Characterization of bean necrotic mosaic virus: a member of a novel evolutionary lineage within the Genus Tospovirus.
ABSTRACT: BACKGROUND:Tospoviruses (Genus Tospovirus, Family Bunyaviridae) are phytopathogens responsible for significant worldwide crop losses. They have a tripartite negative and ambisense RNA genome segments, termed S (Small), M (Medium) and L (Large) RNA. The vector-transmission is mediated by thrips in a circulative-propagative manner. For new tospovirus species acceptance, several analyses are needed, e.g., the determination of the viral protein sequences for enlightenment of their evolutionary history. METHODOLOGY/PRINCIPAL FINDINGS:Biological (host range and symptomatology), serological, and molecular (S and M RNA sequencing and evolutionary studies) experiments were performed to characterize and differentiate a new tospovirus species, Bean necrotic mosaic virus (BeNMV), which naturally infects common beans in Brazil. Based upon the results, BeNMV can be classified as a novel species and, together with Soybean vein necrosis-associated virus (SVNaV), they represent members of a new evolutionary lineage within the genus Tospovirus. CONCLUSION/SIGNIFICANCES: Taken together, these evidences suggest that two divergent lineages of tospoviruses are circulating in the American continent and, based on the main clades diversity (American and Eurasian lineages), new tospovirus species related to the BeNMV-SVNaV clade remain to be discovered. This possible greater diversity of tospoviruses may be reflected in a higher number of crops as natural hosts, increasing the economic impact on agriculture. This idea also is supported since BeNMV and SVNaV were discovered naturally infecting atypical hosts (common bean and soybean, respectively), indicating, in this case, a preference for leguminous species. Further studies, for instance a survey focusing on crops, specifically of leguminous plants, may reveal a greater tospovirus diversity not only in the Americas (where both viruses were reported), but throughout the world.
Project description:Mulberry vein banding associated virus (MVBaV) that infects mulberry plants with typical vein banding symptoms had been identified as a tentative species of the genus Tospovirus based on the homology of N gene sequence to those of tospoviruses. In this study, the complete sequence of the tripartite RNA genome of MVBaV was determined and analyzed. The L RNA has 8905 nucleotides (nt) and encodes the putative RNA-dependent RNA polymerase (RdRp) of 2877 aa amino acids (aa) in the viral complementary (vc) strand. The RdRp of MVBaV shares the highest aa sequence identity (85.9%) with that of Watermelon silver mottle virus (WSMoV), and contains conserved motifs shared with those of the species of the genus Tospovirus. The M RNA contains 4731 nt and codes in ambisense arrangement for the NSm protein of 309 aa in the sense strand and the Gn/Gc glycoprotein precursor (GP) of 1,124 aa in the vc strand. The NSm and GP of MVBaV share the highest aa sequence identities with those of Capsicum chlorosis virus (CaCV) and Groundnut bud necrosis virus (GBNV) (83.2% and 84.3%, respectively). The S RNA is 3294 nt in length and contains two open reading frames (ORFs) in an ambisense coding strategy, encoding a 439-aa non-structural protein (NSs) and the 277-aa nucleocapsid protein (N), respectively. The NSs and N also share the highest aa sequence identity (71.1% and 74.4%, respectively) with those of CaCV. Phylogenetic analysis of the RdRp, NSm, GP, NSs, and N proteins showed that MVBaV is most closely related to CaCV and GBNV and that these proteins cluster with those of the WSMoV serogroup, and that MVBaV seems to be a species bridging the two subgroups within the WSMoV serogroup of tospoviruses in evolutionary aspect, suggesting that MVBaV represents a distinct tospovirus. Analysis of S RNA sequence uncovered the highly conserved 5'-/3'-ends and the coding regions, and the variable region of IGR with divergent patterns among MVBaV isolates.
