Investigation of CaMV-host co-evolution through synonymous codon pattern.

Cauliflower mosaic virus (CaMV) has a double-stranded DNA genome and is globally distributed. The phylogeny tree of 121 CaMV isolates was categorized into two primary groups, with Iranian isolates showing the greatest genetic variations. Nucleotide A demonstrated the highest percentage (36.95%) in the CaMV genome and the dinucleotide odds ratio analysis revealed that TC dinucleotide (1.34 ≥ 1.23) and CG dinucleotide (0.63 ≤ 0.78) are overrepresented and underrepresented, respectively. Relative synonymous codon usage (RSCU) analysis confirmed codon usage bias in CaMV and its hosts. Brassica oleracea and Brassica rapa, among the susceptible hosts of CaMV, showed a codon adaptation index (CAI) value above 0.8. Additionally, relative codon deoptimization index (RCDI) results exhibited the highest degree of deoptimization in Raphanus sativus. These findings suggest that the genes of CaMV underwent codon adaptation with its hosts. Among the CaMV open reading frames (ORFs), genes that produce reverse transcriptase and virus coat proteins showed the highest CAI value of 0.83. These genes are crucial for the creation of new virion particles. The results confirm that CaMV co-evolved with its host to ensure the optimal expression of its genes in the hosts, allowing for easy infection and effective spread. To detect the force behind codon usage bias, an effective number of codons (ENC)-plot and neutrality plot were conducted. The results indicated that natural selection is the primary factor influencing CaMV codon usage bias.

The Carboxyl Terminal Regions of P0 Protein Are Required for Systemic Infections of Poleroviruses.

P0 proteins encoded by poleroviruses Brassica yellows virus (BrYV) and Potato leafroll virus (PLRV) are viral suppressors of RNA silencing (VSR) involved in abolishing host RNA silencing to assist viral infection. However, other roles that P0 proteins play in virus infection remain unclear. Here, we found that C-terminal truncation of P0 resulted in compromised systemic infection of BrYV and PLRV. C-terminal truncation affected systemic but not local VSR activities of P0 proteins, but neither transient nor ectopic stably expressed VSR proteins could rescue the systemic infection of BrYV and PLRV mutants. Moreover, BrYV mutant failed to establish systemic infection in DCL2/4 RNAi or RDR6 RNAi plants, indicating that systemic infection might be independent of the VSR activity of P0. Partially rescued infection of BrYV mutant by the co-infected PLRV implied the functional conservation of P0 proteins within genus. However, although C-terminal truncation mutant of BrYV P0 showed weaker interaction with its movement protein (MP) when compared to wild-type P0, wild-type and mutant PLRV P0 showed similar interaction with its MP. In sum, our findings revealed the role of P0 in virus systemic infection and the requirement of P0 carboxyl terminal region for the infection.

Concatemeric Broccoli reduces mRNA stability and induces aggregates.

Fluorogenic aptamers are an alternative to established methodology for real-time imaging of RNA transport and dynamics. We developed Broccoli-aptamer concatemers ranging from 4 to 128 substrate-binding site repeats and characterized their behavior fused to an mCherry-coding mRNA in transient transfection, stable expression, and in recombinant cytomegalovirus infection. Concatemerization of substrate-binding sites increased Broccoli fluorescence up to a concatemer length of 16 copies, upon which fluorescence did not increase and mCherry signals declined. This was due to the combined effects of RNA aptamer aggregation and reduced RNA stability. Unfortunately, both cellular and cytomegalovirus genomes were unable to maintain and express high Broccoli concatemer copy numbers, possibly due to recombination events. Interestingly, negative effects of Broccoli concatemers could be partially rescued by introducing linker sequences in between Broccoli repeats warranting further studies. Finally, we show that even though substrate-bound Broccoli is easily photobleached, it can still be utilized in live-cell imaging by adapting a time-lapse imaging protocol.

Genomic analysis of the brassica pathogen turnip mosaic potyvirus reveals its spread along the former trade routes of the Silk Road.

