Molecular characterization and incidence of new tospovirus: Soybean Vein Necrosis Virus (SVNV) in Egypt / Caracterização molecular e incidência de novo tospovírus: Vírus da Necrose da Veia da Soja (SVNV) no Egito

Abstract Field survey study was conducted season (2017). Soybeans and weeds were weekly sampled randomly. Thrips adults were identified and counted. Detection of the virus isolate and the natural incidence was determined using; Mechanical transmission, host range, DAS-ELISA, RT-PCR. The natural incidence thrips individuals was detected depending on the SVNV% in thrips individuals and weeds hosts. Ten thrips species were associated with soybean plants in the field. The most abundant species was T. tabaci, average 256.5 average no.of individuals, followed by F. occidentalis (142.5 average no. of individuals), then N. variabilis (86.6/ average no. of individuals). Fourteen thrips species occurred on 5 legumes field crops and 41 weed plant species within soybean field. The highest average number 40.6.of individuals were recorded on Ammi majus. While the lowest one 3.3 average no. of individuals were on Urtica urens. Only 21diagnostic plant species were susceptible to infection with SVNV. G. max and Vigna radiate, were the highest percentage of infection 80% followed by V. unguilata & N. benthamiana, 75%. Egyptian isolate of Soybean vein necrosis virus (SVNV) in this study showed a high degree of similarity and it is closely related to TSWV from Egypt (DQ479968) and TCSV from USA (KY820965) with nucleotide sequence identity of 78%. Four thrips species transmitted SVNV (F. fusca 4.0%, F. schultzei 4.3%, F. tritici 3.3% and N. variabilis 68.0% transmission). Both C. phaseoli and M. sjostedti can acquire the virus but unable to transmit it. The following species; T. tabaci, F. occidentalis, S. dorsallis and T. palmi cannot acquire or transmit SVNV. The incidence of SVNV in the field started by the end of July then increased gradualy from 12.7 to 71.3% by the end of the season. In conclusion, few thrips individuals invaded soybean crops are enough to transmit high rate of SVNV within the crop. Furthermore, several vector species are also abundant on weeds, which are the major sources of soybean viruses transmitted to the crops. This information might be important for control and reduce the incidence of SVNV infection.

Resumo O estudo de pesquisa de campo foi realizado na temporada (2017). A soja e as ervas daninhas foram amostradas semanalmente de forma aleatória. Tripes adultos foram identificados e contados. A detecção do vírus isolado e a incidência natural foram determinadas usando transmissão mecânica, gama de hospedeiros, DAS-ELISA, RT-PCR. A incidência natural de tripes em indivíduos foi detectada dependendo da % de SVNV em tripes e hospedeiros infestantes. Dez espécies de tripes foram associadas a plantas de soja no campo. A espécie mais abundante foi T. tabaci, com média de 256,5 número médio de indivíduos, seguida por F. occidentalis (142,5) e N. variabilis (86,6 / número médio de indivíduos). Catorze espécies de tripes ocorreram em 5 culturas de leguminosas e 41 espécies de plantas daninhas dentro de campos de soja. O maior número médio de 40,6 indivíduos foi registrado em Ammi majus. Enquanto o mais baixo, 3,3 número médio de indivíduos, foi no Urtica urens. Apenas 21 espécies de plantas diagnosticadas foram suscetíveis à infecção com SVNV. G. max e Vigna radiate foram os maiores percentuais de infecção, 80%, seguidos por V. unguilata e N. benthamiana, 75%. O isolado egípcio neste estudo mostrou um alto grau de similaridade e está intimamente relacionado ao TSWV do Egito (DQ479968) e ao TCSV dos EUA (KY820965), com identidade de sequência de nucleotídeos de 78%. Quatro espécies de tripes transmitiram SVNV (F. fusca 4,0%, F. schultzei 4,3%, F. tritici 3,3% e N. variabilis 68,0% de transmissão). Tanto C. phaseoli quanto M. sjostedti podem adquirir o vírus, mas não podem transmiti-lo. As seguintes espécies, T. tabaci, F. occidentalis, S. dorsallis e T. palmi não podem adquirir ou transmitir SVNV. A incidência de SVNV no campo, iniciada no final de julho, aumentou gradativamente de 12,7 para 71,3% no final da temporada. Em conclusão, poucos indivíduos de tripes invadiram a cultura da soja e são suficientes para transmitir alta taxa de SVNV dentro da cultura. Além disso, várias espécies de vetores também abundam em ervas daninhas, que são as principais fontes dos vírus da soja transmitidos às lavouras. Essas informações podem ser importantes para controlar e reduzir a incidência de infecção por SVNV.

