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Brome mosaic virus (BMV) and cocksfoot mottle virus (CfMV) are pathogens of grass species including all economically important cereals. Both viruses have been identified in Poland therefore they create a potential risk to cereal crops. In this study, a duplex—reverse transcription—polymerase chain reaction (duplex-RT-PCR) was developed and optimized for simultaneous detection and differentiation of BMV and CfMV as well as for confirmation of their co-infection. Selected primers CfMVdiag-F/CfMVdiag-R and BMV2-F/BMV2-R amplified 390 bp and 798 bp RT-PCR products within coat protein (CP) region of CfMV and replicase gene of BMV, respectively. Duplex-RT-PCR was successfully applied for the detection of CfMV-P1 and different Polish BMV isolates. Moreover, one sample was found to be co-infected with BMV-ML1 and CfMV-ML1 isolates. The specificity of generated RT-PCR products was verified by sequencing. Duplex-RT-PCR, like conventional RT-PCR, was able to detect two viruses occurring in plant tissues in very low concentration (as low as 4.5 pg/µL of total RNA). In contrast to existing methods, newly developed technique offers a significant time and cost-saving advantage. In conclusion, duplex-RT-PCR is a useful tool which can be implemented by phytosanitary services to rapid detection and differentiation of BMV and CfMV.

Pathogens have evolved various strategies to overcome host immunity for successful infection. Maize chlorotic mottle virus (MCMV) can cause lethal necrosis in maize (Zea mays) when it coinfects with a virus in the Potyviridae family. However, the MCMV pathogenicity determinant remains largely unknown. Here we show that the P31 protein of MCMV is important for viral accumulation and essential for symptom development. Ectopic expression of P31 using foxtail mosaic virus or potato virus X induced necrosis in systemically infected maize or Nicotiana benthamiana leaves. Maize catalases (CATs) were shown to interact with P31 in yeast and in planta. P31 accumulation was elevated through its interaction with ZmCAT1. P31 attenuated the expression of salicylic acid (SA)-responsive pathogenesis-related (PR) genes by inhibiting catalase activity during MCMV infection. In addition, silencing of ZmCATs using a brome mosaic virus-based gene silencing vector facilitated MCMV RNA and coat protein accumulation. This study reveals an important role for MCMV P31 in counteracting host defence and inducing systemic chlorosis and necrosis. Our results have implications for understanding the mechanisms in defence and counter-defence during infection of plants by various pathogens.

There is an increasing interest in the use of plant viruses as vehicles for anti-cancer therapy. In particular, the plant virus brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are novel potential nanocarriers for different therapies in nanomedicine. In this work, BMV and CCMV were loaded with a fluorophore and assayed on breast tumor cells. The viruses BMV and CCMV were internalized into breast tumor cells. Both viruses, BMV and CCMV, did not show cytotoxic effects on tumor cells in vitro. However, only BMV did not activate macrophages in vitro. This suggests that BMV is less immunogenic and may be a potential carrier for therapy delivery in tumor cells. Furthermore, BMV virus-like particles (VLPs) were efficiently loaded with small interfering RNA (siRNA) without packaging signal. The gene silencing was demonstrated by VLPs loaded with siGFP and tested on breast tumor cells that constitutively express the green fluorescent protein (GPF). After VLP-siGFP treatment, GFP expression was efficiently inhibited corroborating the cargo release inside tumor cells and the gene silencing. In addition, BMV VLP carring siAkt1 inhibited the tumor growth in mice. These results show the attractive potential of plant virus VLPs to deliver molecular therapy to tumor cells with low immunogenic response.

The wheat streak mosaic virus (WSMV), vectored by the wheat curl mite, is globally distributed and threatens wheat production worldwide. Since its first occurrence in Serbia in the 1960s, WSMV presence has not been monitored. In 2019, a total of 62 samples of fi ve wheat cultivars from eight locations in Serbia were collected and tested for the presence of nine common wheat viruses: WSMV, barley yellow dwarf virus-PAV, -MAV, -SGV, and -RMV, cereal yellow dwarf virus-RPV, wheat spindle streak virus, brome mosaic virus, and soil-borne wheat mosaic virus, using individual or multiplex RT-PCR. WSMV was detected in 58.1% of the tested samples in seven wheat crops at five different locations. Species-specific primers failed to detect the presence of the other eight tested viruses. For further confirmation of WSMV, RT-PCR with the WS8166F/WS8909R primers covering the coat protein (CP) gene was carried out for both amplification and sequencing. The amplified product of the correct predicted size (750 bp) derived from four selected isolates, 98-19, 99-19, 102-19 and 120-19, was sequenced and deposited in GenBank (MT461299, MT461300, MT461301 and MT461302, respectively). Serbian WSMV isolates showed very high nucleotide identity (98.16-99.02%) and shared a deletion of triplet codon GCA at nucleotide position 8412- 8414 resulting in deletion of glycine amino acid (Gly2761). Phylogenetic analysis conducted on CP gene sequences revealed the existence of four clades, named A, B, C and D, and one recently introduced clade B1. All Serbian wheat WSMV isolates grouped into clade B together with other European isolates and one isolate from Iran. The results of this study provide the first insight into molecular characterisation of Serbian WSMV isolates, indicating their close relationship with other European isolates and existence of a single genotype in the country. Phylogenetic analysis also confirms the dispersal of WSMV isolates throughout Europe from a single locus.

A hallmark feature of (+)-strand RNA viruses of eukaryotic cells is that progeny (+)-strands are accumulated 100-fold over (−)-strands. Previous experimental evidence suggests that, in Brome mosaic virus (BMV), a plant-infecting member of the alphavirus-like superfamily, the addition of RNA3 and, specifically, translation of the wild-type (WT) coat protein (CP) gene contributes to increased accumulation of (+)-strands. It is unclear whether this stimulation of (+)-strand accumulation by CP is due to direct regulation of viral RNA replication or RNA stabilization via encapsidation. Analysis of BMV progeny RNA in Nicotiana benthamiana plants revealed that expression of RNA3 variants that did not express WT CP led to a severe defect in BMV (+)-strand accumulation. The (+)-strand accumulation could be rescued when CP was complemented in trans. To verify whether stimulation of (+)-strand accumulation is coupled with encapsidation, two independent mutations were engineered into CP open reading frames. An N-terminal deletion that prevented CP binding to the viral RNAs resulted in a severe reduction of BMV (+)-strand accumulation but stimulated (−)-strand accumulation over the WT. On the other hand, a C-terminal mutation affecting CP dimerization caused a significant decrease in (+)-strand accumulation but had no detectable effect on (−)-strand accumulation. Nucleotide sequences in the movement protein-coding region were also found to contribute to (+)-strand accumulation, in part by providing packaging signals for efficient RNA3 encapsidation. Overall, these results show that RNA encapsidation is a significant determinant of BMV RNA intracellular accumulation.