Plant pathogens have agricultural impacts on a global scale and resolving the timing and route of their spread can aid crop protection and inform control strategies. However, the evolutionary and phylogeographic history of plant pathogens in Eurasia remains largely unknown because of the difficulties in sampling across such a large landmass. Here, we show that turnip mosaic potyvirus (TuMV), a significant pathogen of brassica crops, spread from west to east across Eurasia from about the 17th century CE. We used a Bayesian phylogenetic approach to analyze 579 whole genome sequences and up to 713 partial sequences of TuMV, including 122 previously unknown genome sequences from isolates that we collected over the past five decades. Our phylogeographic and molecular clock analyses showed that TuMV isolates of the Asian-Brassica/Raphanus (BR) and basal-BR groups and world-Brassica3 (B3) subgroup spread from the center of emergence to the rest of Eurasia in relation to the host plants grown in each country. The migration pathways of TuMV have retraced some of the major historical trade arteries in Eurasia, a network that formed the Silk Road, and the regional variation of the virus is partly characterized by different type patterns of recombinants. Our study presents a complex and detailed picture of the timescale and major transmission routes of an important plant pathogen.

Complete nucleotide sequence of a novel partitivirus from Brassica campestris L. ssp. chinensis.

In the present work, we report the discovery and complete genome sequence of a novel partitivirus identified from Brassica campestris L. ssp. chinensis, which we have named "Brassica campestris chinensis cryptic virus 1" (BCCV1). Next-generation sequencing (NGS) combined with adapter-ligation-mediated amplification allowed assembly of the full-length genome sequence of BCCV1. The genome of BCCV1 contains two dsRNA segments, dsRNA1 (1595 bp) and dsRNA2 (1591 bp), which encode a conserved RNA-dependent RNA polymerase (RdRp) and a putative capsid protein (CP), respectively. Homology searches and phylogenetic analysis of the 479-aa RdRp and 438-aa CP showed that BCCV1 is a new member of the genus Deltapartitivirus, family Partitiviridae. This is the first report of the identification of a member of the family Partitiviridae in Brassica campestris L. ssp. chinensis.

The complete genome sequence of a new mitovirus from the phytopathogenic fungus Colletotrichum higginsianum.

In this study, a novel mitovirus designed "Colletotrichum higginsianum mitovirus 1" (ChMV1) was isolated from the phytopathogenic fungus Colletotrichum higginsianum. The genome of this mitovirus is 2,893 nt in length with an A + U content of 61% and contains a large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp). A BLASTp analysis revealed that the RdRp domain of ChMV1 had 30.25% to 61.72% sequence identity to those of members of the genus Mitovirus and showed the highest degree of similarity (61.72% identity) to Botrytis cinerea mitovirus 3 (BcMV3). Phylogenetic analysis further indicated that ChMV1 is a member in the genus Mitovirus of the family Mitoviridae. To the best of our knowledge, this is the first report of a mitovirus in C. higginsianum.

A 2-kb Mycovirus Converts a Pathogenic Fungus into a Beneficial Endophyte for Brassica Protection and Yield Enhancement.

Mycoviruses are viruses that infect fungi, and hypovirulence-associated mycoviruses have the potential to control fungal diseases. However, it is unclear how mycovirus-mediated hypovirulent strains live and survive in the field, and no mycovirus has been applied for field crop protection. In this study, we found that a previously identified small DNA mycovirus (SsHADV-1) can convert its host, Sclerotinia sclerotiorum, from a typical necrotrophic pathogen to a beneficial endophytic fungus. SsHADV-1 downregulates the expression of key pathogenicity factor genes in S. sclerotiorum during infection. When growing in rapeseed, the SsHADV-1-infected strain DT-8 significantly regulates the expression of rapeseed genes involved in defense, hormone signaling, and circadian rhythm pathways. As a result, plant growth is promoted and disease resistance is enhanced. Field experiments showed that spraying DT-8 at the early flowering stage can reduce the disease severity of rapeseed stem rot by 67.6% and improve yield by 14.9%. Moreover, we discovered that SsHADV-1 could also infect other S. sclerotiorum strains on DT-8-inoculated plants and that DT-8 could be recovered from dead plants. These findings suggest that the mycoviruses may have the ability to shape the origin of endophytism. Our discoveries suggest that mycoviruses may influence the origin of endophytism and may also offer a novel strategy for disease control in which mycovirus-infected strains are used to improve crop health and release mycoviruses into the field.

