A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots

Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.     

Genotoxicity of barley stripe mosaic virus in infected host plants

A comparative study of the effect of barley stripe mosaic virus (BSMV) and gamma irradiation on mitotic divisions in barley (Hordeum vulgare L.) roots was performed by evaluating the mitotic index (MI), micronucleus (MN) frequency and sister chromatid exchanges (SCE). Results indicate that, similarly to gamma irradiation at doses of 100, 150 and 250 Gy, BSMV reduces the mitotic activity, increases the micronucleus frequency and the rate of SCE and promotes the formation of C-metaphases. In root meristematic cells of the three barley cultivars studied (Galactic, Sonor and Unirea), the mitotic index of infected plants was found to be 52.5, 54.48 and 64.17%, respectively, lower than the uninfected control. An increase in frequency of sister chromatid exchanges was observed in all the experimental variants. In treatments involving viral infection alone or in combination with gamma irradiation chromosomes with three and more chromatid exchanges were observed, while their percentage in the control or in treatments with gamma irradiation alone was reduced. The results of the study indicate that in plants derived from irradiated seeds, BSMV produces an effect that is correlated nonlinearly with the radiation dose applied. Cytological analysis of mitotic divisions in barley roots revealed the genotoxicity of BSMV infection.     

The structure of barley stripe mosaic virus double-stranded RNAs

The terminal structures of the double-stranded replicative forms (RFs) of barley stripe mosaic virus (BSMV) RNAs 1–3 have been investigated. All three BSMV RFs have identical right-hand ends but unique left-hand ends. The plus (+) strands of RFs lack the 3′-ultimate A typical for the encapsidated BSMV RNAS. The 3′-termini of the minus (−) strands contain an unpaired G. It was demonstrated that the internal poly(A) tract of BSMV genome has an equivalent poly(U)-counterpart in the RF (−) strands. The possible role of these peculiarities of BSMV RF structure in RNA replication is discussed.     

大麦条纹花叶病毒新疆株血清学及基因同源性检测

在我国新疆从小麦上分离到的大麦条纹花叶病毒(Barley stripe mosaic Virus,BSMV),可以侵染大麦、小麦、玉米等重要粮食作物。主要以带毒种子传播病毒。大麦、小麦种子带毒率达70％以上。BSMV曾一度使世界主要的大麦产区减产25～30％,是影响农业生产的重要病毒。从预防病害流行和探索该种子传RNA病毒作为植物基因工程载体的可     

Fine mapping of the Bsr1 barley stripe mosaic virus resistance gene in the model grass Brachypodium distachyon

The ND18 strain of Barley stripe mosaic virus (BSMV) infects several lines of Brachypodium distachyon, a recently developed model system for genomics research in cereals. Among the inbred lines tested, Bd3-1 is highly resistant at 20 to 25 °C, whereas Bd21 is susceptible and infection results in an intense mosaic phenotype accompanied by high levels of replicating virus. We generated an F(6:7) recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21 and used the RILs, and an F(2) population of a second Bd21 × Bd3-1 cross to evaluate the inheritance of resistance. The results indicate that resistance segregates as expected for a single dominant gene, which we have designated Barley stripe mosaic virus resistance 1 (Bsr1). We constructed a genetic linkage map of the RIL population using SNP markers to map this gene to within 705 Kb of the distal end of the top of chromosome 3. Additional CAPS and Indel markers were used to fine map Bsr1 to a 23 Kb interval containing five putative genes. Our study demonstrates the power of using RILs to rapidly map the genetic determinants of BSMV resistance in Brachypodium. Moreover, the RILs and their associated genetic map, when combined with the complete genomic sequence of Brachypodium, provide new resources for genetic analyses of many other traits.     

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus.

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus (BSMV) has been determined. The sequence is 3289 nucleotides in length and contains four open reading frames (ORFs) which code for proteins of Mr 22,147 (ORF1), Mr 58,098 (ORF2), Mr 17,378 (ORF3), and Mr 14,119 (ORF4). The predicted N-terminal amino acid sequence of the polypeptide encoded by the ORF nearest the 5'-end of the RNA (ORF1) is identical (after the initiator methionine) to the published N-terminal amino acid sequence of BSMV coat protein for 29 of the first 30 amino acids. ORF2 occupies the central portion of the coding region of RNA beta and ORF3 is located at the 3'-end. The ORF4 sequence overlaps the 3'-region of ORF2 and the 5'-region of ORF3 and differs in codon usage from the other three RNA beta ORFs. The coding region of RNA beta is followed by a poly(A) tract and a 238 nucleotide tRNA-like structure which are common to all three BSMV genomic RNAs.     