Field-grown wheat (Triticum aestivum L.) plants can be co-infected by multiple viruses, including wheat streak mosaic virus (WSMV), Triticum mosaic virus (TriMV), brome mosaic virus (BMV), and barley stripe mosaic virus (BSMV). These viruses belong to four different genera in three different families and are, hence, genetically divergent. However, the impact of potential co-infections with two, three, or all four of them on the viruses themselves, as well as the wheat host, has yet to be examined. This study examined bi-, tri-, and quadripartite interactions among these viruses in wheat for disease development and accumulation of viral genomic RNAs, in comparison with single virus infections. Co-infection of wheat by BMV and BSMV resulted in BMV-like symptoms with a drastic reduction in BSMV genomic RNA copies and coat protein accumulation, suggesting an antagonism-like effect exerted by BMV toward BSMV. However, co-infection of either BMV or BSMV with WSMV or TriMV led to more severe disease than singly infected wheat, but with a decrease or no significant change in titers of interacting viruses in the presence of BMV or BSMV, respectively. These results were in stark contrast with exacerbated disease phenotype accompanied with enhanced virus titers caused by WSMV and TriMV co-infection. Co-infection of wheat by WSMV, TriMV, and BMV or BSMV resulted in enhanced synergistic disease accompanied by increased accumulation of TriMV and BMV but not WSMV or BSMV. Quadripartite interactions in co-infected wheat by all four viruses resulted in very severe disease synergism, leading to the death of the most infected plants, but paradoxically, a drastic reduction in BSMV titer. Our results indicate that interactions among different viruses infecting the same plant host are more complex than previously thought, do not always entail increases in virus titers, and likely involve multiple mechanisms. These findings lay the foundation for additional mechanistic dissections of synergistic interactions among unrelated plant viruses.

Virus-induced gene silencing (VIGS) is a rapid and powerful method to evaluate gene function, especially for species like hexaploid wheat that have large, redundant genomes and are difficult and time-consuming to transform. The Brome mosaic virus (BMV)-based VIGS vector is widely used in monocotyledonous species but not wheat. Here we report the establishment of a simple and effective VIGS procedure in bread wheat using BMVCP5, the most recently improved BMV silencing vector, and wheat genes PHYTOENE DESATURASE (TaPDS) and PHOSPHATE2 (TaPHO2) as targets. Time-course experiments revealed that smaller inserts (~100 nucleotides, nt) were more stable in BMVCP5 and conferred higher silencing efficiency and longer silencing duration, compared with larger inserts. When using a 100-nt insert and a novel coleoptile inoculation method, BMVCP5 induced extensive silencing of TaPDS transcript and a visible bleaching phenotype in the 2nd to 5th systemically-infected leaves from nine to at least 28 days post inoculation (dpi). For TaPHO2, the ability of BMVCP5 to simultaneously silence all three homoeologs was demonstrated. To investigate the feasibility of BMV VIGS in wheat roots, ectopically expressed enhanced GREEN FLUORESCENT PROTEIN (eGFP) in a transgenic wheat line was targeted for silencing. Silencing of eGFP fluorescence was observed in both the maturation and elongation zones of roots. BMVCP5 mediated significant silencing of eGFP and TaPHO2 mRNA expression in roots at 14 and 21 dpi, and TaPHO2 silencing led to the doubling of inorganic phosphate concentration in the 2nd through 4th systemic leaves. All 54 wheat cultivars screened were susceptible to BMV infection. BMVCP5-mediated TaPDS silencing resulted in the expected bleaching phenotype in all eight cultivars examined, and decreased TaPDS transcript was detected in all three cultivars examined. This BMVCP5 VIGS technology may serve as a rapid and effective functional genomics tool for high-throughput gene function studies in aerial and root tissues and in many wheat cultivars.

Viral infections universally rely on numerous hijacked host factors to be successful. It is therefore possible to control viral infections by manipulating host factors that are critical for viral replication. Given that host genes may play essential roles in certain cellular processes, any successful manipulations for virus control should cause no or mild effects on host fitness. We previously showed that a group of positive-strand RNA viruses enrich phosphatidylcholine (PC) at the sites of viral replication. Specifically, brome mosaic virus (BMV) replication protein 1a interacts with and recruits a PC synthesis enzyme, phosphatidylethanolamine methyltransferase, Cho2p, to the viral replication sites that are assembled on the perinuclear endoplasmic reticulum (ER) membrane. Deletion of the CHO2 gene inhibited BMV replication by 5-fold; however, it slowed down host cell growth as well. Here, we show that an engineered Cho2p mutant supports general PC synthesis and normal cell growth but blocks BMV replication. This mutant interacts and colocalizes with BMV 1a but prevents BMV 1a from localizing to the perinuclear ER membrane. The mislocalized BMV 1a fails to induce the formation of viral replication complexes. Our study demonstrates an effective antiviral strategy in which a host lipid synthesis gene is engineered to control viral replication without comprising host growth.

Phloem-mobile mRNAs are assumed to contain sequence elements directing RNA to the phloem translocation pathway. One of such elements is represented by tRNA sequences embedded in untranslated regions of many mRNAs, including those proved to be mobile. Genomic RNAs of a number of plant viruses possess a 3'-terminal tRNA-like structures (TLSs) only distantly related to genuine tRNAs, but nevertheless aminoacylated and capable of interaction with some tRNA-binding proteins. Here, we elaborated an experimental system for analysis of RNA phloem transport based on an engineered RNA of Potato virus X capable of replication, but not encapsidation and movement in plants. The TLSs of Brome mosaic virus, Tobacco mosaic virus and Turnip yellow mosaic virus were demonstrated to enable the phloem transport of foreign RNA. A miRNA precursor, pre-miR390b, was also found to render RNA competent for the phloem transport. In line with this, sequences of miRNA precursors were identified in a Cucurbita maxima phloem transcriptome, supporting the hypothesis that, at least in some cases, miRNA phloem signaling can involve miRNA precursors. Collectively, the data presented here suggest that RNA molecules can be directed into the phloem translocation pathway by structured RNA elements such as those of viral TLSs and miRNA precursors.

Brome mosaic virus (BMV) is generally thought to be of little economic importance to crops; consequently, there is little information about its impact on wheat production under field conditions. After repeated detection of BMV in Ohio wheat fields at incidences up to 25%, the virus was isolated, sequenced, characterized, and tested for its impact on soft red winter wheat (SRWW). The Ohio isolate of brome mosaic virus (BMV-OH) was found to be >99% identical to a BMV-Fescue isolate (accession no. DQ530423-25) and capable of systemically infecting multiple monocot and dicot species, including cowpea and soybean, in experimental inoculations. BMV-OH was used in field experiments during the 2016 and 2017 growing seasons to quantify its effect on SRWW grain yield and development when inoculated at Feekes 1, 5, 8, and 10 in two to four cultivars. Cultivar and timing of inoculation had statistically significant (P < 0.05) main and interaction effects on grain yield, wheat growth, and multiple components of yield. Compared with noninoculated controls, BMV-OH reduced grain yield by up to 61% when inoculated at Feekes 1 and by as much as 25, 36, and 31% for inoculations at Feekes 5, 8, and 10, respectively. The magnitude of the yield reduction varied among cultivars and was associated with reductions in grain size and weight or plant population. These findings suggest that BMV could impact wheat productivity in Ohio and will serve as the basis for more large-scale investigations of the effects of this virus in commercial fields.