An efficient virus-induced gene silencing (VIGS) system for functional genomics in Brassicas using a cabbage leaf curl virus (CaLCuV)-based vector.

MAIN CONCLUSION: CaLCuV-based VIGS effectively works in cabbage and contributes to efficient functional genomics research in Brassica crop species. Virus-induced gene silencing (VIGS), a posttranscriptional gene silencing method, is an effective technique for analysing the functions of genes in plants. However, no VIGS vectors have been available for Brassica oleracea until now. Here, tobacco rattle virus (TRV), pTYs and cabbage leaf curl virus (CaLCuV) gene-silencing vectors (PCVA/PCVB) were chosen to improve the VIGS system in cabbage using the phytoene desaturase (PDS) gene as an efficient visual indicator of VIGS. We successfully silenced the expression of PDS and observed photobleaching phenomena in cabbage in response to pTYs and CaLCuV, with the latter being more easy to operate and less expensive. The parameters potentially affecting the silencing efficiency of VIGS by CaLCuV in cabbage, including the targeting fragment strategy, inoculation method and incubation temperature, were then compared. The optimized CaLCuV-based VIGS system involves the following: an approximately 500 bp insert sequence, an Agrobacterium OD600 of 1.0, use of the vacuum osmosis method applied at the bud stage, and an incubation temperature of 22 °C. Using these parameters, we achieved a stable silencing efficiency of 65%. To further test the effectiveness of the system, we selected the Mg-chelatase H subunit (ChlH) gene in cabbage and knocked down its expression, and we observed yellow leaves, as expected. We successfully applied the CaLCuV-based VIGS system to two other representative Brassica crop species, B. rapa and B. nigra, and thus expanded the application scope of this system. Our VIGS system described here will contribute to efficient functional genomics research in Brassica crop species.

A novel formulation technology for baculoviruses protects biopesticide from degradation by ultraviolet radiation.

Biopesticides are biological pest control agents that are viewed as safer alternatives to the synthetic chemicals that dominate the global insecticide market. A major constraint on the wider adoption of biopesticides is their susceptibility to the ultraviolet (UV: 290-400 nm) radiation in sunlight, which limits their persistence and efficacy. Here, we describe a novel formulation technology for biopesticides in which the active ingredient (baculovirus) is micro-encapsulated in an ENTOSTAT wax combined with a UV absorbant (titanium dioxide, TiO2). Importantly, this capsule protects the sensitive viral DNA from degrading in sunlight, but dissolves in the alkaline insect gut to release the virus, which then infects and kills the pest. We show, using simulated sunlight, in both laboratory bioassays and trials on cabbage and tomato plants, that this can extend the efficacy of the biopesticide well beyond the few hours of existing virus formulations, potentially increasing the spray interval and/or reducing the need for high application rates. The new formulation has a shelf-life at 30 °C of at least 6 months, which is comparable to standard commercial biopesticides and has no phytotoxic effect on the host plants. Taken together, these findings suggest that the new formulation technology could reduce the costs and increase the efficacy of baculovirus biopesticides, with the potential to make them commercially competitive alternatives to synthetic chemicals.

A Brassica miRNA Regulates Plant Growth and Immunity through Distinct Modes of Action.