Amino acid sequence analysis of barley stripe mosaic viral proteins: tentative identification of viral serine proteases

Analysis of amino acid sequences of barley stripe mosaic virus (BSMV) proteins revealed the pentapeptide GDSGG, the sequence unique for catalytic centers of serine chymotrypsin-like proteases, in protein p14 encoded by open reading frame 4 of RNA beta. Computer-assisted comparisons revealed a statistically significant similarity between amino acid sequences of p14 and chymotrypsin-like proteases. The catalytic His and Asp residues tentatively identified in p14 together with the Ser residue of the GDSGG sequence, presumably, constitute the "catalytic triad" characteristic of chymotrypsin-like proteases. Based on these observations and on the presence of a potential N-proximal transmembrane domain in p14, this protein may be suggested to be a serine protease involved in processing of the replicase precursor within a membrane-bound replication complex of BSMV.     

Production and some characteristics of monoclonal antibodies against beet necrotic yellow vein virus

Four rat monoclonal antibodies (MAbs) specific for beet necrotic yellow vein virus (BNYVV) were produced. In indirect ELISA, all four MAbs reacted strongly with BNYVV infected plant leaf extracts (19 isolates from eight countries) but they did not react with beet soil-borne virus (BSBV), an unnamed rod-shaped soil-borne beet virus isolate (86 - 109) from Sweden or barley stripe mosaic virus (BSMV). However, two of the MAbs, MAFF 6 and MAFF 7 did not detect BNYVV in ELISA of infected sugar beet roots whereas MAbs MAFF 8 and MAFF 9 did detect virus in root extracts. In electro-blot immunoassay (EBIA), MAFF 6 and MAFF 7 readily detected BNYVV coat protein from leaf extracts whereas MAFF 8 and MAFF 9 reacted only weakly. None of the MAbs reacted with BSBV, 86 - 109, BSMV or plum pox virus in EBIA. MAFF 6 coated BNYVV particles which were trapped from infected leaf or root sap on to electron microscope grids by polyclonal antibodies. MAFF 6 was partially purified from tissue culture supernatant fluid by cation exchange chromatography and the preparation used to coat microtitre plates and successfully trap BNYVV in ELISA of leaf sap extracts.     

Nucleotide sequence of the 3''-terminal tRNA-like structure in barley stripe mosaic virus genome.

This paper describes the sequence of 257 nucleotides from the 3' end of RNA 2 of barley stripe mosaic virus ( BSMV , strain Argentina Mild) including an internal oligo (A) tract localized at a distance of 236 nucleotides from the 3' end, and the 3' terminal tRNA-like structure accepting tyrosine. This sequence is shown to be the same with RNAs 1,2 and 3 of another BSMV strain, Norwich , for at least the first 106 nucleotides from the 3' end. The 3' extremity of BSMV RNA bears some resemblance to tRNATyr from yeast in its primary structure. The possible secondary structures of the tRNA-like sequence in BSMV genome are discussed.     

The barley stripe mosaic virus 58-kilodalton beta(b) protein is a multifunctional RNA binding protein.

The barley stripe mosaic virus (BSMV) beta(b) gene product is the major viral nonstructural protein synthesized during early stages of the infection cycle and is required for systemic movement of the virus. To examine the biochemical properties of beta(b), a histidine tag was engineered at the amino terminus and the protein was purified from BSMV-infected barley tissue by metal affinity chromatography. The beta(b) protein bound ATPs in vitro, with a preference for ATP over dATP, and also exhibited ATPase activity. In addition, beta(b) bound RNA without detectable sequence specificity. However, binding was selective, as the beta(b) protein had a strong affinity for both single-stranded (ss) and double-stranded (ds) RNAs but not for tRNA or DNA substrates. Mutational analyses of beta(b) purified from Escherichia coli indicated that the protein has multiple RNA binding sites. These sites appear to contribute differently, because mutants that were altered in their binding affinities for ss and ds RNA substrates were recovered.     

