Barley Stripe Mosaic Virus RNA Research Articles

Development and optimization of a Barley stripe mosaic virus-mediated gene editing system to improve Fusarium head blight resistance in wheat.

Development and optimization of a Barley stripe mosaic virus-mediated gene editing system to improve Fusarium head blight resistance in wheat.

Hijacking of the nucleolar protein fibrillarin by TGB1 is required for cell-to-cell movement of Barley stripe mosaic virus.

Barley stripe mosaic virus (BSMV) Triple Gene Block1 (TGB1) is a multifunctional movement protein with RNA-binding, ATPase and helicase activities which mainly localizes to the plasmodesmata (PD) in infected cells. Here, we show that TGB1 localizes to the nucleus and the nucleolus, as well as the cytoplasm, and that TGB1 nuclear-cytoplasmic trafficking is required for BSMV cell-to-cell movement. Prediction analyses and laser scanning confocal microscopy (LSCM) experiments verified that TGB1 possesses a nucleolar localization signal (NoLS) (amino acids 95-104) and a nuclear localization signal (NLS) (amino acids 227-238). NoLS mutations reduced BSMV cell-to-cell movement significantly, whereas NLS mutations almost completely abolished movement. Furthermore, neither the NoLS nor NLS mutant viruses could infect Nicotiana benthamiana systemically, although the NoLS mutant virus was able to establish systemic infections of barley. Protein interaction experiments demonstrated that TGB1 interacts directly with the glycine-arginine-rich (GAR) domain of the nucleolar protein fibrillarin (Fib2). Moreover, in BSMV-infected cells, Fib2 accumulation increased by about 60%-70% and co-localized with TGB1 in the plasmodesmata. In addition, BSMV cell-to-cell movement in fib2 knockdown transgenic plants was reduced to less than one-third of that of non-transgenic plants. Fib2 also co-localized with both TGB1 and BSMV RNA, which are the main components of the ribonucleoprotein (RNP) movement complex. Collectively, these results show that TGB1-Fib2 interactions play a direct role in cell-to-cell movement, and we propose that Fib2 is hijacked by BSMV TGB1 to form a BSMV RNP which functions in cell-to-cell movement.

The Barley stripe mosaic virus γb protein promotes chloroplast-targeted replication by enhancing unwinding of RNA duplexes.

RNA viruses encode various RNA binding proteins that function in many steps of viral infection cycles. These proteins function as RNA helicases, methyltransferases, RNA-dependent RNA polymerases, RNA silencing suppressors, RNA chaperones, movement proteins, and so on. Although many of the proteins bind the viral RNA genome during different stages of infection, our knowledge about the coordination of their functions is limited. In this study, we describe a novel role for the Barley stripe mosaic virus (BSMV) γb as an enhancer of αa RNA helicase activity, and we show that the γb protein is recruited by the αa viral replication protein to chloroplast membrane sites of BSMV replication. Mutagenesis or deletion of γb from BSMV resulted in reduced positive strand (+) RNAα accumulation, but γb mutations abolishing viral suppressor of RNA silencing (VSR) activity did not completely eliminate genomic RNA replication. In addition, cis- or trans-expression of the Tomato bushy stunt virus p19 VSR protein failed to complement the γb replication functions, indicating that the direct involvement of γb in BSMV RNA replication is independent of VSR functions. These data support a model whereby two BSMV-encoded RNA-binding proteins act coordinately to regulate viral genome replication and provide new insights into strategies whereby double-stranded viral RNA unwinding is regulated, as well as formation of viral replication complexes.

‘Phytochip’: On-chip detection of phytopathogenic RNA viruses by a new surface plasmon resonance platform

The surface plasmon resonance (SPR) based ‘Phytochip’ was developed to distinguish virus-infected plants from non-infected plants. The system detects DNA–RNA hybridization to show the presence of phytopathogenic viruses such as the RNA virus barley stripe mosaic virus (BSMV) in wheat leaves. To achieve this BSMV and wheat specific oligonucleotides, and a negative control yeast oligonucleotide, were immobilized on a SPR gold surface chip. After optimization of the hybridization parameters with purified wheat samples, wheat infected with BSMV resulted in detectable signals with both the BSMV and the wheat probes. In contrast, a hybridization reaction was not be detected with the negative probe. The method is fast and sensitive with a detection time of 3000s (50min), a detection limit of 14.7pgμl−1 BSMV RNA and a measuring range of 14.7–84pgμl−1 BSMV RNA (1.323–7.56ng BSMV RNA per 90μl sample). These characteristics, combined with the high throughput design, make it suitable for application in plant breeding and virus control.