In plants, high disease resistance often results in a reduction of yield. Therefore, breeding crops with balanced yield and disease resistance has become a major challenge. Recently, microRNA (miRNA)-mediated R gene turnover has been shown to be a protective mechanism used by plants to prevent autoimmunity in the absence of pathogens. However, whether these miRNAs play a role in plant growth and how miRNA-mediated R gene turnover responds to pathogen infection have rarely been explored. Here, we found that a Brassica miRNA, miR1885, targets both an immune receptor gene and a development-related gene for negative regulation through distinct modes of action. MiR1885 directly silences the TIR-NBS-LRR class of R gene BraTNL1 but represses the expression of the photosynthesis-related gene BraCP24 by targeting the Trans-Acting Silencing (TAS) gene BraTIR1 for trans-acting small interfering RNAs (tasiRNAs)-mediated silencing. We found that, under natural conditions, miR1885 was kept at low levels to maintain normal development and basal immunity but peaked during the floral transition to promote flowering. Interestingly, upon Turnip mosaic virus (TuMV) infection, miR1885-dependent trans-acting silencing of BraCP24 was enhanced to speed up the floral transition, whereas miR1885-mediated R gene turnover was overcome by TuMV-induced BraTNL1 expression, reflecting precise regulation of the arms race between plants and pathogens. Collectively, our results demonstrate that a single Brassica miRNA dynamically regulates both innate immunity and plant growth and responds to viral infection, revealing that Brassica plants have developed a sophisticated mechanism in modulating the interplay between growth, immunity, and pathogen infection.

A Turnip Mosaic Virus Determinant of Systemic Necrosis in Nicotiana benthamiana and a Novel Resistance-Breaking Determinant in Chinese Cabbage Identified from Chimeric Infectious Clones.

Infectious clones of Korean turnip mosaic virus (TuMV) isolates KIH1 and HJY1 share 88.1% genomic nucleotides and 96.4% polyprotein amino acid identity, and they induce systemic necrosis or mild mosaic, respectively, in Nicotiana benthamiana. Chimeric constructs between these isolates exchanged the 5', central, and 3' domains of KIH1 (K) and HJY1 (H), where the order of the letters indicates the origin of these domains. KIH1 and chimeras KHH and KKH induced systemic necrosis, whereas HJY1 and chimeras HHK, HKK, and HKH induced mild symptoms, indicating the determinant of necrosis to be within the 5' 3.9 kb of KIH1; amino acid identities of the included P1, Helper component protease, P3, 6K1, and cylindrical inclusion N-terminal domain were 90.06, 98.91, 93.80, 100, and 100%, respectively. Expression of P1 or P3 from a potato virus X vector yielded symptom differences only between P3 of KIH1 and HJY1, implicating a role for P3 in necrosis in N. benthamiana. Chimera KKH infected Brassica rapa var. pekinensis 'Norang', which was resistant to both KIH1 and HJY1, indicating that two separate TuMV determinants are required to overcome the resistance. Ability of diverse TuMV isolates, chimeras, and recombinants to overcome resistance in breeding lines may allow identification of novel resistance genes.

Molecular characterization of a new recombinant brassica yellows virus infecting tobacco in China.

Brassica yellows virus (BrYV), prevalently distributed throughout mainland China and South Korea while triggering serious diseases in cruciferous crops, is proposed to be a new species in the genus Polerovirus within the family Luteoviridae. There are three distinct genotypes (BrYV-A, BrYV-B and BrYV-C) reported in cabbage and radish. Here, we describe a new BrYV isolate infecting tobacco plants in the field, which was named BrYV-NtabQJ. The complete genome sequence of BrYV-NtabQJ is 5741 nt in length, and 89% of the sequence shares higher sequence identities (about 90%) with different BrYV isolates. However, it possesses a quite divergent region within ORF5, which is more close to Beet western yellows virus (BWYV), Beet mild yellowing virus (BMYV) and Beet chlorosis virus (BChV). A significant recombination event was then detected among BrYV-NtabQJ, BrYV-B Beijng isolate (BrYV-BBJ) and BWYV Leonurus sibiricus isolate (BWYV-LS). It is proposed that BrYV-NtabQJ might be an interspecific recombinant between BrYV-BBJ and BWYV-LS, and the recombination might result in the successful aphid transmission of BrYV from cruciferous crops to tobacco. And it also poses new challenges for BrYV diagnosis and the vegetable production.