Project description:A tospovirus causing necrotic streaks on leaves was isolated from Alstroemeria sp. in Colombia. Infected samples reacted positively with tomato spotted wilt virus (TSWV) antiserum during preliminary serological tests. Further analysis revealed a close serological relationship to tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV). A major part of the S-RNA segment, encompassing the nucleocapsid (N) protein gene, the 5' untranslated region and a part of the intergenic region 3' of the N gene, was cloned and sequenced. The deduced N protein sequence showed highest amino acid identity (82%) to that of TCSV, indicating that the virus represents a new tospovirus species, for which the name Alstroemeria necrotic streak virus (ANSV) is coined. Phylogenetic analysis based on the N protein sequence revealed that this Alstroemeria-infecting tospovirus clustered with tospoviruses from the American continent. Frankliniella occidentalis was identified as potential vector species for ANSV.
Project description: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.
Project description:Zucchini lethal chlorosis virus (ZLCV) causes significant losses in the production of cucurbits in Brazil. This virus belongs to the genus Tospovirus (family Bunyaviridae) and seems to be exclusively transmitted by Frankliniella zucchini (Thysanoptera). Tospoviruses have a tripartite and single-stranded RNA genome classified as S (Small), M (Medium) and L (Large) RNAS. Although ZLCV was identified as a member of the genus Tospovirus in 1999, its complete genome had not been sequenced until now.We sequenced the full-length genome of two ZLCV isolates named ZLCV-SP and ZLCV-DF. The phylogenetic analysis showed that ZLCV-SP and ZLCV-DF clustered with the previously reported isolate ZLCV-BR09. Their proteins were closely related, except the non-structural protein (NSm), which was highly divergent (approximately 90 % identity). All viral proteins clustered similarly in our phylogenetic analysis, excluding that these ZLCV isolates have originated from reassortment events of different tospovirus species.Here we report for the first time the complete genome of two ZLCV isolates that were found in the field infecting zucchini and cucumber.
Project description:BACKGROUND: Emerging tospoviruses cause significant yield losses and quality reduction in vegetables, ornamentals, and legumes throughout the world. So far, eight tospoviruses were reported in China. Tomato fruits displaying necrotic and concentric ringspot symptoms were found in Guizhou province of southwest China. FINDING: ELISA experiments showed that crude saps of the diseased tomato fruit samples reacted with antiserum against Tomato zonate spot virus (TZSV). Electron microscopy detected presence of quasi-spherical, enveloped particles of 80-100 nm in such saps. The putative virus isolate was designated 2009-GZT. Mechanical back-inoculation showed that 2009-GZT could infect systemically some solanaceous crop and non-crop plants including Capiscum annuum, Datura stramonium, Nicotiana benthamiana, N. rustica, N. tabacum and Solanum lycopersicum. The 3012 nt full-length sequence of 2009-GZT S RNA shared 68.2% nt identity with that of Calla lily chlorotic spot virus (CCSV), the highest among all compared viruses. This RNA was predicted to encode a non-structural protein (NSs) (459 aa, 51.7 kDa) and a nucleocapsid protein (N) (278 aa, 30.3 kDa). The N protein shared 85.8% amino acid identity with that of CCSV. The NSs protein shared 82.7% amino acid identity with that of Tomato zonate spot virus(TZSV). CONCLUSION: Our results indicate that the isolate 2009-GZT is a new species of Tospovirus, which is named Tomato necrotic spot virus (TNSV). This finding suggests that a detailed survey in China is warranted to further understand the occurrence and distribution of tospoviruses.