The genome of a bunyavirus cannot be defined at the level of the viral particle but only at the scale of the viral population.

Bunyaviruses are enveloped negative or ambisense single-stranded RNA viruses with a genome divided into several segments. The canonical view depicts each viral particle packaging one copy of each genomic segment in one polarity named the viral strand. Several opposing observations revealed nonequal ratios of the segments, uneven number of segments per virion, and even packaging of viral complementary strands. Unfortunately, these observations result from studies often addressing other questions, on distinct viral species, and not using accurate quantitative methods. Hence, what RNA segments and strands are packaged as the genome of any bunyavirus remains largely ambiguous. We addressed this issue by first investigating the virion size distribution and RNA content in populations of the tomato spotted wilt virus (TSWV) using microscopy and tomography. These revealed heterogeneity in viral particle volume and amount of RNA content, with a surprising lack of correlation between the two. Then, the ratios of all genomic segments and strands were established using RNA sequencing and qRT-PCR. Within virions, both plus and minus strands (but no mRNA) are packaged for each of the three L, M, and S segments, in reproducible nonequimolar proportions determined by those in total cell extracts. These results show that virions differ in their genomic content but together build up a highly reproducible genetic composition of the viral population. This resembles the genome formula described for multipartite viruses, with which some species of the order Bunyavirales may share some aspects of the way of life, particularly emerging properties at a supravirion scale.

The Plant Virus Tomato Spotted Wilt Orthotospovirus Benefits Its Vector Frankliniella occidentalis by Decreasing Plant Toxic Alkaloids in Host Plant Datura stramonium.

The transmission of insect-borne viruses involves sophisticated interactions between viruses, host plants, and vectors. Chemical compounds play an important role in these interactions. Several studies reported that the plant virus tomato spotted wilt orthotospovirus (TSWV) increases host plant quality for its vector and benefits the vector thrips Frankliniella occidentalis. However, few studies have investigated the chemical ecology of thrips vectors, TSWV, and host plants. Here, we demonstrated that in TSWV-infected host plant Datura stramonium, (1) F. occidentalis were more attracted to feeding on TSWV-infected D. stramonium; (2) atropine and scopolamine, the main tropane alkaloids in D. stramonium, which are toxic to animals, were down-regulated by TSWV infection of the plant; and (3) F. occidentalis had better biological performance (prolonged adult longevity and increased fecundity, resulting in accelerated population growth) on TSWV-infected D. stramonium than on TSWV non-infected plants. These findings provide in-depth information about the physiological mechanisms responsible for the virus's benefits to its vector by virus infection of plant regulating alkaloid accumulation in the plant.

A novel tomato spotted wilt virus isolate encoding a noncanonical NSm C118F substitution associated with Sw-5 tomato gene resistance breaking.

The nonstructural protein NSm of tomato spotted wilt virus (TSWV) has been identified as the avirulence determinant of the tomato single dominant Sw-5 resistance gene. Although Sw-5 effectiveness has been shown for most TSWV isolates, the emergence of resistance-breaking (RB) isolates has been observed. It is strongly associated with two point mutations (C118Y or T120N) in the NSm viral protein. TSWV-like symptoms were observed in tomato crop cultivars (+Sw-5) in the Baja California peninsula, Mexico, and molecular methods confirmed the presence of TSWV. Sequence analysis of the NSm 118-120 motif and three-dimensional protein modelling exhibited a noncanonical C118F substitution in seven isolates, suggesting that this substitution could emulate the C118Y-related RB phenotype. Furthermore, phylogenetic and molecular analysis of the full-length genome (TSWV-MX) revealed its reassortment-related evolution and confirmed that putative RB-related features are restricted to the NSm protein. Biological and mutational NSm 118 residue assays in tomato (+Sw-5) confirmed the RB nature of TSWV-MX isolate, and the F118 residue plays a critical role in the RB phenotype. The discovery of a novel TSWV-RB Mexican isolate with the presence of C118F substitution highlights a not previously described viral adaptation in the genus Orthotospovirus, and hence, the necessity of further crop monitoring to alert the establishment of novel RB isolates in cultivated tomatoes.