Brassica yellows virus' movement protein upregulates anthocyanin accumulation, leading to the development of purple leaf symptoms on Arabidopsis thaliana.

Poleroviruses are widely distributed and often of great economic importance because they cause a variety of symptoms, such as the rolling of young leaves, leaf color changes, and plant decline, in infected plants. However, the molecular mechanism behind these viral-induced symptoms is still unknown. Here, we verified the pathogenicity of the polerovirus Brassica yellows virus (BrYV) by transforming its full-length amplicon into Arabidopsis thaliana, which resulted in many abnormal phenotypes. To better understand the interactions between BrYV and its host, global transcriptome profiles of the transgenic plants were compared with that of non-transgenic Arabidopsis plants. An association between the BrYV- induced purple leaf symptoms and the activation of anthocyanin biosynthesis was noted. Using the transgenic approach, we found that movement protein of BrYV was responsible for the induction of these coloration symptoms. Collectively, our findings demonstrate the BrYV' pathogenicity and show that the BrYV-induced purple leaf symptom resulted from its movement protein stimulating anthocyanin accumulation.

The Aphid-Transmitted Turnip yellows virus Differentially Affects Volatiles Emission and Subsequent Vector Behavior in Two Brassicaceae Plants.

Aphids are important pests which cause direct damage by feeding or indirect prejudice by transmitting plant viruses. Viruses are known to induce modifications of plant cues in ways that can alter vector behavior and virus transmission. In this work, we addressed whether the modifications induced by the aphid-transmitted Turnip yellows virus (TuYV) in the model plant Arabidopsis thaliana also apply to the cultivated plant Camelina sativa, both belonging to the Brassicaceae family. In most experiments, we observed a significant increase in the relative emission of volatiles from TuYV-infected plants. Moreover, due to plant size, the global amounts of volatiles emitted by C. sativa were higher than those released by A. thaliana. In addition, the volatiles released by TuYV-infected C. sativa attracted the TuYV vector Myzus persicae more efficiently than those emitted by non-infected plants. In contrast, no such preference was observed for A. thaliana. We propose that high amounts of volatiles rather than specific metabolites are responsible for aphid attraction to infected C. sativa. This study points out that the data obtained from the model pathosystem A. thaliana/TuYV cannot be straightforwardly extrapolated to a related plant species infected with the same virus.

Identification of Plant Virus Receptor Candidates in the Stylets of Their Aphid Vectors.

Plant viruses transmitted by insects cause tremendous losses in most important crops around the world. The identification of receptors of plant viruses within their insect vectors is a key challenge to understanding the mechanisms of transmission and offers an avenue for future alternative control strategies to limit viral spread. We here report the identification of two cuticular proteins within aphid mouthparts, and we provide experimental support for the role of one of them in the transmission of a noncirculative virus. These two proteins, named Stylin-01 and Stylin-02, belong to the RR-1 cuticular protein subfamily and are highly conserved among aphid species. Using an immunolabeling approach, they were localized in the maxillary stylets of the pea aphid Acyrthosiphon pisum and the green peach aphid Myzus persicae, in the acrostyle, an organ earlier shown to harbor receptors of a noncirculative virus. A peptide motif present at the C termini of both Stylin-01 and Stylin-02 is readily accessible all over the surface of the acrostyle. Competition for in vitro binding to the acrostyle was observed between an antibody targeting this peptide and the helper component protein P2 of Cauliflower mosaic virus Furthermore, silencing the stylin-01 but not stylin-02 gene through RNA interference decreased the efficiency of Cauliflower mosaic virus transmission by Myzus persicae These results identify the first cuticular proteins ever reported within arthropod mouthparts and distinguish Stylin-01 as the best candidate receptor for the aphid transmission of noncirculative plant viruses.IMPORTANCE Most noncirculative plant viruses transmitted by insect vectors bind to their mouthparts. They are acquired and inoculated within seconds when insects hop from plant to plant. The receptors involved remain totally elusive due to a long-standing technical bottleneck in working with insect cuticle. Here we characterize the role of the two first cuticular proteins ever identified in arthropod mouthparts. A domain of these proteins is directly accessible at the surface of the cuticle of the acrostyle, an organ at the tip of aphid stylets. The acrostyle has been shown to bind a plant virus, and we consistently demonstrated that one of the identified proteins is involved in viral transmission. Our findings provide an approach to identify proteins in insect mouthparts and point at an unprecedented gene candidate for a plant virus receptor.

