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.

Exposure to watermelon bud necrosis virus and groundnut bud necrosis virus alters the life history traits of their vector, Thrips palmi (Thysanoptera: Thripidae).

Thrips palmi transmits the tospoviruses watermelon bud necrosis (WBNV) and groundnut bud necrosis virus (GBNV) in persistent propagative way. Little is known about the T. palmi-WBNV and -GBNV relationship. In this study, we report the effects of WBNV and GBNV infection on the life history traits of T. palmi. Both WBNV and GBNV had some negative effects on the adult life span, fecundity and survival of T. palmi as compared to non-exposed T. palmi. Tospovirus exposure favoured a female-biased ratio in the experimental population.

Molecular interactions between tospoviruses and thrips vectors.

Thrips-transmitted tospoviruses are an emerging and re-emerging threat to crop production worldwide. Tospoviruses are transstadially transmitted from larval to pupal stages of development, with adults serving as the primary inoculators of plants. A unique feature of the transmission cycle is that adults-while they can acquire virus from plants directly-are competent as vectors only if they acquire virus as larvae. Thrips vectors also serve as hosts for the virus, supporting its replication in midgut tissues and salivary glands. There is a tight link between thrips development and virus dissemination in the insect, and recent transcriptome studies point to stage-specific responses that coincide with localization of the virus in the insect body. Transcriptome sequencing of thrips vectors is leading to identification of virus-responsive thrips genes and possibly new targets to disrupt the virus transmission cycle. Accumulation of thrips-omics resources and advancements in functional biology tools will propel new and exciting molecular studies of thrips-tospoviruses interactions.

Non-Structural Protein NSm of Tomato Spotted Wilt Virus Is an Avirulence Factor Recognized by Resistance Genes of Tobacco and Tomato via Different Elicitor Active Sites.

Tomato spotted wilt virus (TSWV) is one of the most destructive viral pathogens of plants. Recently, a single dominant gene conferring complete resistance to TSWV (RTSW) was identified in Nicotina alata and introgressed into cultivated tobacco (N. tabacum). However, whether the TSWV carries an avirulence (Avr) factor directed against RTSW remains obscure. In the present study, we identified the non-structural protein (NSm), the movement protein of TSWV, which is an RTSW-specific Avr factor, by using two different transient expression systems. Using amino acid (aa) substitution mutants, we demonstrated the ability to induce RTSW-mediated hypersensitive response (HR) of NSm is independent of its movement function. Moreover, key substitutions (C118Y and T120N), a 21-aa viral effector epitope, and different truncated versions of NSm, which are responsible for the recognition of the Sw-5b resistance gene of tomato, were tested for their ability to trigger HR to TSWV in tobacco. Together, our results demonstrated that RTSW-mediated resistance is triggered by NSm in the same way as by Sw-5b, however, via different elicitor active sites. Finally, an Avr gene-based diagnostic approach was established and used to determine the presence and effectiveness of resistance genes in tobacco.

Towards area wide management of insect vectored viruses of tomatoes in the Bowen district.

The Bowen region of Northern Queensland is an important winter production area for tomatoes in Australia. There are three economically important viruses in the region that affect tomato, Tomato yellow leaf curl virus (TYLCV), Tomato spotted wilt virus (TSWV) and Potato leafroll virus (PLRV), which are vectored by whiteflies, thrips and aphids, respectively. An area wide management approach is required to lower the primary inoculum throughout the district. To this end, we undertook investigations into the virus incidence and alternative hosts for the virus and vectors in different cropping regions throughout the district, as well as local management options such as insecticide application and possible non-host cover crops for the wet-season break in production. The initial incidence of Potato leafroll virus was very high, most probably due to abnormal weather patterns for the district, and has ceased to be a problem. Tomato yellow leaf curl virus is a continual problem even at the beginning of the season, indicating large reservoir host(s) in the environment. Only four alternative hosts have been identified: Stachytarpheta jamaicensis (TSWV), Solanum americanum (PLRV and TYLCV) Trianthema portulacastrum (TYLCV), and Amaranthus viridis(TLYCV). Different insecticide and application options were trialled for protection against Tomato yellow leaf curl virus, with the best possible option yielding marketable fruit more than ninety percent of a resistant hybrid. A trial of yield vs time of infection of TYLCV found that whitefly exclusion for 6 weeks post-transplant yielded an average increase of nearly three kilograms of marketable fruit per plant. A number of pulse crops have been confirmed as non-hosts of tomato yellow leaf curl for use as cover crops in the wet-season break. Most of the production has moved to dual resistant TYLCV/TSWV hybrids, though an area wide management program still needs to be established to reduce the primary inoculum throughout the district, giving growers more varietal options, especially early in the season.

Transcriptome-wide identification of host genes targeted by tomato spotted wilt virus-derived small interfering RNAs.

