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 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.     

Barley stripe mosaic virus and the frequency of triploids and aneuploids in barley

BSMV infection caused a pronounced increase in the frequency of triploid and aneuploid seeds in eleven barley varieties, but with considerable variation in frequency among varieties. In some of the varieties triploids exceeded three per cent. In virus-free material a few triploids were found in most of the varieties, but the frequency was very low. There was, however, a significant variation among varieties.     

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.     

Comparison of barley stripe mosaic virus strains

BSMV (barley stripe mosaic virus) particles were obtained in a pure state from infected host plant tissues of Hordeum vulgare. The three genomic parities (alpha, beta and gamma) were amplified by PCR using specific primers for each particle; each was cloned. Partial sequence of the alpha, beta and gamma segments was determined for the Egyptian isolate of barley stripe mosaic virus (BSMV AE1). Alignment of nucleotide sequences with that of other known strains of the virus, BSMV type strains (CV17, ND18 and China), and the generation of phylogenetic trees was performed. A low level of homology was detected comparing 467 bp of the a and 643 bp of the segments to that of the other strains, and thus BSMV alpha and beta segments were in separate clusters. However, 1154 bp of the gamma segments of BSMV AE1 showed a high level of homology especially to strain BSMV ND18, as they both formed a distinct cluster. Northern blotting of pure BSMV AE1 virus and H. vulgare-infected tissue were compared using an alpha ND18 specific probe. Western blotting using antibodies specific for the coat protein (CP) and the triple gene block 1 (TGB1) protein, which are both encoded by the beta ND18 segment, still indicated a high level of similarity between proteins produced by BSMV ND18 and AE1. We suggest that the BSMV AE1 isolate is a distinct strain of BSMV which reflects the genetic evolutionary divergence among BSMV strains and members of the Hordeivirus group.     

Triple gene block protein interactions involved in movement of Barley stripe mosaic virus

Barley stripe mosaic virus (BSMV) encodes three movement proteins in an overlapping triple gene block (TGB), but little is known about the physical interactions of these proteins. We have characterized a ribonucleoprotein (RNP) complex consisting of the TGB1 protein and plus-sense BSMV RNAs from infected barley plants and have identified TGB1 complexes in planta and in vitro. Homologous TGB1 binding was disrupted by site-specific mutations in each of the first two N-terminal helicase motifs but not by mutations in two C-terminal helicase motifs. The TGB2 and TGB3 proteins were not detected in the RNP, but affinity chromatography and yeast two-hybrid experiments demonstrated that TGB1 binds to TGB3 and that TGB2 and TGB3 form heterologous interactions. These interactions required the TGB2 glycine 40 and the TGB3 isoleucine 108 residues, and BSMV mutants containing these amino acid substitution were unable to move from cell to cell. Infectivity experiments indicated that TGB1 separated on a different genomic RNA from TGB2 and TGB3 could function in limited cell-to-cell movement but that the rates of movement depended on the levels of expression of the proteins and the contexts in which they are expressed. Moreover, elevated expression of the wild-type TGB3 protein interfered with cell-to-cell movement but movement was not affected by the similar expression of a TGB3 mutant that fails to interact with TGB2. These experiments suggest that BSMV movement requires physical interactions of TGB2 and TGB3 and that substantial deviation from the TGB protein ratios expressed by the wild-type virus compromises movement.     

Efficient virus-induced gene silencing in Arabidopsis

Virus-induced gene silencing (VIGS) is a plant RNA-silencing technique that uses viral vectors carrying a fragment of a gene of interest to generate double-stranded RNA, which initiates the silencing of the target gene. Several viral vectors have been developed for VIGS and they have been successfully used in reverse genetics studies of a variety of processes occurring in plants. This approach has not been widely adopted for the model dicotyledonous species Arabidopsis (Arabidopsis thaliana), possibly because, until now, there has been no easy protocol for effective VIGS in this species. Here, we show that a widely used tobacco rattle virus-based VIGS vector can be used for silencing genes in Arabidopsis ecotype Columbia-0. The protocol involves agroinfiltration of VIGS vectors carrying fragments of genes of interest into seedlings at the two- to three-leaf stage and requires minimal modification of existing protocols for VIGS with tobacco rattle virus vectors in other species like Nicotiana benthamiana and tomato (Lycopersicon esculentum). The method described here gives efficient silencing in Arabidopsis ecotype Columbia-0. We show that VIGS can be used to silence genes involved in general metabolism and defense and it is also effective at knocking down expression of highly expressed transgenes. A marker system to monitor the progress and efficiency of VIGS is also described.     

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.     

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.     