Project description:Tomato spotted wilt virus (TSWV), belonging to the genus Tospovirus of the family Bunyaviridae, causes significant economic damage to several vegetables and ornamental plants worldwide. Similar to those of all other negative-strand RNA viruses, the nucleocapsid (N) protein plays very important roles in its viral life cycle. N proteins protect genomic RNAs by encapsidation and form a viral ribonucleoprotein complex (vRNP) with some RNA-dependent RNA polymerases. Here we show the crystal structure of the N protein from TSWV. Protomers of TSWV N proteins consist of three parts: the N arm, C arm, and core domain. Unlike N proteins of other negative-strand RNA viruses, the TSWV N protein forms an asymmetric trimeric ring. To form the trimeric ring, the N and C arms of the N protein interact with the core domains of two adjacent N proteins. By solving the crystal structures of the TSWV N protein with nucleic acids, we showed that an inner cleft of the asymmetric trimeric ring is an RNA-binding site. These characteristics are similar to those of N proteins of other viruses of the family Bunyaviridae Based on these observations, we discuss possibilities of a TSWV encapsidation model.IMPORTANCE Tospoviruses cause significant crop losses throughout the world. Particularly, TSWV has an extremely wide host range (>1,000 plant species, including dicots and monocots), and worldwide losses are estimated to be in excess of $1 billion annually. Despite such importance, no proteins of tospoviruses have been elucidated so far. Among TSWV-encoded proteins, the N protein is required for assembling the viral genomic RNA into the viral ribonucleoprotein (vRNP), which is involved in various steps of the life cycle of these viruses, such as RNA replication, virus particle formation, and cell-to-cell movement. This study revealed the structure of the N protein, with or without nucleic acids, of TSWV as the first virus of the genus Tospovirus, so it completed our view of the N proteins of the family Bunyaviridae.
Project description:BACKGROUND:Localization and interaction studies of viral proteins provide important information about their replication in their host plants. Tospoviruses (Family Bunyaviridae) are economically important viruses affecting numerous field and horticultural crops. Iris yellow spot virus (IYSV), one of the tospoviruses, has recently emerged as an important viral pathogen of Allium spp. in many parts of the world. We studied the in vivo localization and interaction patterns of the IYSV proteins in uninfected and infected Nicotiana benthamiana and identified the interacting partners. PRINCIPAL FINDINGS:Bimolecular fluorescence complementation (BiFC) analysis demonstrated homotypic and heterotypic interactions between IYSV nucleocapsid (N) and movement (NSm) proteins. These interactions were further confirmed by pull-down assays. Additionally, interacting regions of IYSV N and NSm were identified by the yeast-2-hybrid system and ?-galactosidase assay. The N protein self-association was found to be mediated through the N- and C-terminal regions making head to tail interaction. Self-interaction of IYSV NSm was shown to occur through multiple interacting regions. In yeast-2-hybrid assay, the N- and C-terminal regions of IYSV N protein interacted with an N-terminal region of IYSV NSm protein. CONCLUSION/SIGNIFICANCE:Our studies provide new insights into localization and interactions of IYSV N and NSm proteins. Molecular basis of these interactions was studied and is discussed in the context of tospovirus assembly, replication, and infection processes.
Project description:Thrips-borne tospoviruses cause severe damage to crops worldwide. In this investigation, tobacco lines transgenic for individual WLm constructs containing the conserved motifs of the L RNA-encoded RNA-dependent RNA polymerase (L) gene of Watermelon silver mottle virus (WSMoV) were generated by Agrobacterium-mediated transformation. The WLm constructs included: (i) translatable WLm in a sense orientation; (ii) untranslatable WLmt with two stop codons; (iii) untranslatable WLmts with stop codons and a frame-shift; (iv) untranslatable antisense WLmA; and (v) WLmhp with an untranslatable inverted repeat of WLm containing the tospoviral S RNA 3'-terminal consensus sequence (5'-ATTGCTCT-3') and an NcoI site as a linker to generate a double-stranded hairpin transcript. A total of 46.7-70.0% transgenic tobacco lines derived from individual constructs showed resistance to the homologous WSMoV; 35.7-100% plants of these different WSMoV-resistant lines exhibited broad-spectrum resistance against four other serologically unrelated tospoviruses Tomato spotted wilt virus, Groundnut yellow spot virus, Impatiens necrotic spot virus and Groundnut chlorotic fan-spot virus. The selected transgenic tobacco lines also exhibited broad-spectrum resistance against five additional tospoviruses from WSMoV and Iris yellow spot virus clades, but not against RNA viruses from other genera. Northern analyses indicated that the broad-spectrum resistance is mediated by RNA silencing. To validate the L conserved region resistance in vegetable crops, the constructs were also used to generate transgenic tomato lines, which also showed effective resistance against WSMoV and other tospoviruses. Thus, our approach of using the conserved motifs of tospoviral L gene as a transgene generates broad-spectrum resistance against tospoviruses at the genus level.