RNA silencing mechanism functions as a major defense against invading viruses. The caveat in the RNA silencing mechanism is that the effector small interfering RNAs (siRNAs) act on any RNA transcripts with sequence complementarity irrespective of target's origin. A subset of highly expressed viral small interfering RNAs (vsiRNAs) derived from the tomato spotted wilt virus (TSWV; Tospovirus: Bunyaviridae) genome was analyzed for their propensity to downregulate the tomato transcriptome. A total of 11898 putative target sites on tomato transcripts were found to exhibit a propensity for down regulation by TSWV-derived vsiRNAs. In total, 2450 unique vsiRNAs were found to have potential cross-reacting capability with the tomato transcriptome. VsiRNAs were found to potentially target a gamut of host genes involved in basal cellular activities including enzymes, transcription factors, membrane transporters, and cytoskeletal proteins. KEGG pathway annotation of targets revealed that the vsiRNAs were mapped to secondary metabolite biosynthesis, amino acids, starch and sucrose metabolism, and carbon and purine metabolism. Transcripts for protein processing, hormone signalling, and plant-pathogen interactions were the most likely targets from the genetic, environmental information processing, and organismal systems, respectively. qRT-PCR validation of target gene expression showed that none of the selected transcripts from tomato cv. Marglobe showed up regulation, and all were down regulated even upto 20 folds (high affinity glucose transporter). However, the expression levels of transcripts from cv. Red Defender revealed differential regulation as three among the target transcripts showed up regulation (Cc-nbs-lrr, resistance protein, AP2-like ethylene-responsive transcription factor, and heat stress transcription factor A3). Accumulation of tomato target mRNAs of corresponding length was proved in both tomato cultivars using 5' RACE analysis. The TSWV-tomato interaction at the sRNA interface points to the ability of tomato cultivars to overcome vsiRNA-mediated targeting of NBS-LRR class R genes. These results suggest the prevalence of vsiRNA-induced RNA silencing of host transcriptome, and the interactome scenario is the first report on the interaction between tospovirus genome-derived siRNAs and tomato transcripts, and provide a deeper understanding of the role of vsiRNAs in pathogenicity and in perturbing host machinery.

Three decades of managing Tomato spotted wilt virus in peanut in southeastern United States.

Southeastern states namely Georgia, Florida, and Alabama produce two-thirds of the peanuts in the United States. Thrips-transmitted Tomato spotted wilt virus (TSWV), which causes spotted wilt disease, has been a major impediment to peanut production for the past three decades. The cultivars grown in the 1980s were extremely susceptible to TSWV. Early yield losses extended to tens of millions of dollars each year (up to 100% loss in many fields). This situation led to the creation of an interdisciplinary team known as "SWAT: Spotted Wilt Action Team". Initial efforts focused on risk mitigation using a combination of chemical and cultural management practices along with a strong investment in breeding programs. Beginning in the mid 1990s, cultivars with field resistance were developed and integrated with cultural and chemical management options. A Risk Mitigation Index (Peanut Rx) was made available to growers to assess risks, and provide options for mitigating risks such as planting field resistant cultivars with in-furrow insecticides, planting after peak thrips incidence, planting in twin rows, and increasing seeding rates. These efforts helped curtail losses due to spotted wilt. The Peanut Rx continues to be refined every year based on new research findings. Breeding efforts, predominantly in Georgia and Florida, continue to develop cultivars with incremental field resistance. The present-day cultivars (third-generation TSWV-resistant cultivars released after 2010) possess substantially greater field resistance than second-generation (cultivars released from 2000 to 2010) and first-generation (cultivars released from 1994 to 2000) TSWV resistant cultivars. Despite increased field resistance, these cultivars are not immune to TSWV and succumb under high thrips and TSWV pressure. Therefore, field resistant cultivars cannot serve as a 'stand-alone' option and have to be integrated with other management options. The mechanism of resistance is also unknown in field resistant cultivars. Recent research in our laboratory evaluated field resistant cultivars against thrips and TSWV. Results revealed that some resistant cultivars suppressed thrips feeding and development, and they accumulated fewer viral copies than susceptible cultivars. Transcriptomes developed with the aid of Next Generation Sequencing revealed differential gene expression patterns following TSWV infection in susceptible than field resistant cultivars. Results revealed that the upregulation of transcripts pertaining to constitutive and induced plant defense proteins in TSWV resistant cultivars was more robust over susceptible cultivars. On the flipside, the long-term effects of using such resistant cultivars on TSWV were assessed by virus population genetics studies. Initial results suggest lack of positive selection pressure on TSWV, and that the sustainable use of resistant cultivars is not threatened. Follow up research is being conducted. Improvements in TSWV management have enhanced sustainability and contributed to increased yields from <2800kg/ha before 1995 to ∼5000kg/ha in 2015.

Development of apple latent spherical virus-based vaccines against three tospoviruses.