Movement of a Barley Stripe Mosaic Virus Chimera with a Tobacco Mosaic Virus Movement Protein

The tobacco mosaic virus (TMV) 30K movement protein (MP) gene was inserted into a full-length cDNA clone of barley stripe mosaic virus (BSMV) RNAβ replacing the triple gene block (TGB). The resulting recombinant ND-MPT genome, consisting of infectious wt transcripts of BSMV RNAs α and γ, together with the hybrid RNAβ transcript, was inoculated onto test plants to study the functional compatibility between the BSMV TGB-adapted genetic system and the tobamovirus transport gene. ND-MPT infected the inoculated leaves ofNicotiana benthamianaandChenopodium amaranticolor,which are common hosts for the parental viruses; the size, growth rate, and morphology of local lesions onC. amaranticolorwere influenced by the foreign MP gene. However, the hybrid virus failed to infect barley,N. tabacum(var. Samsun), andN. clevelandii,the selective hosts. Thus, the TMV MP was able to functionally substitute for the BSMV TGB-coded MPs, i.e., the 30K MP functioned independently of any other BSMV sequences. However, the TMV MP gene promoted the cell-to-cell movement in a host-dependent manner.

Poa semilatent virus, a hordeivirus having no internal polydisperse poly(A) in the 3' non-coding region of the RNA genome.

RNA from the Hungarian isolate of poa semilatent virus (PSLV) directed in vitro synthesis of 120K, 75K, 25K (coat protein) and 20K polypeptides. In vitro translation of PSLV RNA was blocked by the cap analogue, m7Gpp, thus suggesting that the virus RNA was capped. PSLV RNA could be aminoacylated with [14C]tyrosine in vitro. The sequence of 1.5 kb from the 3' end of the PSLV RNA gamma component revealed two open reading frames (ORFs) separated by a uridine-rich intergenic region. The putative product of the incomplete 5'-proximal ORF showed a close amino acid sequence similarity with the C-terminal segment of the gamma a protein (putative RNA replicase) encoded in the barley stripe mosaic virus (BSMV) RNA gamma, and the 20K product of the 3'-proximal ORF was found to be related to the 17K gamma b product of BSMV. The sequence of 0.8 kb from the 3' end of PSLV RNA beta encompassed two (incomplete) overlapping ORFs whose putative products are related to the beta c and beta d proteins encoded in the similarly arranged ORFs of BSMV RNA beta. Nucleotide sequence homology between the respective parts of the two hordeivirus genomes was restricted to the ORF for gamma a, the spacer between the ORFs for gamma a and gamma b, and the 3' non-coding region, particularly the 95 nucleotide segment at the 3' end representing a tRNA-like structure. Despite limited sequence conservation beyond this segment, the entire 3' non-coding region of PSLV RNA could be folded in a tight pseudoknotted structure closely resembling that of BSMV RNA. Surprisingly, the 'signature' sequence typical for BSMV RNA, internal polydisperse poly(A) intercalated between the coding part of the 3' tRNA-like structure, was not detected in the PSLV genome. Instead, the virus RNA contained several oligoadenylate stretches spaced by other residues, close to the junction of its coding and 3' non-coding portions.

Mutational analysis of barley stripe mosaic virus rna β

Barley stripe mosaic virus (BSMV), the type member of the hordeivirus group, has a plus-stranded genome comprising RNA species designated α, β and γ Although RNA β is essential for infection of whole plants, it is dispensable for infection of barley protoplasts. We have used a full-length cDNA clone of RNA β from which infectious in vitro transcripts can be derived to construct a number of mutations in its four genes. Mutations introduced into the βb βc or βd genes eliminated infectivity of the RNA. The coat protein and the RNA sequences encoding the coat protein were completely dispensable for infection of barley plants by BSMV, and no detrimental effect on systemic movement of the virus was observed. However, besides eliminating coat protein expression in vivo, mutations within the coat protein gene and the first intercistronic region affected a number of other phenotypes: (1) expression of a downstream gene (βb), (2) stability of the genomic RNA during virus multiplication in planta, (3) the requirement for a trans-acting BSMV protein (γb), (4) symptomatology and disease development in infected barley plants, and (5) host range.