Efficacy of Combination Treatment with Sodium Metasilicate and Sodium Hypochlorite for Inactivation of Norovirus on Fresh Vegetables.

In recent years, fresh vegetables have frequently been associated with the foodborne transmission of enteric viruses, such as human norovirus (NoV). Therefore, several studies have focused on developing methods to inactivate foodborne viruses for preventing outbreaks of foodborne illnesses. Sodium hypochlorite (NaOCl) is commonly used as a disinfectant, but results in undesirable effects on the appearance and taste of foods and can generate toxic byproducts when it exceeds the allowable concentration. Here, we evaluated the efficacy of a range of NaOCl concentrations (50-1000 ppm) for reducing the amounts of human NoV (NoV GII.4) on lettuce (Lactuca sativa), celery (Apium graveolens L.), and white cabbage (Brassica oleracea ssp. capitata). In addition, the combination treatment of NaOCl and sodium metasilicate (SMS, 0.1-0.5%) pentahydrate was evaluated for its ability to decrease the populations of NoV GII.4 in the three food samples. An immunomagnetic separation procedure combined with reverse transcription quantitative polymerase chain reaction was used for virus detection. For lettuce, celery, and cabbage, the NoV GII.4 recovery rates were 57.3% ± 6.5%, 52.5% ± 1.7%, and 60.3% ± 3.9%, respectively, using a glycine/NaCl elution buffer (0.25 M glycine/0.14 M NaCl, pH 9.5). The reductions of NoV GII.4 were 3.17, 3.06, and 3.27 log10 genomic copies/µL for lettuce, celery, and cabbage, respectively, at 1000 ppm NaOCl, while a reduction of ∼3 log10 genomic copies/µL was obtained when the samples were treated with a combination of 100 ppm NaOCl and 0.4% SMS pentahydrate. Taken together, these results demonstrated that combined treatment with NaOCl and SMS pentahydrate was an efficient strategy to reduce the concentration of NaOCl for control of NoV GII.4 contamination in fresh vegetables.

Correlation between Changes in Microbial/Physicochemical Properties and Persistence of Human Norovirus during Cabbage Kimchi Fermentation.

Recently, cabbage kimchi has occasionally been associated with the foodborne diseases of enteric viruses such as human norovirus (HuNoV). This study aimed to evaluate the correlation between microbial/physicochemical properties and persistence of HuNoV in experimentally contaminated cabbage kimchi fermented and stored at 4°C or 10°C for 28 days. Changes in organic acid content, lactic acid bacteria (LAB), acidity, pH, and salinity were analyzed. The recovery of structurally intact HuNoV was examined for up to 28 days post-inoculation, using a NoV GII.4 monoclonal antibody-conjugated immuno-magnetic separation method combined with quantitative real-time reverse transcription polymerase chain reaction. On day 0, LAB loads were 4.70 log10 colony forming units/g and HuNoV GII.4 titers were 2.57 log10 genomic copies/µl, at both temperatures. After 28 days, intact HuNoV titers decreased to 1.58 (4°C) and 1.04 (10°C) log10 genomic copies/µl, whereas the LAB density increased. This correlated with a gradual increase in lactic acid and acetic acid at both temperatures. Our findings support a statistical correlation between changes in physicochemical properties and the recovery of structurally intact HuNoV GII.4. Moreover, we determined that the production of organic acid and low pH could affect HuNoV GII.4 titers in cabbage kimchi during fermentation. However, HuNoV GII.4 was not completely eliminated by microbial/physicochemical factors during fermentation, although HuNoV GII.4 was reduced. Based on this, we speculate that the persistence of HuNoV GII.4 may be affected by the continually changing conditions during kimchi fermentation.