Virus-induced gene silencing (VIGS) is a rapid and powerful method to evaluate gene function, especially for species like hexaploid wheat that have large, redundant genomes and are difficult and time-consuming to transform. The Brome mosaic virus (BMV)-based VIGS vector is widely used in monocotyledonous species but not wheat. Here we report the establishment of a simple and effective VIGS procedure in bread wheat using BMVCP5, the most recently improved BMV silencing vector, and wheat genes PHYTOENE DESATURASE (TaPDS) and PHOSPHATE2 (TaPHO2) as targets. Time-course experiments revealed that smaller inserts (~100 nucleotides, nt) were more stable in BMVCP5 and conferred higher silencing efficiency and longer silencing duration, compared with larger inserts. When using a 100-nt insert and a novel coleoptile inoculation method, BMVCP5 induced extensive silencing of TaPDS transcript and a visible bleaching phenotype in the 2nd to 5th systemically-infected leaves from nine to at least 28 days post inoculation (dpi). For TaPHO2, the ability of BMVCP5 to simultaneously silence all three homoeologs was demonstrated. To investigate the feasibility of BMV VIGS in wheat roots, ectopically expressed enhanced GREEN FLUORESCENT PROTEIN (eGFP) in a transgenic wheat line was targeted for silencing. Silencing of eGFP fluorescence was observed in both the maturation and elongation zones of roots. BMVCP5 mediated significant silencing of eGFP and TaPHO2 mRNA expression in roots at 14 and 21 dpi, and TaPHO2 silencing led to the doubling of inorganic phosphate concentration in the 2nd through 4th systemic leaves. All 54 wheat cultivars screened were susceptible to BMV infection. BMVCP5-mediated TaPDS silencing resulted in the expected bleaching phenotype in all eight cultivars examined, and decreased TaPDS transcript was detected in all three cultivars examined. This BMVCP5 VIGS technology may serve as a rapid and effective functional genomics tool for high-throughput gene function studies in aerial and root tissues and in many wheat cultivars.

Nanotechnology involves the development of potential applications such as engineered nanovehicles derived from nonpathogenic plant viruses and bacteriophages for drug packaging and payload delivery. Such plant virus-based engineered nanovehicles (PVENVs) are more advantageous compared with synthetic nanoparticles because of their biocompatibility and biodegradability. The viral coat protein in conjugation with small organic molecules or short peptides provides binder molecules that bind proteins with high affinity, selectivity, and prolonged half-life. PVENVs have been developed using several plant viruses, such as cowpea chlorotic mottle virus, potato virus X, cowpea mosaic virus, tobacco mosaic virus, and Brome mosaic virus; these plant viruses are widely used to engineer nanomaterials conjugated with a wide range of biocompatible molecules. This review focuses on the potential of PVENVs having vast applications in the medical field and agricultural sector, thus employing a renewable and environmentally safe method for producing PVENVs.

Non-enveloped RNA viruses pervade all domains of life. In a cell, they co-assemble from viral RNA and capsid proteins. Virus-like particles can form in vitro where virtually any non-cognate polyanionic cargo can be packaged. How only viral RNA gets selected for packaging in vivo, in presence of myriad other polyanionic species, has been a puzzle. Through a combination of charge detection mass spectrometry and cryo-electron microscopy, it is determined that co-assembling brome mosaic virus (BMV) coat proteins and nucleic acid oligomers results in capsid structures and stoichiometries that differ from the icosahedral virion. These previously unknown shell structures are strained and less stable than the native one. However, they contain large native structure fragments that can be recycled to form BMV virions, should a viral genome become available. The existence of such structures suggest the possibility of a previously unknown regulatory pathway for the packaging process inside cells.

The members of the family Bromoviridae have spherical or bacilliform virions with tri-segmented, single-stranded genomic RNAs, packaged in separate particles. Six genera including Alfamovirus, Anulavirus, Bromovirus, Cucumovirus, Ilarvirus, and Oleavirus are part of the family. RNA1 and RNA2 code for the replicase whereas RNA3 codes for movement and coat proteins. Genomic RNAs are infectious, but some species also require CP for infectivity. Members can encapsidate/accumulate sub-genomic RNAs, and/or defective or satellite RNAs. RNA replication occurs inside membranous spherules, and the role of host factors in RNA replication have been documented. Frequent RNA-RNA recombination and segment reassortment processes were observed among bromovirids. Transmission occurs mechanically, via pollen, seeds or insects. Host range varies from narrow to wide, infecting herbaceous plants, shrubs and trees, with some members causing major epidemics.

We report the asymmetric reconstruction of the single-stranded RNA (ssRNA) content in one of the three otherwise identical virions of a multipartite RNA virus, brome mosaic virus (BMV). We exploit a sample consisting exclusively of particles with the same RNA content—specifically, RNAs 3 and 4—assembled in planta by agrobacterium-mediated transient expression. We find that the interior of the particle is nearly empty, with most of the RNA genome situated at the capsid shell. However, this density is disordered in the sense that the RNA is not associated with any particular structure but rather, with an ensemble of secondary/tertiary structures that interact with the capsid protein. Our results illustrate a fundamental difference between the ssRNA organization in the multipartite BMV viral capsid and the monopartite bacteriophages MS2 and Qβ for which a dominant RNA conformation is found inside the assembled viral capsids, with RNA density conserved even at the center of the particle. This can be understood in the context of the differing demands on their respective lifecycles: BMV must package separately each of several different RNA molecules and has been shown to replicate and package them in isolated, membrane-bound, cytoplasmic complexes, whereas the bacteriophages exploit sequence-specific “packaging signals” throughout the viral RNA to package their monopartite genomes.

Ohio is a leading producer of soft red winter wheat in the United States. Many viruses impact wheat production, but there is a lack of contemporary information on the distribution and potential impact of wheat viruses in Ohio. To address this knowledge gap, we created a comprehensive dataset of viruses identified by high-throughput sequencing (HTS) and their incidence in field sites sampled across the state. Samples were collected from 103 field sites in surveys conducted in 2012, 2016, and 2017 and subjected to RNA HTS, reverse transcription (RT) PCR, or enzyme-linked immunosorbent assay to assess virus sequence diversity, prevalence, and incidence within fields. Partial and complete virus sequences were assembled and detection validated by RT-PCR. Assembled sequences were compared with previously known virus sequences, and novel sequences were validated by Sanger sequencing. The viruses detected most often included barley yellow dwarf virus (BYDV), cereal yellow dwarf virus (CYDV), wheat streak mosaic virus (WSMV), and wheat spindle streak mosaic virus (WSSMV). These viruses were detected at 67, 69, 55, and 28% of the field sites sampled, with mean incidences of 18, 19, 20, and 49%, respectively, within fields where they were detected. Brome mosaic virus (BMV) and cocksfoot mottle virus (CfMV) were also viruses of potential importance detected in Ohio, found in 26 and 17% of the field sites sampled, respectively. Based on results from logistic regression analyses, the presence of BYDV, CYDV, WSMV, and WSSMV was associated with the presence of volunteer wheat, BYDV and CfMV with monocots as the previous crop, and BMV with the presence of nearby corn fields (P < 0.10). For six viruses, there was evidence of spatial clustering in at least one field site and the variance of mean incidence was higher at the county level than at the regional spatial level. This finding suggests that county- and site-specific factors influenced the incidence and spatial pattern of some viruses. The results of this study provide a snapshot of viruses present in Ohio wheat and insights into their biology, potential risks to wheat production, and possible management strategies.

Brome mosaic virus (BMV) is an isometric, non-enveloped, positive-strand RNA virus and a well-studied, representative member of the alphavirus-like super-family of human, animal, and plant viruses. BMV has been extensively studied as a model to examine some of the common features shared by all positive-strand RNA viruses. This article provides insights into virion assembly, encapsidation, gene expression, recombination, RNA replication, and virus-host interactions. These studies have not only advanced understanding of BMV, but have also revealed insights and principles extending to many other viruses and to general cellular biology.