Nicotiana Velutina Mosaic Virus: Evidence for a Bipartite Genome Comprising 3 kb and 8 kb RNAs

DNA complementary to Nicotiana velutina mosaic virus (NVMV) RNA was cloned and five segments larger than 0.9 kb were used in Northern blot hybridization analysis to identify two virus-specific RNAs, approximately 8 kb (RNA 1) and 3 kb (RNA 2) in size. The clones selected as probes did not hybridize with RNA from various tobamoviruses, or from beet necrotic yellow vein (BNYVV) and peanut clump furoviruses. In an attempt to determine the taxonomic position of the virus, about 75% of the NVMV RNA 2 was sequenced and four open reading frames (ORFs) were identified. ORFs 1, 2 and 3 encode proteins of Mr 20K, 39K and 13K, whereas ORF 4 was incomplete. ORFs 2, 3 and 4 overlapped in an arrangement closely resembling the triple gene block identified in BNYVV RNA 2, barley stripe mosaic virus (BSMV) RNA 2, potato virus X and potato virus M RNA. The presumed coat protein gene of NVMV RNA 2 (ORF 1) is situated to the 5' side of the triple gene block as for BNYVV and BSMV RNA 2. Amino acid homologies were detected among the 13K and 14K proteins of NVMV RNA 2, BNYVV RNA 2 and BSMV RNA 2. Significant homology was also detected between the 39K protein of NVMV RNA 2 and the 42K protein of BNYVV RNA 2, with a motif specific for ATP- and GTP-binding (NTP-binding motif), and a conserved viral DNA polymerase domain. The presence of a triple gene block in NVMV RNA 2 indicates that NVMV has affinities with members of the hordei-, furo-, potex- and carlavirus groups but not with the tobamovirus group. The divided RNA genome of NVMV, and the sizes of the two RNAs suggest that NVMV is most closely allied to the furoviruses, but the unique nature of its different biological properties and lack of any serological relationships with furoviruses lead us to conclude that NVMV has no clear relatedness to any taxonomic group of plant viruses.

Infectious Barley stripe mosaic virus RNA transcribed in Vitro from full-length genomic cDNA clones

Full-length genomic cDNA clones of the Type and ND18 strains of barley stripe mosaic virus (BSMV) were transcribed in vitro using T7 RNA polymerase. The combination of RNAs α, β, and γ synthesized in the presence of 5′ cap analogs was infectious after inoculation onto barley plants, conclusively demonstrating the tripartite nature of the BSMV genome. Transcripts synthesized in the absence of cap analogs were not infectious. A γ-specific subgenomic RNA which is normally present in BSMV virions was not required to establish a systemic infection. In vitro transcripts of variant cDNA clones which were isolated from the ND18 strain, containing either a simple nucleotide substitution or a 372-nucleotide duplication similar to one found in the genome of the Type strain, were also found to be biologically active. Two dicotyledonous hosts which have a differential response to infection with the Type and ND18 strains of BSMV were identified and these phenotypes were shown to be faithfully reproduced by inoculation with in vitro transcripts derived from the appropriate full-length cDNA clones.

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

SummaryThe 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.

Site-specific cleavage and religation of viral RNAs I. Infectivity of barley stripe mosaic virus RNA religated from functionally active segments and restoration of the internal poly(A) tract in progeny

The genomic RNAs of barley stripe mosaic virus (BSMV) were used in experiments involving site-specific cleavage to yield noninfective but functionally active fragments and subsequent restoration of infectious viral RNA by means of religation. The specific fragments were obtained after site-specific cleavage of BSMV RNAs with RNase H in the presence of oligo(dT) 10 complementary to the internal poly(A) track in BSMV genomic RNAs: (i) long (L) 5′-terminal fragments, lacking the tyrosine-accepting activity, but capable of directing in vitro a full set of BSMV-specific polypeptides ( A. A. Agranovsky, V. V. Dolja, and J. G. Atabekov, 1982, Virology 119, 51–58) and (ii) short (Sh) fragments containing a tRNA-like structure accepting tyrosine. L- and Sh-fragments of BSMV RNAs were religated by T4 RNA ligase producing infectious BSMV RNA. The religated BSMV RNA was shown to lack the internal poly(A) track present in authentic BSMV RNA. Nevertheless the typical poly(A) spectra were restored in the progeny RNA from religated, poly(A) - deficient BSMV RNA.

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.

