana and Solanum lycopersicum infected by polygonum ringspot virus (PolRSV) (Tospovirus, Bunyaviridae). VsRNAs were abundant in both hosts, but a different size profile was observed, with an abundance peak at 21 in N. benthamiana and at 22 nt in tomato. VsRNAs mapping to the PolRSV L genomic segment were under-represented in both hosts, while S and M segments were differentially and highly targeted in N. benthamiana and tomato, respectively. Differences in preferential targeting of single ORFs were observed, with over-representation of NSs ORF-derived reads in N. benthamiana. Intergenic regions (IGRs)-mapping vsRNAs were under-represented, while enrichment of vsRNAs reads mapping to the NSs positive sense strand was observed in both hosts. Comparison with a previous study on tomato spotted wilt virus (TSWV) under the same experimental conditions, showed that the relative accumulation of PolRSV-specific and endogenous sRNAs was similar to the one observed for silencing suppressor-deficient TSWV strains, suggesting possible different properties of PolRSV NSs silencing suppressor compared to that of TSWV.

Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing.

RNA silencing is a sequence-specific gene regulation mechanism that in plants also acts antiviral. In order to counteract antiviral RNA silencing, viruses have evolved RNA silencing suppressors (RSS). In the case of tospoviruses, the non-structural NSs protein has been identified as the RSS. Although the tomato spotted wilt virus (TSWV) tospovirus NSs protein has been shown to exhibit affinity to long and small dsRNA molecules, its ability to suppress the non-cell autonomous part of RNA silencing has only been studied to a limited extent. Here, the NSs proteins of TSWV, groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), representatives for three distinct tospovirus species, have been studied on their ability and strength to suppress local and systemic silencing. A system has been developed to quantify suppression of GFP silencing in Nicotiana benthamiana 16C lines, to allow a comparison of relative RNA silencing suppressor strength. It is shown that NSs of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSsTYRV was just as strong as those by NSsTSWV and NSsGRSV, even though NSsTYRV was expressed in lower amounts. Using the system established, a set of selected NSsTSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream the biogenesis of siRNAs and their sequestration. The findings are discussed in light of the affinity of NSs for small and long dsRNA, and recent mutant screen of NSsTSWV to map domains required for RSS activity and triggering of Tsw-governed resistance.

Small RNA profiles of wild-type and silencing suppressor-deficient tomato spotted wilt virus infected Nicotiana benthamiana.

Tospoviruses are plant-infecting viruses belonging to the family Bunyaviridae. We used a collection of wild-type, phylogenetically distinct tomato spotted wilt virus isolates and related silencing-suppressor defective mutants to study the effects on the small RNA (sRNA) accumulation during infection of Nicotiana benthamiana. Our data showed that absence of a functional silencing suppressor determined a marked increase of the total amount of viral sRNAs (vsRNAs), and specifically of the 21 nt class. We observed a common under-representation of vsRNAs mapping to the intergenic region of S and M genomic segments, and preferential mapping of the reads against the viral sense open reading frames, with the exception of the NSs gene. The NSs-mutant strains showed enrichment of NSm-derived vsRNA compared to the expected amount based on gene size. Analysis of 5' terminal nucleotide preference evidenced a significant enrichment in U for the 21 nt- and in A for 24 nt-long endogenous sRNAs in all the samples. Hotspot analysis revealed a common abundant accumulation of reads at the 5' end of the L segment, mostly in the antiviral sense, for the NSs-defective isolates, suggesting that absence of the silencing suppressor can influence preferential targeting of the viral genome.

Two Novel Motifs of Watermelon Silver Mottle Virus NSs Protein Are Responsible for RNA Silencing Suppression and Pathogenicity.

The NSs protein of Watermelon silver mottle virus (WSMoV) is the RNA silencing suppressor and pathogenicity determinant. In this study, serial deletion and point-mutation mutagenesis of conserved regions (CR) of NSs protein were performed, and the silencing suppression function was analyzed through agroinfiltration in Nicotiana benthamiana plants. We found two amino acid (aa) residues, H113 and Y398, are novel functional residues for RNA silencing suppression. Our further analyses demonstrated that H113 at the common epitope (CE) ((109)KFTMHNQ(117)), which is highly conserved in Asia type tospoviruses, and the benzene ring of Y398 at the C-terminal ß-sheet motif ((397)IYFL(400)) affect NSs mRNA stability and protein stability, respectively, and are thus critical for NSs RNA silencing suppression. Additionally, protein expression of other six deleted (ΔCR1-ΔCR6) and five point-mutated (Y15A, Y27A, G180A, R181A and R212A) mutants were hampered and their silencing suppression ability was abolished. The accumulation of the mutant mRNAs and proteins, except Y398A, could be rescued or enhanced by co-infiltration with potyviral suppressor HC-Pro. When assayed with the attenuated Zucchini yellow mosaic virus vector in squash plants, the recombinants carrying individual seven point-mutated NSs proteins displayed symptoms much milder than the recombinant carrying the wild type NSs protein, suggesting that these aa residues also affect viral pathogenicity by suppressing the host silencing mechanism.