Environmental waters, e.g. rivers, lakes and irrigation water, are a good source of many plant viruses. The pathogens can infect plants getting through damaged root hairs or small wounds that appear during plant growth. First results demonstrated common incidence of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) in water samples collected from irrigation ditches and drainage canals surrounding fields in Southern Greater Poland. Principal objective of this work was to examine if environmental water might be the source of viruses infective to cereals. The investigation was focused on mechanically transmitted pathogens. Virus identification was performed by biological, electron microscopic, serological and molecular methods. Preliminary assays demonstrated Brome mosaic virus (BMV) infections in symptomatic plants inoculated with 9 out of the 17 tested concentrated water samples. The final identification was confirmed by molecular methods for selected isolates named: BMV-Ch1, -DBS, -N, -R and -S. Partial coding sequences of polymerase 1a (RNA1) and 2a (RNA2) and complete nucleotide sequence of coat protein (CP) gene (RNA3) of each BMV isolate were determined and compared with corresponding sequences of other known BMV isolates. Results confirmed the highest amino acid sequence homology in the fragment of polymerase 2a (99.2% – 100%) and the most divergence in CP (96.2% - 100%). This is the first report on the detection of an infective cereal virus in aqueous environment.

In the brome mosaic virus (BMV) virion, the coat protein (CP) selectively contacts the RNA motifs that regulate translation and RNA replication (Hoover et al., 2016. J. Virol. 90, 7748). We hypothesize that the unstructured N-terminal arm (NTA) of the BMV CP can specifically recognize RNA motifs. Using ion mobility spectrometry-mass spectrometry, we demonstrate that peptides containing the NTA of the CP were found to preferentially bind to an RNA hairpin motif that directs the initiation of BMV RNA synthesis. RNA binding causes the peptide to change from heterogeneous structures to a single family of structures. Fluorescence anisotropy, fluorescence quenching and size exclusion chromatography experiments all confirm that the NTA can specific recognize the RNA motif. The peptide introduced into plants along with BMV virion increased accumulation of the BMV CP and accelerated the rate of minus-strand RNA synthesis. The intrinsically disordered BMV NTA could thus specifically recognize BMV RNAs to affect viral infection.

Virus-induced gene silencing (VIGS) is used extensively for gene function studies in plants. VIGS is inexpensive and rapid compared with silencing conducted through stable transformation, but many virus-silencing vectors, especially in grasses, induce only transient silencing phenotypes. A major reason for transient phenotypes is the instability of the foreign gene fragment (insert) in the vector during VIGS. Here, we report the development of a Brome mosaic virus (BMV)-based vector that better maintains inserts through modification of the original BMV vector RNA sequence. Modification of the BMV RNA3 sequence yielded a vector, BMVCP5, that better maintained phytoene desaturase and heat shock protein70-1 (HSP70-1) inserts in Nicotiana benthamiana and maize (Zea mays). Longer maintenance of inserts was correlated with greater target gene silencing and more extensive visible silencing phenotypes displaying greater tissue penetration and involving more leaves. The modified vector accumulated similarly to the original vector in N. benthamiana after agroinfiltration, thus maintaining a high titer of virus in this intermediate host used to produce virus inoculum for grass hosts. For HSP70, silencing one family member led to a large increase in the expression of another family member, an increase likely related to the target gene knockdown and not a general effect of virus infection. The cause of the increased insert stability in the modified vector is discussed in relationship to its recombination and accumulation potential. The modified vector will improve functional genomic studies in grasses, and the conceptual methods used to improve the vector may be applied to other VIGS vectors.

Brome mosaic virus (BMV) has been successfully loaded with different types of nanoparticles. However, studies concerning its application as a nanoparticle carrier demand high-purity virions in large amounts. Existing BMV purification protocols rely on multiple differential ultracentrifugation runs of the initially purified viral preparation. Herein, we describe an alternative method for BMV purification based on ion-exchange chromatography and size-exclusion chromatography (SEC) yielding 0.2 mg of virus from 1 g of plant tissue. Our method is of similar efficiency to previously described protocols and can easily be scaled up. The method results in high-quality BMV preparations as confirmed by biophysical analyses, including cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), static light scattering (SLS), and circular dichroism (CD) measurements and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy. Our results revealed that purified BMV capsids are stable and monodisperse and can be used for further downstream applications. In this work, we also characterize secondary structure and size fluctuations of the BMV virion at different pH values.

Brome mosaic virus (BMV) infects many different species within the Poaceae family. A new strain of BMV, named F-BMV, was identified in a tall fescue (Festuca arundinacea Schreb.) plant. Here, we report the identification and characterization of tall fescue plants resistant to F-BMV, and the effects of F-BMV infection on their growth and development. Susceptible plants infected with F-BMV produced 40% fewer tillers and 42% less dry matter compared with virus-resistant plants in a greenhouse study. In the field, susceptible plants infected with F-BMV produced 25% fewer tillers, 36% less dry matter, 10% less plant height, and 40% lower seed yield compared with virus-resistant plants. In a field evaluation of a tall fescue mapping population, the virus symptom scores were negatively correlated with production of dry matter (r = -0.55), plant height (r = -0.55), and seed yield (r = -0.33). Thus, F-BMV has the potential to cause significant economic damage to susceptible tall fescue plants. These results indicate that the virus can present a serious challenge for long-term maintenance of valuable plant materials. A survey of tall fescue plants from Arkansas, Oklahoma, and Oregon indicated that the prevalence F-BMV in the field was very low.

Analysis of the prevalence and bioecological characteristics of the brome mosaic virus (BMV), which is a widespread polyphage virus that causes significant economic damage to cereals. Experimental studies were carried out in the laboratory of the All-Russian Science-Research Institute of plant Quarantine (Moscow region), visual examinations of crops and sampling of winter wheat in the tillering phase were carried out in the farms of the Rostov region, the Chechen Republic, in the Moscow region (Russian State Agrarian University -Moscow Timiryazev Agricultural Academy) in 2019- 2020. Route surveys of fields in each region were carried out in the spring. The visually estimated prevalence of viroses on average did not exceed 0.5%. The detection and identification of the brome mosaic virus in plants with symptoms of mosaic and mottling was carried out by the method of classical PCR with visualization of the results by gel electrophoresis. Brome mosaic virus was detected by RT-PCR in winter wheat samples from the Rostov region. No brome mosaic virus was detected in winter wheat plants from the Chechen Republic, Moscow region, although the leaves showed virus-like symptoms.

The four brome mosaic virus (BMV) RNAs (RNA1 to RNA4) are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for the differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidate each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of posttranslational modifications. Treatment with phosphatase was found to not significantly affect the stability of the virions containing RNA1 but significantly impacted the stability of the virions that encapsidated BMV RNA2 and RNA3/4. Cryo-electron microscopy reconstruction revealed dramatic structural changes in the capsid and the encapsidated RNA. A phosphomimetic mutation in the flexible N-terminal arm of the CP increased BMV RNA replication and virion production. The degree of phosphorylation modulated the interaction of CP with the encapsidated RNA and the release of three of the BMV RNAs. UV cross-linking and immunoprecipitation methods coupled to high-throughput sequencing experiments showed that phosphorylation of the BMV CP can impact binding to RNAs in the virions, including sequences that contain regulatory motifs for BMV RNA gene expression and replication. Phosphatase-treated virions affected the timing of CP expression and viral RNA replication in plants. The degree of phosphorylation decreased when the plant hosts were grown at an elevated temperature. These results show that phosphorylation of the capsid modulates BMV infection. IMPORTANCE How icosahedral viruses regulate the release of viral RNA into the host is not well understood. The selective release of viral RNA can regulate the timing of replication and gene expression. Brome mosaic virus (BMV) is an RNA virus, and its three genomic RNAs are encapsidated in separate virions. Through proteomic, structural, and biochemical analyses, this work shows that posttranslational modifications, specifically, phosphorylation, on the capsid protein regulate the capsid-RNA interaction and the stability of the virions and affect viral gene expression. Mutational analysis confirmed that changes in modification affected virion stability and the timing of viral infection. The mechanism for modification of the virion has striking parallels to the mechanism of regulation of chromatin packaging by nucleosomes.