Mutations of the Adh1 gene in maize following infection with barley stripe mosaic virus

Summary Mutations at the Adh1 locus in maize were selected from plants infected with barley stripe mosaic virus (BSMV). Pollen from the infected inbred line 1s2p, which is homozygous for Adh1-S (abbreviated S), Adh2-P, c and r was treated with allyl alcohol and applied to silks of a tester stock homozygous for Adh1-F, Adh2-N, C and R. From these pollinations 356 kernels arose on the F₁ ears. Of these eight showed no activity of the S allele in scutellar samples while two exhibited low levels. Five of the putative mutant kernels germinated and two of these contained the contamination markers Adh2-P, c and r. The newly arisen mutations were designated S5446 and S5453. S5453 exhibited an abnormally low level of ADH activity in the F₁ scutellum. In the F₂ generation the mutant reverted at a high frequency with only about 5% of the S5453 alleles expressing low levels. DNA blotting and hybridization analyses showed no alterations in the restriction patterns of S5453 when compared to the progenitor S allele. S5446 which exhibited no ADH activity in the F₁ scutellum is unstable in the pollen; reversion frequencies approaching 10^-2 were observed in samples from some plants. Restriction digestion patterns of DNA from this mutant revealed the presence of a 3.3 kb insertion at Adh. The insert does not appear to contain sequences homologous to the BSMV genome but rigorous analyses remain to be carried out. It is hypothesized that BSMV infection may mobilize endogenous but dormant transposable elements in maize.     

Effects of barley yellow mosaic disease resistant gene rym1 on the infection by strains of Barley yellow mosaic virus and Barley mild mosaic virus

Although a Chinese landrace of barley, Mokusekko 3, is completely resistant to all strains of Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), and is known to have at least two resistant genes, rym1 and rym5, only rym5 has been utilized for BaYMV resistant barley breeding in Japan. In order to clarify the effect of rym1 on BaYMV and BaMMV, and to utilize the gene for resistant barley breeding, the susceptibilities of only rym1 carrying breeding lines against BaYMV and BaMMV were investigated. In the assessment of resistance to BaYMV-I, 341 F(2) populations derived from a cross between the resistant line Y4 with only rym1 and the susceptible cv Haruna Nijo shows that the segregation loosely fits a 1R:3S ratio (0.05 > P > 0.01), suggesting that the resistance is controlled by a single recessive gene, rym1. Further, none of the F(3) lines derived from the nine resistant F(2) plants showed any disease symptoms in the field infected by BaYMV-I. The same nine F(3) lines showed almost the same agronomic characters in the field infected by BaYMV-III as those in the uninfected field, apart from the symptom of showing numerous mosaics. This result indicates that the gene rym1 has an acceptable level of resistance to BaYMV-III. In the assessment of resistance to BaYMV-II, BaMMV-Ka1 and -Na1, an artificial infection method was adopted and the susceptibilities to those viruses were investigated. Although the control varieties, Ko A and Haruna Nijo, were infected with all of them, the rym1 gene carrying BC(2)F(3) lines were completely resistant to all strains. In summary, rym1 is completely resistant to BaYMV-I, -II, BaMMV-Ka1 and -Na1, and has an acceptable level of resistance to BaYMV-III. This study concludes with a discussion of the reason why the important resistance gene rym1 was eliminated along with resistant cultivars during breeding for resistance to BaYMV.     

Immunohistochemical localization of barley stripe mosaic virions in infected wheat cells

Barley stripe mosaic virus particles were localized in ultrathin sections with colloidal gold-labeled specific IgG or antiserum followed by gold-labeled goat anti-rabbit IgG. On the average, 1.5 gold particles were attached per virus rod. A statistical analysis of counts of gold and virus particles showed that the staining procedure was highly reproducible from experiment to experiment and after several independently prepared colloidal gold solutions. The procedure should be useful for the intracellular localization of any protein to which an antibody can be prepared.     

A study of barley stripe mosaic virus (BSMV) genome

Summary The cell cycle mutant, cdc9, in the yeast Saccharomyces cerevisiae is defective in DNA ligase be deficient in the repair of DNA damaged by methyl methane sulphonate. On the other hand survival of cdc9 after irradiation by -rays is little diferent from that of the wild-type, even after a period of stress at the restrictive temperature. The mutant cdc9 is not allelic with any known rad or mms mutants.     

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.     

Alterations of NADPH:protochlorophyllide oxidoreductase quantity and lipid composition in etiolated barley seedlings infected by Barley stripe mosaic virus (BSMV)

To understand the phenomenon by which infection of seed-transmitted Barley stripe mosaic virus (BSMV) alters membrane structures and inhibits protochlorophyllide biosynthesis of dark-grown barley ( Hordeum vulgare L.) plants, we analysed the presence of NADPH:protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) and the galactolipid content and fatty acid composition. The amount of POR in etioplasts of infected leaves, compared with non-infected leaves, was reduced, as measured by immunoelectron microscopy and Western blot. These results are in agreement with the previously described reduction of the ratio of the photoactive 650 nm to non-photoactive 630 nm absorbing protochlorophyllide forms ( Harsányi et al. , 2002 . Physiol. Plant 114 , 149–155). The galactolipid content was lower in infected leaves. Monogalactosyl-diacylglycerol (MGDG) content was reduced to 40% and digalactosyl-diacylglycerol to 55% of control plants on a fresh weight basis. In infected plants, the proportion of linolenic acid decreased in both galactolipids. The lower amount of highly unsaturated fatty acids and the reduced abundance of MGDG correlated well with the previously detected reduction in the membrane ratio of prolamellar body (PLB) to prothylakoid ( Harsányi et al. , 2002 . Physiol. Plant 114 , 149–155). The reduced amount of POR and the above described alterations in the lipid composition resulted in a disturbed structure of PLBs. As a consequence, pigment synthesis and the greening process were inhibited in infected cells, in turn explaining the appearance of chlorotic stripes of BSMV-infected barley leaves. Our results show that BSMV infection can be detected at a very early stage of leaf development.