Detection of Four Seed-borne Plant Viruses by the Enzyme-Linked Immunosorbent Assay (ELISA)

This study was conducted to evaluate the sensitivity of the ELISA technique in detecting four economically important viruses, namely barley stripe mosaic (BSMV), cucumber green mottle mosaic (CGMMV), bean common mosaic (BCMV), and squash mosaic (BSMV) viruses in single seeds as well as in batches of barley, cucumber, bean and squash seeds, respectively. Results indicated the suitability of the technique in detecting the above viruses in single germinated seeds or embryos. Accordingly, seed transmission rates of BSMV, CGMMV, BCMV and SqMV were found to be 67 %, 17%, 17% and 12%, respectively. In artificially contrived mixtures of infected: healthy seeds or embryos, BSMV, CGMMV, BCMV and SqMV were successfully detected at ratios of 1 : 500, 1 : 25, 1 : 10 and 1 : 10, respectively. Sensitivity of detection was increased in the ease of BSMV by using germinated rather than ground dry BSMV-infected barly seeds; and in the case of SqMV, by using whole germinating emybryos rather than coleoptiles only. Trials on re-using the enzyme-γ-globulin conjugate indicated that CGMMV conjugate used once can be re-used with little loss in reactivity.     

Probable reassortment of genomic elements among elongated RNA-containing plant viruses

Summary The relationships of genome organization among elongated (rod-shaped and filamentous) plant viruses have been analyzed. Sequences in coding and noncoding regions of barley stripe mosaic virus (BSMV) RNAs 1, 2, and 3 were compared with those of the monopartite RNA genomes of potato virus X (PVX), white clover mosaic virus (WClMV), and tobacco mosaic virus, the bipartite genome of tobacco rattle virus (TRV), the quadripartite genome of beet necrotic yellow vein virus (BNYVV), and icosahedral tricornaviruses. These plant viruses belong to a supergroup having 5-capped genomic RNAs. The results suggest that the genomic elements in each BSMV RNA are phylogenetically related to those of different plant RNA viruses. RNA 1 resembles the corresponding RNA 1 of tricornaviruses. The putative proteins encoded in BSMV RNA 2 are related to the products of BNYVV RNA 2, PVX RNA, and WClMV RNA. Amino acid sequence comparisons suggest that BSMV RNA 3 resembles TRV RNA 1. Also, it can be proposed that in the case of monopartite genomes, as a rule, every gene or block of genes retains phylogenetic relationships that are independent of adjacent genomic elements of the same RNA. Such differential evolution of individual elements of one and the same viral genome implies a prominent role for gene reassortment in the formation of viral genetic systems.     

Barley stripe mosaic virus-induced gene silencing in a monocot plant

RNA silencing of endogenous plant genes can be achieved by virus-mediated, transient expression of homologous gene fragments. This powerful, reverse genetic approach, known as virus-induced gene silencing (VIGS), has been demonstrated only in dicot plant species, where it has become an important tool for functional genomics. Barley stripe mosaic virus (BSMV) is a tripartite, positive-sense RNA virus that infects many agriculturally important monocot species including barley, oats, wheat and maize. To demonstrate VIGS in a monocot host, we modified BSMV to express untranslatable foreign inserts downstream of the gammab gene, in either sense or antisense orientations. Phytoene desaturase (PDS) is required for synthesizing carotenoids, compounds that protect chlorophyll from photo-bleaching. A partial PDS cDNA amplified from barley was 90, 88 and 74% identical to PDS cDNAs from rice, maize and Nicotiana benthamiana, respectively. Barley infected with BSMV expressing barley, rice or maize PDS fragments became photo-bleached and accumulated phytoene (the substrate for PDS) in a manner similar to plants treated with the chemical inhibitor of PDS, norflurazon. In contrast, barley infected with wild-type BSMV, or BSMV expressing either N. benthamiana PDS or antisense green fluorescent protein (GFP), did not photo-bleach or accumulate phytoene. Thus BSMV silencing of the endogenous PDS was homology-dependent. Deletion of the coat protein enhanced the ability of BSMV to silence PDS. This is the first demonstration of VIGS in a monocot, and suggests that BSMV can be used for functional genomics and studies of RNA-silencing mechanisms in monocot plant species.     

The complete nucleotide sequence of the potexvirus white clover mosaic virus.