Project description:TAXONOMY:Iris yellow spot virus (IYSV) is in the genus Tospovirus, family Bunyaviridae, with a single-stranded, tri-segmented RNA genome with an ambisense genome organization. Members of the other genera in the family infect predominantly vertebrates and insects. GEOGRAPHICAL DISTRIBUTION:IYSV is present in most Allium-growing regions of the world. PHYSICAL PROPERTIES:Virions are pleomorphic particles of 80-120 nm in size. The particle consists of RNA, protein, glycoprotein and lipids. GENOME:IYSV shares the genomic features of other tospoviruses: a segmented RNA genome of three RNAs, referred to as large (L), medium (M) and small (S). The L RNA codes for the RNA-dependent RNA polymerase (RdRp) in negative sense. The M RNA uses an ambisense coding strategy and codes for the precursor for the GN /GC glycoprotein in the viral complementary (vc) sense and a non-structural protein (NSm) in the viral (v) sense. The S RNA also uses an ambisense coding strategy with the coat protein (N) in vc sense and a non-structural protein (NSs) in the v sense. TRANSMISSION:The virus is transmitted by Thrips tabaci Lindeman (Order: Thysanoptera; Family: Thripidae; onion thrips) and with less efficiency by Frankliniella fusca Hinds (tobacco thrips). HOST: IYSV has a relatively broad host range, including cultivated and wild onions, garlic, chives, leeks and several ornamentals. Some weeds are naturally infected by IYSV and may serve as alternative hosts for the virus. SYMPTOMS:IYSV symptoms in Allium spp. are yellow- to straw-coloured, diamond-shaped lesions on leaves and flowering scapes. Diamond-shaped lesions are particularly pronounced on scapes. As the disease progresses, the lesions coalesce, leading to lodging of the scapes. In seed crops, this could lead to a reduction in yield and quality. Early to mid-season infection in bulb crops results in reduced vigour and bulb size. CONTROL:Resistant varieties are not available, but a limited number of accessions with field tolerance have been identified. Integrated disease management tactics, including sanitation, crop rotation, thrips management, maintenance of optimal plant vigour, soil fertility, irrigation and physical separation of bulb and seed crops, can mitigate the effect of the disease. Virus code: 00.011.0.85.009 Useful link: http://www.alliumnet.com/.
Project description:Chenopodium quinoa is a natural local lesion host of numerous plant viruses, including tospoviruses (family Bunyaviridae). Groundnut chlorotic fan-spot tospovirus (GCFSV) has been shown to consistently induce local lesions on the leaves of C. quinoa 4 days post-inoculation (dpi). To reveal the whole genome of GCFSV and its interactions with C. quinoa, RNA-seq was performed to determine the transcriptome profiles of C. quinoa leaves. The high-throughput reads from infected C. quinoa leaves were used to identify the whole genome sequence of GCFSV and its single nucleotide polymorphisms. Our results indicated that GCFSV is a phylogenetically distinct tospovirus. Moreover, 27,170 coding and 29,563 non-coding sequences of C. quinoa were identified through de novo assembly, mixing reads from mock and infected samples. Several key genes involved in the modulation of hypersensitive response (HR) were identified. The expression levels of 4,893 deduced complete genes annotated using the Arabidopsis genome indicated that several HR-related orthologues of pathogenesis-related proteins, transcription factors, mitogen-activated protein kinases, and defense proteins were significantly expressed in leaves that formed local lesions. Here, we also provide new insights into the replication progression of a tospovirus and the molecular regulation of the C. quinoa response to virus infection.