The effects of the E3 ubiquitin-protein ligase UBR7 of Frankliniella occidentalis on the ability of insects to acquire and transmit TSWV.

The interactions between plant viruses and insect vectors are very complex. In recent years, RNA sequencing data have been used to elucidate critical genes of Tomato spotted wilt ortho-tospovirus (TSWV) and Frankliniella occidentalis (F. occidentalis). However, very little is known about the essential genes involved in thrips acquisition and transmission of TSWV. Based on transcriptome data of F. occidentalis infected with TSWV, we verified the complete sequence of the E3 ubiquitin-protein ligase UBR7 gene (UBR7), which is closely related to virus transmission. Additionally, we found that UBR7 belongs to the E3 ubiquitin-protein ligase family that is highly expressed in adulthood in F. occidentalis. UBR7 could interfere with virus replication and thus affect the transmission efficiency of F. occidentalis. With low URB7 expression, TSWV transmission efficiency decreased, while TSWV acquisition efficiency was unaffected. Moreover, the direct interaction between UBR7 and the nucleocapsid (N) protein of TSWV was investigated through surface plasmon resonance and GST pull-down. In conclusion, we found that UBR7 is a crucial protein for TSWV transmission by F. occidentalis, as it directly interacts with TSWV N. This study provides a new direction for developing green pesticides targeting E3 ubiquitin to control TSWV and F. occidentalis.

Interspecies/Intergroup Complementation of Orthotospovirus Replication and Movement through Reverse Genetics Systems.

Orthotospoviruses, the plant-infecting bunyaviruses, cause serious diseases in agronomic crops and pose major threats to global food security. The family of Tospoviridae contains more than 30 members that are classified into two geographic groups, American-type and Euro/Asian-type orthotospovirus. However, the genetic interaction between different species and the possibility, during mixed infections, for transcomplementation of gene functions by orthotospoviruses from different geographic groups remains underexplored. In this study, minireplicon-based reverse genetics (RG) systems have been established for Impatiens necrotic spot virus (INSV) (an American-type orthotospovirus) and for Calla lily chlorotic spot virus and Tomato zonate spot virus (CCSV and TZSV) (two representative Euro/Asian orthotospoviruses). Together with the earlier established RG system for Tomato spotted wilt virus (TSWV), a type species of the Orthotospovirus American-clade, viral replicase/movement proteins were exchanged and analyzed on interspecies transcomplementation. Whereas the homologous RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) protein supported the replication of orthotospoviruses from both geographic groups, heterologous combinations of RdRp from one group and N from the other group were unable to support the replication of viruses from both groups. Furthermore, the NSm movement protein (MP), from both geographic groups of orthotospoviruses, was able to transcomplement heterologous orthotospoviruses or a positive-strand Cucumber mosaic virus (CMV) in their movement, albeit with varying efficiency. MP from Rice stripe tenuivirus (RSV), a plant-infecting bunyavirus that is distinct from orthotospoviruses, or MP from CMV also moves orthotospoviruses. Our findings gain insights into the genetic interaction/reassortant potentials for the segmented plant orthotospoviruses. IMPORTANCE Orthotospoviruses are agriculturally important negative-strand RNA viruses and cause severe yield-losses on many crops worldwide. Whereas the emergence of new animal-infecting bunyaviruses is frequently associated with genetic reassortants, this issue remains underexposed with the plant-infecting orthotospovirus. With the development of reverse genetics systems for orthotospoviruses from different geographic regions, the interspecies/intergroup replication/movement complementation between American- and Euro/Asian-type orthotospoviruses were investigated. Genomic RNAs from American orthotospoviruses can be replicated by the RdRp and N from those of Euro/Asia-group orthotospoviruses, and vice versa. However, their genomic RNAs cannot be replicated by a heterologous combination of RdRp from one geographic group and N from another geographic group. Cell-to-cell movement of viral entity is supported by NSm from both geographic groups, with highest efficiency by NSm from viruses belonging to the same group. Our findings provide important insights into the genetic interaction and exchange ability of viral gene functions between different species of orthotospovirus.