The complete nucleotide sequence (5845 nucleotides) of the genomic RNA of the potexvirus white clover mosaic virus (WC1MV) has been determined from a set of overlapping cDNA clones. Forty of the most 5'-terminal nucleotides of WC1MV showed homology to the 5' sequences of other potexviruses. The genome contained five open reading frames which coded for proteins of Mr 147, 417, Mr 26,356, Mr 12,989, Mr 7,219 and Mr 20,684 (the coat protein). The Mr 147,417 protein had domains of amino acid sequence homology with putative polymerases of other RNA viruses. The Mr 26,356 and Mr 12,989 proteins had homology with proteins of the hordeivirus barley stripe mosaic virus RNA beta and the furovirus beet necrotic yellow vein virus (BNYVV) RNA-2. A portion of the Mr 26,356 protein was also conserved in the cylindrical inclusion proteins of two potyviruses. The Mr 7,219 protein had homology with the 25K putative fungal transmission factor of BNYVV RNA-3.     

An empirical study of the evolution of virulence under both horizontal and vertical transmission

According to current thinking, a parasite's transmission mode will be a major determinant of virulence, defined as the harm induced by parasites to their hosts. With horizontal transmission, virulence will increase as a byproduct of a trade-off between fitness gained through increased among-host transmission (infectivity) and fitness lost through increased virulence. With vertical transmission, virulence will decrease because a parasite's reproductive potential will be maximized only by decreasing harm to the host, allowing parasite transmission to more host offspring. To test both predictions, we transmitted barley stripe mosaic virus (BSMV) horizontally and then vertically in its host, barley (Hordeum vulgare). After four generations of horizontal transmission, we observed a nearly twofold increase in horizontal infectivity and nearly tripled virulence. After three generations of subsequent vertical transmission, we observed a modest (16%) increase in vertical transmissibility and a large (40%) reduction in virulence. Increased horizontal transmission is often due to increased pathogen replication which, in turn, causes increased virulence. However, we found no correlation between within-host virus concentration and virulence, indicating that the observed changes in virulence were not due to changes in viral titer. Finally, horizontally transmitted BSMV had reduced vertical transmission and vertically transmitted BSMV had reduced horizontal infectivity. These two observations suggest that, in nature, in different host populations with varying opportunities for horizontal and vertical transmission, different viral strains may be favored.     

The Barley stripe mosaic virus system used for virus-induced gene silencing in cereals differentially affects susceptibility to fungal pathogens in wheat

Barley stripe mosaic virus (BSMV) has emerged as a vector for virus-induced gene silencing (VIGS) in cereals, having been used to study a number of genes involved in resistance in both wheat and barley. However, the effects of the BSMV vector on plant physiology and disease resistance in plants remains unexplored. The BSMV inoculation control vector, BSMV:GFP was shown to cause severe viral symptoms in wheat, displaying chlorosis, leaf curling and growth inhibition typical of the symptoms seen in BSMV-infected barley. These viral symptoms were accompanied by induction of genes implicated in defense against pathogens, namely PR1, PR4, PR5, PR10 and PAL. Subsequent inoculation of BSMV:GFP-infected wheat with a wheat pathotype of Magnaporthe oryzae, the blast pathogen, resulted in decreased susceptibility. Penetration of epidermal cells and subsequent multiple cell colonization by M. oryzae was significantly reduced. This increased restriction of pathogen growth observed for BSMV:GFP infections with and without the viral coat protein gene. However, prior infection with BSMV:GFP had no effect on the development of a compatible isolate of Blumeria graminis f. sp. tritici, the causal agent of powdery mildew.     

Molecular analysis of the capsid protein gene of a german isolate of barley mild mosaic virus

Summary Barley mild mosaic virus (BaMMV) is one of the agents causing the barley yellow mosaic disease. The sequence corresponding to the 3end of the BaMMV RNA1 of a German isolate was sequenced and the coding sequence for the 251 amino acid containing capsid protein was determined. Comparison of this sequence to other potyviral sequences and to the corresponding sequence of two Japanese isolates of BaMMV was done. The three different isolates of BaMMV show a high degree of similarity.Abbrevations BaMMV barley mild mosaic virus - BaYMV barley yellow mosaic virus; bp: base pair - IPTG isopropyl -D thiogalactopyranoside - kb kilo base - NTR nontranslated region - ORF open reading frame - PVDF polyvinylidene difluoride