Application of gamma radiation for the reduction of norovirus and the quality stability in optimally ripened cabbage kimchi.

Optimally ripened commercial cabbage kimchi is considered the main cause of enteric norovirus (NoV) outbreaks in Korea. This study investigated the effect of 1-10kGy gamma radiation on the inactivation of murine norovirus-1 (MNV-1; initial inoculum of 5-6log10PFU/ml), used as a human NoV surrogate, in kimchi. The effects of gamma radiation on the pH and acidity were also examined to address the index of quality and fermentation, respectively. Titers of MNV-1 significantly reduced (p<0.05) in kimchi subjected to increasing gamma radiation doses: MNV-1 titers in kimchi after 1, 3, 5, 7, and 10kGy were 4.82 (0.34-log10 reduction), 4.45 (0.71-log10 reduction), 4.18 (0.98-log10 reduction), 3.71 (1.45-log10 reduction), and 3.40 (1.76-log10 reduction) log10 PFU/ml, respectively. However, the values of pH (4.5-4.6) and acidity (0.6-0.7%) were not significantly different between non-irradiated and irradiated kimchi (p>0.05). The D-value (1-log reduction) for MNV-1 in kimchi, calculated using first-order kinetics, was 5.75kGy (R2=0.98, RMSE=0.10). Therefore, this study suggests that the use of ≥5.75kGy gamma radiation in the kimchi manufacturing industry could be very effective in reducing NoV contamination by >90% (1 log), without causing changes in quality and fermentation.

Insights in luteovirid structural biology guided by chemical cross-linking and high resolution mass spectrometry.

Interactions among plant pathogenic viruses in the family Luteoviridae and their plant hosts and insect vectors are governed by the topology of the viral capsid, which is the sole vehicle for long distance movement of the viral genome. Previous application of a mass spectrometry-compatible cross-linker to preparations of the luteovirid Potato leafroll virus (PLRV; Luteoviridae: Polerovirus) revealed a detailed network of interactions between viral structural proteins and enabled generation of the first cross-linking guided coat protein models. In this study, we extended application of chemical cross-linking technology to the related Turnip yellows virus (TuYV; Luteoviridae: Polerovirus). Remarkably, all cross-links found between sites in the viral coat protein found for TuYV were also found in PLRV. Guided by these data, we present two models for the TuYV coat protein trimer, the basic structural unit of luteovirid virions. Additional cross-links found between the TuYV coat protein and a site in the viral protease domain suggest a possible role for the luteovirid protease in regulating the structural biology of these viruses.

The genome sequence of Brassica campestris chrysovirus 1, a novel putative plant-infecting tripartite chrysovirus.

A putative chrysovirus recovered from Brassica campestris var. purpurea and provisionally named "Brassica campestris chrysovirus 1" (BrcCV1) was sequenced. The genome of the putative BrcCV1 consists of three double-stranded RNAs (dsRNAs) comprising 3,639 (dsRNA 1), 3,567 (dsRNA 2) and 3,337 (dsRNA 3) base pairs, respectively, each containing a single open reading frame (ORF 1-3). The putative proteins encoded by ORF 1-3 show homologies to RdRp, CP and chryso-P3 of approved or tentative chrysoviruses. In addition, the three dsRNAs of BrcCV1 contain highly conserved 5' and 3' untranslated regions (UTRs) in a way similar to known chrysoviruses. In a phylogenetic tree based on the conserved amino acid sequences of the RdRps of chrysoviruses, totiviruses and partitiviruses, the putative BrcCV1 formed a separate clade with Raphanus sativus chrysovirus 1 (RasCV1), a putative trisegmented, plant-infecting chrysovirus, in the family Chrysoviridae.