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.

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.

A call to arms: novel strategies for thrips and tospovirus control.

Thrips and the tospoviruses they transmit are some of the most significant threats to food and ornamental crop production globally. Control of the insect and virus is challenging and new strategies are needed. Characterizing the thrips-virus interactome provides new targets for disrupting the transmission cycle. Viral and insect determinants of vector competence are being defined, including the viral attachment protein and its structure as well as thrips proteins that interact with and respond to tospovirus infection. Additional thrips control strategies such as RNA interference need further refinement and field-applicable delivery systems, but they show promise for the knockdown of essential genes for thrips survival and virus transmission. The identification of a toxin that acts to deter thrips oviposition on cotton also presents new opportunities for control of this important pest.

Matrine-Type Alkaloids with Anti-Tomato Spotted Wilt Virus Activity from the Root of Sophora tonkinensis Gagnep.

As a continuation of our research on the development of pesticide active quinolizidine alkaloids (QAs) from the family Fabaceae, the chemical constituents of the root of Sophora tonkinensis Gagnep. were systematically investigated. Seventeen new matrine-type alkaloids (1-17), including one new naturally occurring compound (17), along with 20 known ones were isolated from the EtOH extract of S. tonkinensis. Notably, compound 5 possessed an unprecedented 6/6/5/4/6/6 hexacyclic system. Their structures were confirmed via comprehensive spectroscopic data analysis (IR, UV, NMR, HRESIMS), ECD calculation, and X-ray crystallography. Biological tests indicated that compounds 1, 4, 10, 12, 13, and 30 displayed significant anti-tomato spotted wilt virus (TSWV) activities compared with the positive control ningnanmycin. Moreover, compound 12 strongly inhibited the expression of the TSWV N, NSs, and NSm genes and TSWV NSs protein in plant host. Furthermore, compounds 4, 10, 12, 20, and 22 exhibited moderate insecticidal activities against TSWV thrip vector (Frankliniella occidentalis).

Response Surface Methodology for Optimization of Multiplex-PCR Protocols for Detection of TYLCV, TSWV and Fol Molecular Markers: Analytical Performance Evaluation.

Molecular markers linked to disease resistance genes which affect economically important crops are of great interest. In the case of tomato, a major focus on resistance breeding to multiple fungal and viral pathogens such as Tomato yellow leaf curl virus (TYLCV), Tomato spotted wilt virus (TSWV) and Fusarium oxysporum f. sp. lycopersici (Fol), have led to the introgression of several resistance genes; therefore, molecular markers have become important in molecular-assisted selection (MAS) of tomato varieties resistant to those pathogens. However, assays that allow simultaneous evaluation of resistant genotypes, such as multiplex PCR, need to be optimized and evaluated to demonstrate their analytical performance, as many factors can affect them. This work aimed to generate multiplex PCR protocols for the joint detection of the molecular markers associated with pathogen resistance genes in tomato plants that are sensitive, specific and repeatable. For the optimization a central composite design of a response surface methodology (RSM-CCD) was used. For analytical performance evaluation, specificity/selectivity and sensibility (limit of detection and dynamic range) were analyzed. Two protocols were optimized: the first one with a desirability of 1.00, contained two markers (At-2 and P7-43) linked to I- and I-3-resistant genes. The second one with a desirability of 0.99, contained markers (SSR-67, SW5 and P6-25) linked to I-, Sw-5-, and Ty-3-resistant genes. For protocol 1, all the commercial hybrids (7/7) were resistant to Fol, and for protocol 2, two hybrids were resistant to Fol, one to TSWV and one to TYLCV with good analytical performance. In both protocols, the varieties considered susceptible to the pathogens, no-amplicon or susceptible amplicons, were observed. The optimized multiplex PCR protocols showed dynamic ranges from 5.97 up to 161.3 ng DNA. The limit of detection was 17.92 ng and 53.76 ng DNA for protocols 1 and 2, respectively, giving 100% positive results in the test replicates. This method allowed to develop optimized multiplex PCR protocols with few assays which translates into less time and resources, without sacrificing method performance.