Brome mosaic virus (BMV) is a well-known plant virus representing single-stranded RNA (ssRNA) positive-sense viruses. It has been widely used as a model in multiple studies concerning plant virus biology, epidemiology and the application of viral capsids in nanotechnology. Herein, we describe a method for BMV purification based on ion-exchange and size-exclusion chromatography. The presented method is of similar efficiency to previously described protocols relying on differential centrifugation and can easily be scaled up. The resulting BMV capsids are stable and monodisperse and can be used for further applications.

Although RNA viruses evolved the mechanisms of specific encapsidation, miss-packaging of cellular RNAs has been reported in such RNA virus systems as flock house virus or cucumber necrosis virus. To find out if brome mosaic virus (BMV), a tripartite RNA virus, can package cellular RNAs, BMV was propagated in barley and in Nicotiana benthamiana hosts, purified by cesium chloride (CsCl) gradient ultracentrifugation followed by nuclease treatment to remove any contaminating cellular (host) RNAs. The extracted virion RNA was then sequenced by using next-generation sequencing (NGS RNA-Seq) with the Illumina protocol. Bioinformatic analysis revealed the content of host RNAs ranging from 0.07% for BMV extracted from barley to 0.10% for the virus extracted from N. benthamiana. The viruses from two sources appeared to co-encapsidate different patterns of host-RNAs, including ribosomal RNAs (rRNAs), messenger RNAs (mRNAs) but also mitochondrial and plastid RNAs and, interestingly, transposable elements, both transposons and retrotransposons. Our data reveal that BMV virions can carry host RNAs, having a potential to mediate horizontal gene transfer (HGT) in plants.

Positive-strand RNA viruses generally assemble RNA replication complexes on rearranged host membranes. Alphaviruses, other members of the alpha-like virus superfamily, and many other positive-strand RNA viruses invaginate host membrane into vesicular RNA replication compartments, known as spherules, whose interior is connected to the cytoplasm. Brome mosaic virus (BMV) and its close relative, cowpea chlorotic mottle virus (CCMV), form spherules along the endoplasmic reticulum. BMV spherule formation and RNA replication can be fully reconstituted in S. cerevisiae, enabling many studies identifying host factors and viral interactions essential for these processes. To better define and understand the conserved, core pathways of bromovirus RNA replication, we tested the ability of CCMV to similarly support spherule formation and RNA replication in yeast. Paralleling BMV, we found that CCMV RNA replication protein 1a was the only viral factor necessary to induce spherule membrane rearrangements and to recruit the viral 2a polymerase (2apol) to the endoplasmic reticulum. CCMV 1a and 2apol also replicated CCMV and BMV genomic RNA2, demonstrating core functionality of CCMV 1a and 2apol in yeast. However, while BMV and CCMV 1a/2apol strongly replicate each others' genomic RNA3 in plants, neither supported detectable CCMV RNA3 replication in yeast. Moreover, in contrast to plant cells, in yeast CCMV 1a/2apol supported only limited replication of BMV RNA3 (

Background Although the draft genome of sorghum is available, the understanding of gene function is limited due to the lack of extensive mutant resources. Virus-induced gene silencing (VIGS) is an alternative to mutant resources to study gene function. This study reports an improved and efficient method for Brome mosaic virus (BMV)-based VIGS in sorghum. Methods Sorghum plants were rub-inoculated with sap prepared by grinding 2 g of infected Nicotiana benthamiana leaf in 1 ml 10 mM potassium phosphate buffer (pH 6.8) and 100 mg of carborundum abrasive. The sap was rubbed on two to three top leaves of sorghum. Inoculated plants were covered with a dome to maintain high humidity and kept in the dark for two days at 18 °C. Inoculated plants were then transferred to 18 °C growth chamber with 12 h/12 h light/dark cycle. Results This study shows that BMV infection rate can be significantly increased in sorghum by incubating plants at 18 °C. A substantial variation in BMV infection rate in sorghum genotypes/varieties was observed and BTx623 was the most susceptible. Ubiquitin (Ubiq) silencing is a better visual marker for VIGS in sorghum compared to other markers such as Magnesium Chelatase subunit H (ChlH) and Phytoene desaturase (PDS). The use of antisense strand of a gene in BMV was found to significantly increase the efficiency and extent of VIGS in sorghum. In situ hybridization experiments showed that the non-uniform silencing in sorghum is due to the uneven spread of the virus. This study further demonstrates that genes could also be silenced in the inflorescence of sorghum. Conclusion In general, sorghum plants are difficult to infect with BMV and therefore recalcitrant to VIGS studies. However, by using BMV as a vector, a BMV susceptible sorghum variety, 18 °C for incubating plants, and antisense strand of the target gene fragment, efficient VIGS can still be achieved in sorghum. Electronic supplementary material The online version of this article (10.1186/s12870-018-1344-z) contains supplementary material, which is available to authorized users.

Brome mosaic virus (BMV) (the Russian strain) infects monocot plants and has been studied extensively in barley and wheat. Here, we report BMV can systemically infect rice (Oryza sativa var. japonica), including cultivars in which the genomes have been determined. The BMV capsid protein can be found throughout the inoculated plants. However, infection in rice exhibits delayed symptom expression or no symptoms when compared with wheat (Triticum aestivum). The sequences of BMV RNAs isolated from rice did not reveal any nucleotide changes in RNA1 or RNA2, while RNA3 had only one synonymous nucleotide change from the inoculum sequence. Preparations of purified BMV virions contained RNA1 at a significantly reduced level relative to the other two RNAs. Analysis of BMV RNA replication in rice revealed that minus-strand RNA1 was replicated at a reduced rate when compared with RNA2. Thus, rice appears to either inhibit RNA1 replication or lacks a sufficient amount of a factor needed to support efficient RNA1 replication.

Magnaporthe oryzae is a devastating plant pathogen, which has a detrimental impact on rice production worldwide. Despite its agronomical importance, some newly-emerging pathotypes often overcome race-specific disease resistance rapidly. It is thus desirable to develop a novel strategy for the long-lasting resistance of rice plants to ever-changing fungal pathogens. Brome mosaic virus (BMV)-induced RNA interference (RNAi) has emerged as a useful tool to study host-resistance genes for rice blast protection. Planta-generated silencing of targeted genes inside biotrophic pathogens can be achieved by expression of M. oryzae-derived gene fragments in the BMV-mediated gene silencing system, a technique termed host-induced gene silencing (HIGS). In this study, the effectiveness of BMV-mediated HIGS in M. oryzae was examined by targeting three predicted pathogenicity genes, MoABC1, MoMAC1 and MoPMK1. Systemic generation of fungal gene-specific small interfering RNA (siRNA) molecules induced by inoculation of BMV viral vectors inhibited disease development and reduced the transcription of targeted fungal genes after subsequent M. oryzae inoculation. Combined introduction of fungal gene sequences in sense and antisense orientation mediated by the BMV silencing vectors significantly enhanced the efficiency of this host-generated trans-specific RNAi, implying that these fungal genes played crucial roles in pathogenicity. Collectively, our results indicated that BMV-HIGS system was a great strategy for protecting host plants against the invasion of pathogenic fungi.