Differences in polyadenylate length between individual barley stripe mosaic virus RNA species

Barley stripe mosaic virus (BSMV) RNA which has been shown to contain an internal polyadenylate sequence (Agranovsky, Dolja, Gorbulev, Kozlov, and Atabekov, “Virology”, 113, 114–187, 1981; Agranovsky, Dolja, and Atabekov, “Virology,” 119, 51–58, 1982) was resolved into oligo(dT)-cellulose-bound [poly(A) +] and unbound [poly(A) −] fractions. Individual RNAs of all strains tested (i.e., those with two-, three-, or four-components) differentially responded to affinity chromatography. Thus, in all cases the poly(A) + fraction was dramatically enriched in RNA 1 whereas RNA 2 was found mostly in the poly(A) − fraction. RNA 3 was distributed approximately equally between poly(A) + and poly(A) − fractions. The bulk of RNA 4 was in the poly(A) − fraction. The poly(A) length spectra in total, poly(A) +, and poly(A) − BSMV RNA fractions were compared. The length of poly(A) in total BSMV RNA was 8 to 40 residues. Fragments of 19 to 40 residues and probably longer were predominant in the poly(A) + RNA fraction. The poly(A) − fraction contained tracts having not more than approximately 25 adenylate residues, with the bulk of the tracts having 12 or fewer residues.

Sequence relations and coding properties of a subgenomic RNA isolated from barley stripe mosaic virus

A subgenomic (SG) RNA (∼800 nucleotides) is a minor component of barley stripe mosaic virus RNAs. The SG-RNAs isolated from the Type and North Dakota 18 (ND18) strains of BSMV have sequence homology with RNA 2 of the (“ pseudo-two component”) Type strain, which has two electrophoretic components, but only limited homology is evident with RNA 2 of the ND18 and Norwich strains, which have three electrophoretic components (“ three component” strain). Instead, eDNAs from SG-RNA hybridize most efficiently with RNA 3 of the ND18 and Norwich strains. In wheat germ extracts the SG-RNAs direct the synthesis of two polypeptides with apparent molecular weights of 20 to 21 × 10 3. However, these two polypeptides were difficult to detect by polyacrylamide gel electrophoresis of extracts from Type- or ND18-infected barley and so appear not to accumulate during infection.

In vivo aminoacylation of brome mosaic and barley stripe mosaic virus RNAs

The genomic RNAs of several plant viruses are now known to be able to bind a specific amino acid at their 3′ ends in a tRNA-like manner. Where the nucleotide sequence of the 3′-terminal region is known, it can be folded into an extensively base-paired secondary structure having the essential features of tRNAs1,2. The high degree of nucleotide sequence conservation of the 3′-terminal regions of the four RNAs of the three known bromoviruses3, and the loss of infectivity of brome mosaic virus (BMV) RNA after chemical modification of its 3′ end4, strongly suggest that the tRNA-like structure has a biological function in infection2. To elucidate this it has been important to determine whether viral RNAs are aminoacylated within infected host cells. We studied BMV and barley stripe mosaic virus (BSMV), which belong to very different groups but which have genomic RNAs that are capable of both accepting tyrosine and infecting barley protoplasts. We report here that single-stranded RNAs of both viruses are aminoacylated in vivo, but that the encapsidated RNAs of BMV are not.

Investigation of the complexity of barley stripe mosaic virus RNAs with recombinant dna clones

RNA isolated from the Type, ND18, and Norwich strains of barley stripe mosaic virus (BSMV) was electrophoresed in agarose gels, transferred to nitrocellulose, and hybridized with BSMV-specific complementary DNA (cDNA) or recombinant DNA clones derived from ND18 RNA. Genomic RNA components 1 ( M r = 1.43 × 10 6) and 2 ( M r = 1.24 × 10 6) were resolved in all three strains, but RNA 3 ( M r = 1.1 × 10 6) was seen only in the ND18 and Norwich strains. A low-molecular-weight RNA ( M r = 0.27 × 10 6), thought to be a subgenomic (SG) RNA, was also detected in RNA preparations from all three strains by staining with toluidine blue or ethidium bromide and by hybridizing with cDNA or selected recombinant DNA probes. Three classes of recombinant DNA clones, designated pBSM1, pBSM2, and pBSM3, were identified by hybridization of nick-translated recombinant DNA to electrophoretically separated viral RNAs. Clones in the pBSM1 class hybridized only to RNA 1 of all three strains and class pBSM2 clones hybridized only to RNA 2 of all three strains. Class pBSM3 clones hybridized to RNA 3 of the ND18 and Norwich strains and to RNA 2 of the Type strain, but not to RNA 2 of ND18 or Norwich. Based on the sizes of the BSMV-specified inserts in clones designated pBSM1a, pBSM2a, and pBSM3a, we estimate that a minimum of 44, 63, and 63% of the nucleotide sequences of ND18 and Norwich RNAs 1, 2, and 3, respectively, are unique. Furthermore, because the combined size of the inserts in pBSM2a and pBSM3a is approximately 15% greater than the estimated size of RNA 2, it is probable that the second RNA component of the Type strain actually consists of two RNA species which are similar in size but have different sequences. The SG RNA component is viral specific and contains sequences common to clones derived from RNA 3.