Apple latent spherical virus (ALSV) is characterized by its relatively broad host range, latency in most host plants, and ability to induce gene silencing in host plants. Herein, we focus on the above characteristic of ALSV and describe our development of ALSV vector vaccines against three tospoviruses, namely, Impatiens necrotic spot virus (INSV), Iris yellow spot virus (IYSV), and Tomato spotted wilt virus (TSWV). DNA fragments of 201 nt of three tospovirus S-RNAs (silencing suppressor (NSS) and nucleocapsid protein (N) coding regions for each tospovirus) were inserted into an ALSV-RNA2 vector to obtain six types of ALSV vector vaccines. Nicotiana benthamiana plants at the five-leaf stage were inoculated with each ALSV vector vaccine and challenged with the corresponding tospovirus species. Tospovirus-induced symptoms and tospovirus replication after challenge were significantly suppressed in plants preinoculated with all ALSV vector vaccines having the N region fragment, indicating that strong resistance was acquired after infection with ALSV vector vaccines. On the other hand, cross protection was not significant in plants preinoculated with ALSV vectors having the NSs region fragment. Similarly, inoculation with an ALSV-RNA1 vector having the N region fragment in the 3'-noncoding region, but not the NSs region fragment, induced cross protection, indicating that cross protection is via RNA silencing, not via the function of the protein derived from the N region fragment. Our approach, wherein ALSV vectors and selected target inserts are used, enables rapid establishment of ALSV vector vaccines against many pathogenic RNA viruses with known sequences.

Proteomic analysis of Frankliniella occidentalis and differentially expressed proteins in response to tomato spotted wilt virus infection.

Tomato spotted wilt virus (TSWV) is transmitted by Frankliniella occidentalis in a persistent propagative manner. Despite the extensive replication of TSWV in midgut and salivary glands, there is little to no pathogenic effect on F. occidentalis. We hypothesize that the first-instar larva (L1) of F. occidentalis mounts a response to TSWV that protects it from pathogenic effects caused by virus infection and replication in various insect tissues. A partial thrips transcriptome was generated using 454-Titanium sequencing of cDNA generated from F. occidentalis exposed to TSWV. Using these sequences, the L1 thrips proteome that resolved on a two-dimensional gel was characterized. Forty-seven percent of the resolved protein spots were identified using the thrips transcriptome. Real-time quantitative reverse transcriptase PCR (RT-PCR) analysis of virus titer in L1 thrips revealed a significant increase in the normalized abundance of TSWV nucleocapsid RNA from 2 to 21 h after a 3-h acquisition access period on virus-infected plant tissue, indicative of infection and accumulation of virus. We compared the proteomes of infected and noninfected L1s to identify proteins that display differential abundances in response to virus. Using four biological replicates, 26 spots containing 37 proteins were significantly altered in response to TSWV. Gene ontology assignments for 32 of these proteins revealed biological roles associated with the infection cycle of other plant- and animal-infecting viruses and antiviral defense responses. Our findings support the hypothesis that L1 thrips display a complex reaction to TSWV infection and provide new insights toward unraveling the molecular basis of this interaction.

Biological and molecular analyses of the acibenzolar-S-methyl-induced systemic acquired resistance in flue-cured tobacco against Tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV) is an economically important virus of flue-cured tobacco. Activation of systemic acquired resistance (SAR) by acibenzolar-S-methyl (ASM) in flue-cured tobacco was studied under greenhouse conditions by challenge inoculation with a severe isolate of TSWV. ASM restricted virus replication and movement, and as a result reduced systemic infection. Activation of resistance was observed within 2 days after treatment with ASM and a high level of resistance was observed at 5 days onward. Expression of the pathogenesis-related (PR) protein gene, PR-3, and different classes of PR proteins such as PR-1, PR-3, and PR-5 were detected at 2 days post-ASM treatment which inversely correlated with the reduction in the number of local lesions caused by TSWV. Tobacco plants treated with increased quantities of ASM (0.25, 0.5, 1.0, 2.0, and 4.0 g a.i./7,000 plants) showed increased levels of SAR as indicated by the reduction of both local and systemic infections by TSWV. The highest level of resistance was at 4 g a.i., but this rate of ASM also caused phytotoxicity resulting in temporary foliar spotting and stunting of plants. An inverse correlation between the TSWV reduction and phytotoxicity was observed with the increase of ASM concentration. ASM at the rate of 1 to 2 g a.i./7,000 plants activated a high level of resistance and minimized the phytotoxicity. Use of gibberellic acid in combination with ASM reduced the stunting caused by ASM. Present findings together with previous field experiments demonstrate that ASM is a potential option for management of TSWV in flue-cured tobacco.

The emerging problem of tospovirus infection and nonconventional methods of control.

The emergence of tospoviruses as a significant problem in the cultivation of many crops around the world makes it vital to develop strategies to restrain these viruses. So far, only a few natural resistance genes suitable for introduction into plant breeding programs have been identified, prompting the exploitation of alternative ways of introducing virus resistance into crop plants, such as genetic modifications.