Three-dimensional analysis of membrane structures associated with tomato spotted wilt virus infection.

To study viral infection, the direct structural visualization of the viral life cycle consisting of virus attachment, entry, replication, assembly and transport is essential. Although conventional electron microscopy (EM) has been extremely helpful in the investigation of virus-host cell interactions, three-dimensional (3D) EM not only provides important information at the nanometer resolution, but can also create 3D maps of large volumes, even entire virus-infected cells. Here, we determined the ultrastructural details of tomato spotted wilt virus (TSWV)-infected plant cells using focused ion beam scanning EM (FIB-SEM). The viral morphogenesis and dynamic transformation of paired parallel membranes (PPMs) were analyzed. The endoplasmic reticulum (ER) membrane network consisting of tubules and sheets was related to viral intracellular trafficking and virion storage. Abundant lipid-like bodies, clustering mitochondria, cell membrane tubules, and myelin-like bodies were likely associated with viral infection. Additionally, connecting structures between neighboring cells were found only in infected plant tissues and showed the characteristics of tubular structure. These novel connections that formed continuously in the cell wall or were wrapped by the cell membranes of neighboring cells appeared frequently in the large-scale 3D model, suggesting additional strategies for viral trafficking that were difficult to distinguish using conventional EM.

Molecular Characteristics of Subgenomic RNAs and the Cap-Dependent Translational Advantage Relative to Corresponding Genomic RNAs of Tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV) causes severe viral diseases on many economically important plants of Solanaceae. During the infection process of TSWV, a series of 3'-truncated subgenomic RNAs (sgRNAs) relative to corresponding genomic RNAs were synthesized, which were responsible for the expression of some viral proteins. However, corresponding genomic RNAs (gRNAs) seem to possess the basic elements for expression of these viral proteins. In this study, molecular characteristics of sgRNAs superior to genomic RNAs in viral protein expression were identified. The 3' ends of sgRNAs do not cover the entire intergenic region (IGR) of TSWV genomic RNAs and contain the remarkable A-rich characteristics. In addition, the 3' terminal nucleotides of sgRNAs are conserved among different TSWV isolates. Based on the eIF4E recruitment assay and subsequent northern blot, it is suggested that the TSWV sgRNA, but not gRNA, is capped in vivo; this is why sgRNA is competent for protein expression relative to gRNA. In addition, the 5' and 3' untranslated region (UTR) of sgRNA-Ns can synergistically enhance cap-dependent translation. This study further enriched the understanding of sgRNAs of ambisense RNA viruses.

Tomato spotted wilt orthotospovirus shifts sex ratio toward males in the western flower thrips, Frankliniella occidentalis, by down-regulating a FSCB-like gene.

BACKGROUND: Plant viruses can facilitate their transmission by modulating the sex ratios of their insect vectors. Previously, we found that exposure to tomato spotted wilt orthotospovirus (TSWV) in the western flower thrips, Frankliniella occidentalis, led to a male-biased sex ratio in the offspring. TSWV, a generalist pathogen with a broad host range, is transmitted primarily by F. occidentalis in a circulative-propagative manner. Here, we integrated proteomic tools with RNAi to comprehensively investigate the genetic basis underlying the shift in vector sex ratio induced by the virus. RESULTS: Proteomic analysis exhibited 104 differentially expressed proteins between F. occidentalis adult males with and without TSWV. The expression of the fiber sheath CABYR-binding-like (FSCB) protein, namely FoFSCB-like, a sperm-specific protein associated with sperm capacitation and motility, was decreased by 46%. The predicted FoFSCB-like protein includes 10 classic Pro-X-X-Pro motifs and 42 phosphorylation sites, which are key features for sperm capacitation. FoFSCB-like expression was gradually increased during the development and peaked at the pupal stage. After exposure to TSWV, FoFSCB-like expression was substantially down-regulated. Nanoparticle-mediated RNAi substantially suppressed FoFSCB-like expression and led to a significant male bias in the offspring. CONCLUSION: These combined results suggest that down-regulation of FoFSCB-like in virus-exposed thrips leads to a male-biased sex ratio in the offspring. This study not only advances our understanding of virus-vector interactions, but also identifies a potential target for the genetic management of F. occidentalis, the primary vector of TSWV, by manipulating male fertility. © 2022 Society of Chemical Industry.