Possible involvement of eEF1A in Tomato spotted wilt virus RNA synthesis.

Tomato spotted wilt virus (TSWV) is a negative-strand RNA virus in the family Bunyaviridae and propagates in both insects and plants. Although TSWV can infect a wide range of plant species, host factors involved in viral RNA synthesis of TSWV in plants have not been characterized. In this report, we demonstrate that the cell-free extract derived from one of the host plants can activate mRNA transcriptional activity of TSWV. Based on activity-guided fractionation of the cell-free extract, we identified eukaryotic elongation factor (eEF) 1A as a possible host factor facilitating TSWV transcription and replication. The RNA synthesis-supporting activity decreased in the presence of an eEF1A inhibitor, suggesting that eEF1A plays an important role in RNA synthesis of TSWV.

The movement protein (NSm) of Tomato spotted wilt virus is the avirulence determinant in the tomato Sw-5 gene-based resistance.

The avirulence determinant triggering the resistance conferred by the tomato gene Sw-5 against Tomato spotted wilt virus (TSWV) is still unresolved. Sequence comparison showed two substitutions (C118Y and T120N) in the movement protein NSm present only in TSWV resistance-breaking (RB) isolates. In this work, transient expression of NSm of three TSWV isolates [RB1 (T120N), RB2 (C118Y) and non-resistance-breaking (NRB)] in Nicotiana benthamiana expressing Sw-5 showed a hypersensitive response (HR) only with NRB. Exchange of the movement protein of Alfalfa mosaic virus (AMV) with NSm supported cell-to-cell and systemic transport of the chimeric AMV RNAs into N. tabacum with or without Sw-5, except for the constructs with NBR when Sw-5 was expressed, although RB2 showed reduced cell-to-cell transport. Mutational analysis revealed that N120 was sufficient to avoid the HR, but the substitution V130I was required for systemic transport. Finally, co-inoculation of RB and NRB AMV chimeric constructs showed different prevalence of RB or NBR depending on the presence or absence of Sw-5. These results indicate that NSm is the avirulence determinant for Sw-5 resistance, and mutations C118Y and T120N are responsible for resistance breakdown and have a fitness penalty in the context of the heterologous AMV system.

Analysis of Tomato spotted wilt virus NSs protein indicates the importance of the N-terminal domain for avirulence and RNA silencing suppression.

Recently, Tomato spotted wilt virus (TSWV) nonstructural protein NSs has been identified unambiguously as an avirulence (Avr) determinant for Tomato spotted wilt (Tsw)-based resistance. The observation that NSs from two natural resistance-breaking isolates had lost RNA silencing suppressor (RSS) activity and Avr suggested a link between the two functions. To test this, a large set of NSs mutants was generated by alanine substitutions in NSs from resistance-inducing wild-type strains (NSs(RI) ), amino acid reversions in NSs from resistance-breaking strains (NSs(RB)), domain deletions and swapping. Testing these mutants for their ability to suppress green fluorescent protein (GFP) silencing and to trigger a Tsw-mediated hypersensitive response (HR) revealed that the two functions can be separated. Changes in the N-terminal domain were found to be detrimental for both activities and indicated the importance of this domain, additionally supported by domain swapping between NSs(RI) and NSs(RB). Swapping domains between the closely related Tospovirus Groundnut ringspot virus (GRSV) NSs and TSWV NSs(RI) showed that Avr functionality could not simply be transferred between species. Although deletion of the C-terminal domain rendered NSs completely dysfunctional, only a few single-amino-acid mutations in the C-terminus affected both functions. Mutation of a GW/WG motif (position 17/18) rendered NSs completely dysfunctional for RSS and Avr activity, and indicated a putative interaction between NSs and Argonaute 1 (AGO1), and its importance in TSWV virulence and viral counter defence against RNA interference.