Reactive oxygen species (ROS), including superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical, act as signaling molecules to transduce biotic and abiotic stimuli into stress adaptations in plants. A respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) is responsible for O2- production to inhibit pathogen infection during plant innate immunity. RBOH-derived O2- can be immediately converted into H2O2 by the action of superoxide dismutase. Interestingly, we recently showed that red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, hijacks the host's ROS-generating machinery during infection. An RCNMV replication protein associates with NbRBOHB and triggers intracellular ROS bursts. These bursts are required for robust viral RNA replication. However, what types of ROS are required for viral replication is currently unknown. Here, we found that RCNMV replication was sensitive to an O2- scavenger but insensitive to an H2O2 scavenger. Interestingly, replication of another plant (+)RNA virus, brome mosaic virus, was sensitive to both types of scavengers. These results indicate a virus-specific pattern requirement of O2- and H2O2 for (+)RNA virus replication and suggest a conserved nature of the roles of ROS in (+)RNA virus replication.

As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant-virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of N. benthamiana (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOH-derived ROS was demonstrated for the replication of another (+)RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+)RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication.

Rapid detection and diagnosis of plant virus infection is one of the most important steps in preventing damages caused by viral diseases. Bromoviruses and cucumoviruses belong to the family Bromoviridae, which is one of the most important families of plant viruses, and infect a broad range of host plants including various economically important crops. In this study, an RT-PCR assay was developed for the universal detection of bromoviruses and cucumoviruses using a set of primers designed to target the conserved sequences in viral RNA1. The assay detected three species of Cucumovirus (Cucumber mosaic virus (CMV), Peanut stunt virus (PSV) and Tomato aspermy virus (TAV)) and two species of Bromovirus (Brome mosaic virus (BMV) and Cowpea chlorotic mottle virus (CCMV)) with high specificity and sensitivity. The assay developed in this study is predicted to have the potential to detect all major members of the genera Bromovirus and Cucumovirus and to be used as a routine diagnostic assay.

The three subsets of virions that comprise the Brome mosaic virus (BMV) were previously thought to be indistinguishable. This work tested the hypothesis that distinct capsid-RNA interactions in the BMV virions allow different rates of viral RNA release. Several results support distinct interactions between the capsid and the BMV genomic RNAs. First, the deletion of the first eight residues of the BMV coat protein (CP) resulted in the RNA1-containing particles having altered morphologies, while those containing RNA2 were unaffected. Second, subsets of the BMV particles separated by density gradients into a pool enriched for RNA1 (B1) and for RNA2 and RNA3/4 (B2.3/4) were found to have different physiochemical properties. Compared to the B2.3/4 particles, the B1 particles were more sensitive to protease digestion and had greater resistivity to nanoindentation by atomic force microscopy and increased susceptibility to nuclease digestion. Mapping studies showed that portions of the arginine-rich N-terminal tail of the CP could interact with RNA1. Mutational analysis in the putative RNA1-contacting residues severely reduced encapsidation of BMV RNA1 without affecting the encapsidation of RNA2. Finally, during infection of plants, the more easily released RNA1 accumulated to higher levels early in the infection. IMPORTANCE Viruses with genomes packaged in distinct virions could theoretically release the genomes at different times to regulate the timing of gene expression. Using an RNA virus composed of three particles, we demonstrated that the RNA in one of the virions is released more easily than the other two in vitro . The differential RNA release is due to distinct interactions between the viral capsid protein and the RNAs. The ease of RNA release is also correlated with the more rapid accumulation of that RNA in infected plants. Our study identified a novel role for capsid-RNA interactions in the regulation of a viral infection.

The yeast Saccharomyces cerevisiae is an advanced model organism that has emerged as an effective host to gain insights into the intricate interactions of viruses with host cells. RNA viruses have limited coding potential and need to coopt numerous host cellular factors to facilitate their replication. To identify the host factors subverted by viruses, high-throughput genomics and global proteomics approaches have been performed with plant viruses such as brome mosaic virus (BMV) and tomato bushy stunt virus (TBSV). Accordingly, several hundred susceptibility and restriction factors for BMV and TBSV have been identified using yeast as a model host. Amazingly, host factors affecting viral genetic recombination and evolution have also been identified in genome-wide screens in yeast. The roles of many yeast host factors involved in various steps of the viral replication process have been validated by exploiting the orthologous genes in plant hosts. This Opinion summarizes the advantages of using simple viruses and yeast model host to advance our general understanding of virus–host interactions. The knowledge gained on host factors could lead to novel specific or broad-range resistance and antiviral tools against viruses.

Brome mosaic virus (BMV) packages its genomic and subgenomic RNAs into three separate viral particles. BMV purified from barley, wheat, and tobacco have distinct relative abundances of the encapsidated RNAs. We seek to identify the basis for the host-dependent differences in viral RNA encapsidation. Sequencing of the viral RNAs revealed recombination events in the 3' untranslated region of RNA1 of BMV purified from barley and wheat, but not from tobacco. However, the relative amounts of the BMV RNAs that accumulated in barley and wheat are similar and RNA accumulation is not sufficient to account for the difference in RNA encapsidation. Virions purified from barley and wheat were found to differ in their isoelectric points, resistance to proteolysis, and contacts between the capsid residues and the RNA. Mass spectrometric analyses revealed that virions from the three hosts had different post-translational modifications that should impact the physiochemical properties of the virions. Another major source of variation in RNA encapsidation was due to the purification of BMV particles to homogeneity. Highly enriched BMV present in lysates had a surprising range of sizes, buoyant densities, and distinct relative amounts of encapsidated RNAs. These results show that the encapsidated BMV RNAs reflect a combination of host effects on the physiochemical properties of the viral capsids and the enrichment of a subset of virions. The previously unexpected heterogeneity in BMV should influence the timing of the infection and also the host innate immune responses.

Viral nanoparticles (VNPs) are becoming versatile tools in platform technology development. Their well-defined structures as well as their programmability through chemical and genetic modification allow VNPs to be engineered for potential imaging and therapeutic applications. In this article, we report the application of a variety of bioconjugation chemistries to the plant VNP Brome mosaic virus (BMV). Functional BMV nanoparticles displaying multiple copies of fluorescent dyes, PEG molecules, chemotherapeutic drug moieties, targeting proteins and cell penetrating peptides were formulated. This opens the door for the application of BMV in nanomedicine.

Both genome-wide transcriptomic surveys of the mRNA expression profiles and virus-induced gene silencing-based molecular studies of target gene during virus-plant interaction involve the precise estimation of the transcript abundance. Quantitative real-time PCR (qPCR) is the most widely adopted technique for mRNA quantification. In order to obtain reliable quantification of transcripts, identification of the best reference genes forms the basis of the preliminary work. Nevertheless, the stability of internal controls in virus-infected monocots needs to be fully explored. In this work, the suitability of ten housekeeping genes (ACT, EF1α, FBOX, GAPDH, GTPB, PP2A, SAND, TUBβ, UBC18 and UK) for potential use as reference genes in qPCR were investigated in five different monocot plants (Brachypodium, barley, sorghum, wheat and maize) under infection with different viruses including Barley stripe mosaic virus (BSMV), Brome mosaic virus (BMV), Rice black-streaked dwarf virus (RBSDV) and Sugarcane mosaic virus (SCMV). By using three different algorithms, the most appropriate reference genes or their combinations were identified for different experimental sets and their effectiveness for the normalisation of expression studies were further validated by quantitative analysis of a well-studied PR-1 gene. These results facilitate the selection of desirable reference genes for more accurate gene expression studies in virus-infected monocots.