Seed Transmission of Tomato Spotted Wilt Orthotospovirus in Peppers.

Tomato spotted wilt orthotospovirus (TSWV) severely damaged agricultural production in many places around the world. It is generally believed that TSWV transmits among plants via their insect vector. In this study, we provide evidence on the seed-borne transmission of TSWV in pepper (Capsicum annuum L.) plants. RT-PCR, RT-qPCR, and transmission electron microscopy data demonstrate the seed transmission ability of TSWV in peppers. Endosperm, but not the embryo, is the abundant virus-containing seed organ. TSWV can also be detected in the second generation of newly germinated seedlings from virus-containing seed germination experiments. Our data are useful for researchers, certification agencies, the seed industry, and policy makers when considering the importance of TSWV in vegetable production all over the world.

Sex-biased proteomic response to tomato spotted wilt virus infection of the salivary glands of Frankliniella occidentalis, the western flower thrips.

Successful transmission of tomato spotted wilt virus (TSWV) by Frankliniella occidentalis requires robust infection of the salivary glands (SGs) and virus delivery to plants during salivation. Feeding behavior and transmission efficiency are sexually-dimorphic traits of this thrips vector species. Proteins secreted from male and female SG tissues, and the effect of TSWV infection on the thrips SG proteome are unknown. To begin to discern thrips factors that facilitate virus infection of SGs and transmission by F. occidentalis, we used gel- and label-free quantitative and qualitative proteomics to address two hypotheses: (i) TSWV infection modifies the composition and/or abundance of SG-expressed proteins in adults; and (ii) TSWV has a differential effect on the male and female SG proteome and secreted saliva. Our study revealed a sex-biased SG proteome for F. occidentalis, and TSWV infection modulated the SG proteome in a sex-dependent manner as evident by the number, differential abundance, identities and generalized roles of the proteins. Male SGs exhibited a larger proteomic response to the virus than female SGs. Intracellular processes modulated by TSWV in males indicated perturbation of SG cytoskeletal networks and cell-cell interactions, i.e., basement membrane (BM) and extracellular matrix (ECM) proteins, and subcellular processes consistent with a metabolic slow-down under infection. Several differentially-abundant proteins in infected male SGs play critical roles in viral life cycles of other host-virus pathosystems. In females, TSWV modulated processes consistent with tissue integrity and active translational and transcriptional regulation. A core set of proteins known for their roles in plant cell-wall degradation and protein metabolism were identified in saliva of both sexes, regardless of virus infection status. Saliva proteins secreted by TSWV-infected adults indicated energy generation, consumption and protein turnover, with an enrichment of cytoskeletal/BM/ECM proteins and tricarboxylic acid cycle proteins in male and female saliva, respectively. The nonstructural TSWV protein NSs - a multifunctional viral effector protein reported to target plant defenses against TSWV and thrips - was identified in female saliva. This study represents the first description of the SG proteome and secretome of a thysanopteran and provides many candidate proteins to further unravel the complex interplay between the virus, insect vector, and plant host.

Bunyaviral N Proteins Localize at RNA Processing Bodies and Stress Granules: The Enigma of Cytoplasmic Sources of Capped RNA for Cap Snatching.

Most cytoplasmic-replicating negative-strand RNA viruses (NSVs) initiate genome transcription by cap snatching. The source of host mRNAs from which the cytoplasmic NSVs snatch capped-RNA leader sequences has remained elusive. Earlier reports have pointed towards cytoplasmic-RNA processing bodies (P body, PB), although several questions have remained unsolved. Here, the nucleocapsid (N) protein of plant- and animal-infecting members of the order Bunyavirales, in casu Tomato spotted wilt virus (TSWV), Rice stripe virus (RSV), Sin nombre virus (SNV), Crimean-Congo hemorrhagic fever virus (CCHFV) and Schmallenberg virus (SBV) have been expressed and localized in cells of their respective plant and animal hosts. All N proteins localized to PBs as well as stress granules (SGs), but extensively to docking stages of PB and SG. TSWV and RSV N proteins also co-localized with Ran GTPase-activating protein 2 (RanGAP2), a nucleo-cytoplasmic shuttling factor, in the perinuclear region, and partly in the nucleus when co-expressed with its WPP domain containing a nuclear-localization signal. Upon silencing of PB and SG components individually or concomitantly, replication levels of a TSWV minireplicon, as measured by the expression of a GFP reporter gene, ranged from a 30% reduction to a four-fold increase. Upon the silencing of RanGAP homologs in planta, replication of the TSWV minireplicon was reduced by 75%. During in vivo cap-donor competition experiments, TSWV used transcripts destined to PB and SG, but also functional transcripts engaged in translation. Altogether, the results implicate a more complex situation in which, besides PB, additional cytoplasmic sources are used during transcription/cap snatching of cytoplasmic-replicating and segmented NSVs.