Structure of the 3′ extremity of barley stripe mosaic virus RNA: Evidence for internal poly(A) and a 3′-terminal tRNA-like structure

The results of a previous study suggested that the poly(A) sequence in barley stripe mosaic virus (BSMV) RNA is intercalated between a 3′-terminal tyrosine-accepting structure and the 5′-terminal coding part of the BSMV genome. Here we show that poly(A) + and poly(A) − fractions of BSMV RNA can be cleaved into two fragments specifically at the position of poly(A) or oligo(A) sequence with RNase H from Escherichia coli in the presence of oligo(dT) 10. The shorter fragment (Sh) retains the ability of intact viral RNA to be aminoacylated, i.e., it represents the 3′-terminal part of BSMV RNA. Electrophoretic analysis of Sh-RNA reveals three closely positioned subspecies with an average length of about 210 nucleotides. The long 5′-terminal RNA fragment (L) produced by RNase H treatment has electrophoretic mobility similar to that of intact BSMV RNA, but displays neither amino acid-accepting ability nor infectivity. Nevertheless, L-RNA possesses the same messenger activity as the intact viral RNA and codes for the same pattern of polypeptides in rabbit reticulocyte lysate in vitro translation assays.

Aminoacylation of barley stripe mosaic virus RNA: Polyadenylate-containing RNA has a 3′-terminal tyrosine-accepting structure

Barley stripe mosaic virus (BSMV) RNA which was previously reported to contain poly(A) sequences (Agranovsky et al., 1978) can be specifically esterified with tyrosine in vitro in the presence of an aminoacyl-tRNA synthetase fraction from wheat embryos. All the three RNA components of the BSMV strain with a three-component genome (Norwich) and both RNA components of a two-component strain (Russian) can be tyrosylated. The poly(A)-containing (bound to oligo(dT)-cellulose) and poly(A)-deficient (not bound to oligo(dT)-cellulose) fractions of BSMV RNA display a similar amino acid-accepting ability. The nucleotide sequence which accepts tyrosine is coupled with the intact genomic polyadenylated BSMV RNA. The viral RNA isolated after sucrose density gradient centrifugation under drastic denaturing conditions retains its aminoacylating activity, which suggests that this activity is not due to the presence in a BSMV RNA preparation of a tyrosine tRNA associated with BSMV RNA. Inhibition of aminoacylation of the 3′-oxidized (treated with sodium metaperiodate) BSMV RNA suggests that the tyrosine-accepting structure is localized at the 3′ terminus of BSMV RNA molecules. It is shown that segments of different lengths obtained upon random fragmentation can be tyrosylated. The 3′-terminal (tyrosine-accepting) poly(A) + segments can be isolated. The shortest segments of viral RNA capable of being aminoacylated [i.e., containing both tRNA-like structure and poly(A)] consists of approximately 150–200 nucleotides. The analysis of the oligonucleotides derived from individual BSMV RNA components labeled with 32P at the 3′ end revealed two types of 3′-terminal sequences different from poly(A). It is suggested that a poly(A) sequence is intercalated between a 3′-terminal tyrosine-accepting structure and the 5′-terminal portion of poly(A) + BSMV RNA.

Homology in the 3′-terminal regions of different barley stripe mosaic virus RNA species

Preparations of [ 3H]DNA complementary to the individual species of barley stripe mosaic virus (BSMV) RNA (cDNA) about 1000 nucleotides long were obtained using avian myeloblastosis virus reverse transcriptase and oligo(dT) 10 primer. These cDNAs were used in kinetic hybridization experiments to study the sequence homology between the 3′-terminal regions (not including 150–200 nucleotides adjacent to the 3′-end) of individual BSMV RNA species. These regions in RNA 2 and 3 are highly homologous and have no sequences in common with the respective region in RNA 1.

Size determination of poly(A) sequences in barley stripe mosaic virus RNA

The length of poly(A) fragments in barley stripe mosaic virus (BSMV) RNA was determined. Poly(A) fragments obtained from total BSMV RNA samples treated with a mixture of pancreatic RNase and T1 RNase were labeled at the 5′-end by [γ- 32 P]ATP and analyzed in 20% polyacrylamide gels containing 7 m urea. It was found that the length of BSMV RNA poly(A) fragments was 8 to 30 nucleotide residues, with 19 to 28 nucleotide residues fragments being predominant.