Positive-strand RNA viruses are known to rearrange the endomembrane network to make it more conducive for replication, maturation, or egress. Our previous transmission electron microscopic (TEM) analysis showed that ectopic expression of wild-type (wt) capsid protein (CP) of Brome mosaic virus (BMV) has an intrinsic property of modifying the endoplasmic reticulum (ER) to induce vesicles similar to those present in wt BMV infection. In this study, we evaluated the functional significance of CP-mediated vesicle induction to the BMV infection cycle in planta . Consequently, the cytopathologic changes induced by wt CP or its mutants defective in virion assembly due to mutations engineered in either N- or C-proximal domains were comparatively analyzed by TEM in two susceptible ( Nicotiana benthamiana and Chenopodium quinoa ) and one nonhost ( N. clevelandii ) plant species. The results showed that in susceptible hosts, CP-mediated ER-derived vesicle induction is contingent on the expression of encapsidation-competent CP. In contrast, unlike in N. benthamiana and C. quinoa , transient expression of wt CP in nonhost N. clevelandii plants eliminated vesicle induction. Additionally, comparative source-to-sink analysis of virus spread in leaves of N. benthamiana and N. clevelandii coexpressing wt BMV and Cucumber mosaic virus (CMV) showed that despite trans -encapsidation, CMV failed to complement the defective cell-to-cell movement of BMV. The significance and relation of CP-mediated vesicle induction to virus cell-to-cell movement are discussed.

Positive-strand RNA viruses depend on recruited host factors to control critical replication steps. Previously, it was shown that replication of evolutionarily diverse positive-strand RNA viruses, such as hepatitis C virus and brome mosaic virus, depends on host decapping activators LSm1-7, Pat1, and Dhh1 (J. Diez et al., Proc. Natl. Acad. Sci. U. S. A. 97:3913–3918, 2000; A. Mas et al., J. Virol. 80:246 –251, 2006; N. Scheller et al., Proc. Natl. Acad. Sci. U. S. A. 106:13517–13522, 2009). By using a system that allows the replication of the insect Flock House virus (FHV) in yeast, here we show that LSm1-7, Pat1, and Dhh1 control the ratio of subgenomic RNA3 to genomic RNA1 production, a key feature in the FHV life cycle mediated by a long-distance base pairing within RNA1. Depletion of LSM1 , PAT1 , or DHH1 dramatically increased RNA3 accumulation during replication. This was not caused by differences between RNA1 and RNA3 steady-state levels in the absence of replication. Importantly, coimmunoprecipitation assays indicated that LSm1-7, Pat1, and Dhh1 interact with the FHV RNA genome and the viral polymerase. By using a strategy that allows dissecting different stages of the replication process, we found that LSm1-7, Pat1, and Dhh1 did not affect the early replication steps of RNA1 recruitment to the replication complex or RNA1 synthesis. Furthermore, their function on RNA3/RNA1 ratios was independent of the membrane compartment, where replication occurs and requires ATPase activity of the Dhh1 helicase. Together, these results support that LSm1-7, Pat1, and Dhh1 control RNA3 synthesis. Their described function in mediating cellular mRNP rearrangements suggests a parallel role in mediating key viral RNP transitions, such as the one required to maintain the balance between the alternative FHV RNA1 conformations that control RNA3 synthesis.

All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting the CHO2 gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes.

Every year, leaf mosaic, yellow streaks, and mottling can be commonly observed in wheat fields worldwide. A number of different factors can cause these symptoms, including nitrogen deficiency, winter injury, and virus diseseas. Today, total number of 44 viruses has been aassociated with the crops and 43 viruses can infect wheat under the experimental conditions. Virus infections in wheat range from latent to lethal. Although not consistently present, diseased fields may yield 5 to 10% less grain. Viruses become economically important when they appear in epidemic proportions. One complex of viruses, Barley yellow dwarf virus (BYDV, Luteovirus, Luteoviridae) is now considered as possibly the most serious disease of wheat and other small grains worldwide. Two other widespread viruses are Wheat streak mosaic virus (WSMV, Tritimovirus, Potyviridae and Soil-borne wheat mosaic virus (SBWMV, Furovirus, Virgaviridae). However, there are additional mite-transmitted viruses, aphidtransmitted viruses, and soilborne viruses that may be important. The emphasis in this article will be on viruses infecting wheat in Serbia: BYDV, WSMV and Brome mosaic virus (BMV, Bromovirus, Bromoviridae). The most practical disease control strategies against wheat viruses are cultivar resistance to the virus and/or to the vector, chemical control of vectors, crop rotation and sanitations. Svake godine u usevima pšenice širom sveta uobičajeno je da se uoče simptomi mozaika, žute crticavosti i šarenila lišća. Mnogobrojni faktori mogu da izazovu ovakvu diskoloraciju lišca, uključujući nedostatak azota, kalijuma ili gvožđa, oštećenja od mraza ili virusna oboljenja. Pšenica je prirodni domaćin 44 i eksperimentalni domaćin još 43 različita virusa. Virusi izazivaju na pšenici zaraze različitog intenziteta, od latentnih do letalnih zaraza. Iako nisu stalno prisutni u usevu, zaraze pojedinih godina mogu znatno da smanje prinos, od 5 do 10%. Virusi postaju ekonomski značajni patogeni pšenice kada se jave u epidemijskim razmerama. Najvažnija oboljenja pšenice i drugih žitarica izaziva jedan kompleks virusa, nazvan virus žute patuljavosti ječma (Barley yellow dwarf virus, BYDV, Luteovirus, Luteoviridae). Pored njega, najraširenijim se smatraju virus crticastog mozaika pšenice (Wheat streak mosaic virus, WSMV, Tritimovirus, Potyviridae) i virus mozaika pšenice koji se prenosi zemljišnim pseudogljivama (Soil-borne wheat mosaic virus, SBWMV, Furovirus, Virgaviridae). Pored njih, ekonomski značajnim virusima, smatraju se i drugi virusi koji se prenose grinjama, biljnim vašima ili zemljišnim pseudogljivama. U radu ce biti detaljno opisani virusi koji su prisutni na pšenici u Srbiji: BYDV, WSMV i virus mozaika bromusa (Brome mosaic virus, BMV, Bromovirus, Bromoviridae). Takođe, biće prodiskutovane i najbolje strategije kontrole, kao što su otpornost sorti prema virusima i/ili prema vektorima, hemijska kontrola vektora, plodored i sanitarne mere.