Cap-snatching as a possible contributor to photosynthesis shut-off.

Cap-snatching is a mechanism applied by segmented, negative strand (-) RNA viruses (NSVs) to initiate genome transcription. So far, the cap donor source of cytoplasmic-replicating NSVs has remained elusive. Recently, studies pointed to processing body (P body, PB) as the potential source for providing capped RNAs but conclusive evidence is still lacking. To attempt identifying these sources, here the 5' non-viral leader sequences of Tomato spotted wilt virus (TSWV) N mRNAs were analysed by high-throughput sequencing (HTS) from plants subjected to normal and heat-stress conditions, and subsequently mapped on host donor transcripts. The majority of non-viral heterogenous, host-derived leader sequences ranged in size between ~10-20 nt and contained A or AG residues at the cleavage site and the presence of certain sequence motifs. Mapping the capped-leader sequences to the 5' UTR region of genes encoded by the Nicotiana tabacum genome, identified 348 donor genes and which were specifically enriched in cellular photosynthesis pathway. Nineteen of those were clearly expressed differentially at normal condition versus heat-stress conditions. Although the results did not point towards snatching of capped-RNA leader sequences from certain cytoplasmic RNA granules in particular, they indicated photosynthesis downregulation (and development of disease symptoms) partially result from cap-snatching.

Viruses of Economic Impact on Tomato Crops in Mexico: From Diagnosis to Management-A Review.

Tomato is the most economically important vegetable crop worldwide and the second most important for Mexico. However, viral diseases are among the main limiting factors that affect the productivity of this crop, causing total losses in some cases. This review provides key information and findings on the symptoms, distribution, transmission, detection, and management of diseases caused by viruses of major importance in tomato crops in Mexico. Currently, about 25 viruses belonging to nine different families have been reported infecting tomato in Mexico, but not all of them cause economically significant diseases. Viruses of economic importance include tomato brown rugose fruit virus (ToBRFV), tomato spotted wilt virus (TSWV), tomato yellow leaf curl virus (TYLCV), pepino mosaic virus (PepMV), and tomato marchitez virus (ToMarV). The topics discussed here will provide updated information about the status of these plant viruses in Mexico as well as diverse management strategies that can be implemented according to the specific circumstances of each viral pathosystem. Additionally, a list of tomato-affecting viruses not present in Mexico that are continuous threats to the crop health is included.

Design, Synthesis, Anti-Tomato Spotted Wilt Virus Activity, and Mechanism of Action of Thienopyrimidine-Containing Dithioacetal Derivatives.

Currently, there is insufficient viricide to effectively control tomato spotted wilt virus (TSWV). To address this pending issue, a series of thienopyrimidine-containing dithioacetal derivatives were prepared and tested for their anti-TSWV activities. A subsequent three-dimensional quantitative structure-activity relationship was constructed to indicate the development of optimal compound 35. The obtained compound 35 had excellent anti-TSWV curative, protective, and inactivating activities (63.0, 56.6, and 74.1%, respectively), and the EC50 values of protective and inactivating activities of compound 35 were 252.8 and 113.5 mg/L, respectively, better than those of ningnanmycin (284.8 and 144.7 mg/L) and xiangcaoliusuobingmi (624.9 and 300.0 mg/L). In addition, the anti-TSWV activity of compound 35 was associated with defense-related enzyme activities, enhanced photosynthesis, and reduced stress response, thereby enhancing disease resistance.