Plant RNA viruses are intracellular infectious agents with limited coding capacity. Therefore, these viruses have developed sophisticated ways to co-opt numerous cellular factors to facilitate the viral infectious cycle. To understand virus-host interactions, it is necessary to identify all the host components that are co-opted for viral infections. Development of yeast (Saccharomyces cerevisiae) as a host greatly facilitated the progress in our understanding of plant virus, such as brome mosaic virus (BMV) and tomato bushy stunt virus (TBSV), interactions with the host cells. Systematic genome-wide screens using yeast genomic libraries have led to the identification of a large number of host factors affecting (+)RNA virus replication. In combination with proteomic approaches, both susceptibility and restriction factors for BMV and TBSV have been identified using yeast. More detailed biochemical and cellular studies then led to the dissection of molecular functions of many host factors that promote each step of the viral replication process. The development of in vitro systems with TBSV, such as yeast cell-free extract and purified active replicase assays, together with proteomics, lipidomics and artificial vesicle-based assays helped to comprehend the complex nature of virus replication. Subsequently, comparable pro- or antiviral functions of several of the characterized yeast host factors have been validated in plant hosts. Overall, yeast is an advanced model organism that has emerged as an attractive host to gain insights into the intricate interactions of plant viruses with host cells. This chapter describes our current understanding of virus-host interactions, based mostly on TBSV-yeast system. Many of the pioneering findings with TBSV are likely applicable to other plant and animal viruses and their interactions with their hosts. The gained knowledge on host factors could lead to novel specific or broad-range antiviral tools against viruses.

NAC (NAM/ATAF/CUC) transcription factors have important functions in regulating plant growth, development, and abiotic and biotic stress responses. Here, we characterized two rice pathogen-responsive NAC transcription factors, ONAC122 and ONAC131. We determined that these proteins localized to the nucleus when expressed ectopically and had transcriptional activation activities. ONAC122 and ONAC131 expression was induced after infection by Magnaporthe grisea, the causal agent of rice blast disease, and the M. grisea-induced expression of both genes was faster and higher in the incompatible interaction compared with the compatible interaction during early stages of infection. ONAC122 and ONAC131 were also induced by treatment with salicylic acid, methyl jasmonate or 1-aminocyclopropane-1-carboxylic acid (a precursor of ethylene). Silencing ONAC122 or ONAC131 expression using a newly modified Brome mosaic virus (BMV)-based silencing vector resulted in an enhanced susceptibility to M. grisea. Furthermore, expression levels of several other defense- and signaling-related genes (i.e. OsLOX, OsPR1a, OsWRKY45 and OsNH1) were down-regulated in plants silenced for ONAC122 or ONAC131 expression via the BMV-based silencing system. Our results suggest that both ONAC122 and ONAC131 have important roles in rice disease resistance responses through the regulated expression of other defense- and signaling-related genes.

Many scientists, if not all, feel that their particular plant virus should appear in any list of the most important plant viruses. However, to our knowledge, no such list exists. The aim of this review was to survey all plant virologists with an association with Molecular Plant Pathology and ask them to nominate which plant viruses they would place in a 'Top 10' based on scientific/economic importance. The survey generated more than 250 votes from the international community, and allowed the generation of a Top 10 plant virus list for Molecular Plant Pathology. The Top 10 list includes, in rank order, (1) Tobacco mosaic virus, (2) Tomato spotted wilt virus, (3) Tomato yellow leaf curl virus, (4) Cucumber mosaic virus, (5) Potato virus Y, (6) Cauliflower mosaic virus, (7) African cassava mosaic virus, (8) Plum pox virus, (9) Brome mosaic virus and (10) Potato virus X, with honourable mentions for viruses just missing out on the Top 10, including Citrus tristeza virus, Barley yellow dwarf virus, Potato leafroll virus and Tomato bushy stunt virus. This review article presents a short review on each virus of the Top 10 list and its importance, with the intent of initiating discussion and debate amongst the plant virology community, as well as laying down a benchmark, as it will be interesting to see in future years how perceptions change and which viruses enter and leave the Top 10.

The capsids of most plant viruses are simple and robust structures consisting of multiple copies of one or a few types of protein subunit arranged with either icosahedral or helical symmetry. In many cases, capsids can be produced in large quantities either by the infection of plants or by the expression of the subunit(s) in a variety of heterologous systems. In view of their relative simplicity, stability and ease of production, plant virus particles or virus-like particles (VLPs) have attracted attention as potential reagents for applications in bionanotechnology. As a result, plant virus particles have been subjected to both genetic and chemical modification, have been used to encapsulate foreign material and have, themselves, been incorporated into supramolecular structures.

This article demonstrates the encapsulation of cubic iron oxide nanoparticles (NPs) by Brome mosaic virus capsid shells and the formation, for the first time, of virus-based nanoparticles (VNPs) with cubic cores. Cubic iron oxide NPs functionalized with phospholipids containing poly(ethylene glycol) tails and terminal carboxyl groups exhibited exceptional relaxivity in magnetic resonance imaging experiments, which opens the way for in vivo MRI studies of systemic virus movement in plants. Preliminary data on cell-to-cell and long-distance transit behavior of cubic iron oxide NPs and VNPs in Nicotiana benthamiana leaves indicate that VNPs have specific transit properties, i.e., penetration into tissue and long-distance transfer through the vasculature in N. benthamiana plants, even at low temperature (6 °C), while NPs devoid of virus protein coats exhibit limited transport by comparison. These particles potentially open new opportunities for high-contrast functional imaging in plants and for the delivery of therapeutic antimicrobial cores into plants.

Cell-penetrating peptides (CPP) can translocate across the cell membrane and have been extensively studied for the delivery of proteins, nucleic acids, and therapeutics in mammalian cells. However, characterizations of CPP in plants have only recently been initiated. We showed that the intact virion and a recombinant capsid protein (CaP) from a plant-infecting nonenveloped icosahedral RNA virus, Brome mosaic virus (BMV), can penetrate the membranes of plant protoplasts but are trapped by the extracellular matrix. Furthermore, a 22-residue peptide derived from the N-terminal region of the CaP (CPNT) can enter barley protoplasts and cells of intact barley and Arabidopsis roots. An inhibitor of the macropinocytosis reduced CPNT entry, while treatment with NiCl2 changed the cellular localization of CPNT. CPNT increased uptake of the green flourescent protein (GFP) into the cell when covalently fused to GFP or when present in trans of GFP. The BMV CPNT overlaps with the sequence known to bind BMV RNA, and it can deliver BMV RNAs into cells, resulting in viral replication, as well as deliver double-stranded RNAs that can induce gene silencing.

An isolate of Brome mosaic virus (BMV) was originally isolated from agropyron repens and maintained in Hordeum vulgare. The full-length genome of this isolate (BMV-CZ) was sequenced. Phylogenetic analysis revealed that BMV-CZ shared a minimum of 95.6% sequence identity, localized in the 5′-UTR of RNA-1 with the other BMV isolates from the database, and a maximum of divergence of 30.8% with Broad bean mottle virus localized in the 5′-UTR of RNA-3. This is the first sequence report of full-length BMV from the Czech Republic.

The Brome mosaic virus (BMV) coat protein (CP) accompanies the three BMV genomic RNAs and the subgenomic RNA into and out of cells in an infection cycle. In addition to serving as a protective shell for all of the BMV RNAs, CP plays regulatory roles during the infection process that are mediated through specific binding of RNA elements in the BMV genome. One regulatory RNA element is the B box present in the 5' untranslated region (UTR) of BMV RNA1 and RNA2 that play important roles in the formation of the BMV replication factory, as well as the regulation of translation. A second element is within the tRNA-like 3' UTR of all BMV RNAs that is required for efficient RNA replication. The BMV CP can also encapsidate ligand-coated metal nanoparticles to form virus-like particles (VLPs). This update summarizes the interaction between the BMV CP and RNAs that can regulate RNA synthesis, translation and RNA encapsidation, as well as the formation of VLPs.