Barley Stripe Mosaic Virus Strains Research Articles

Construction of an Infectious Poa semilatent virus cDNA Clone and Comparisons of Hordeivirus Cytopathology and Pathogenicity.

Poa semilatent virus (PSLV), Lychnis ringspot virus (LRSV), and Barley stripe mosaic virus (BSMV) are members of the genus Hordeivirus in the family Virgaviridae. However, the biological properties and molecular genetics of PSLV have not been compared with other hordeiviruses. Here, we have constructed an infectious cDNA clone of the PSLV Canadian strain and provided evidence that PSLV differs from BSMV and LRSV. First, unlike the other two hordeiviruses that replicate in chloroplasts, PSLV induces dramatic structural changes in peroxisome during its infection in barley. The αa replication protein also localizes to peroxisomes, suggesting that PSLV replication occurs in peroxisomes. Second, PSLV encodes a γb protein that shares 19 to 23% identity with those of other hordeiviruses, and its activity as a viral suppressor of RNA (VSR) silencing is distinct from those of BSMV and LRSV. Substitution of the BSMV γb protein with that of PSLV or LRSV revealed a negative correlation between VSR activity and symptom severity of the recombinant BSMV derivatives. Intriguingly, the Ser-Lys-Leu (SKL) peroxisome-targeting signals differ among γb proteins of various hordeiviruses, including some BSMV strains. The presence of the C-terminal SKL motif in the γb protein impairs its silencing suppressor activity and influences symptoms. Finally, we developed a PSLV-based virus-induced gene silencing vector that induced strong and effective silencing phenotypes of endogenous genes in barley, wheat, and millet. Our results shed new light on hordeivirus pathogenesis and evolution, and provide an alternative tool for genomics studies of model hosts and economically important monocots.

RNA-seq analysis of Brachypodium distachyon responses to Barley stripe mosaic virus infection

Barley stripe mosaic virus (BSMV) is the type member of the genus Hordeivirus. Brachypodium distachyon line Bd3-1 shows resistance to the BSMV ND18 strain, but is susceptible to an ND18 double mutant (β NDTGB1R390K, T392K) in which lysine is substituted for an arginine at position 390 and for threonine at position 392 of the triple gene block 1 (TGB1) protein. In order to understand differences in gene expression following infection with ND18 and double mutant ND18, Bd3-1 seedlings were subjected to RNA-seq analyses at 1, 6, and 14days post inoculation (dpi). The results revealed that basal immunity genes involved in cellulose synthesis and pathogenesis-related protein biosynthesis were enhanced in incompatible interactions between Bd3-1 and ND18. Most of the differentially expressed transcripts are related to trehalose biosynthesis, ethylene, jasmonic acid metabolism, protein phosphorylation, protein ubiquitination, transcriptional regulation, and transport process, as well as pathogenesis-related protein biosynthesis. In compatible interactions between Bd3-1 and ND18 mutant, Bd3-1 developed weak basal resistance responses to the virus. Many genes involved in cellulose biosynthesis, protein amino acid phosphorylation, protein biosynthesis, protein glycosylation, glycolysis and cellular macromolecular complex assembly that may be related to virus replication, assembly and movement were up-regulated. Some genes involved in oxidative stress responses were also up-regulated at 14dpi. BSMV ND18 mutant infection suppressed expression of genes functioning in regulation of transcription, protein kinase, cellular nitrogen compound biosynthetic process and photosynthesis. Differential expression patterns between compatible and incompatible interactions in Bd3-1 to the two BSMV strains provide important clues for understanding mechanism of resistance to BMSV in the model plant Brachypodium.

Development of a one-step immunocapture real-time RT-PCR assay for the detection of barley stripe mosaic virus strains in barley seedlings

A one-step immunocapture real-time RT-PCR (IC-real-time RT-PCR) was developed for efficient detection of barley stripe mosaic virus (BSMV) in barley seedlings. The novel detection system was designed using a primer set targeting the conserved region in the triple gene block 2 (TGB2) to expand its capacity to detect all BSMV strains. This assay was evaluated for its efficiency in detecting BSMV in barley seedlings. Using the immunocapture sample preparation, real-time RT-PCR was able to detect BSMV in samples, which were indicated as negative by ELISA. The sensitivity of detection in the real-time RT-PCR was as low as 50 fg/µl of total viral RNA under optimal reaction conditions. This level of sensitivity indicated that the one-step IC-real-time RT-PCR developed in the present study could be used for routine plant and seed health assays.

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.

Mapping of the seed transmission determinants of barley stripe mosaic virus.

The specific mechanism(s) by which some plant viruses are transmitted through seed, while others are excluded, is not known. Using infectious barley stripe mosaic virus (BSMV) RNAs transcribed in vitro from full-length cDNA clones, the viral genetic determinants of seed transmission have been mapped. Both pseudorecombinant and chimeric viruses were constructed from BSMV strains ND18 (seed transmitted) and CV17 (not seed transmitted). The markedly different seed transmissibility of these two strains facilitated the identification of RNAgamma as the location of the primary determinants of seed transmission phenotype. RNAbeta also played a role in seed transmission, but to a lesser extent than RNAgamma. Major genetic determinants of seed transmission on RNAgamma included the 5' untranslated leader, a 369-nt repeat in the gammaa gene, and the gammab gene. Important determinants of symptom phenotype mapped to the RNAgamma leader and the gammab gene as well. Some heterologous combinations of the RNAgamma leader and the gammab gene resulted in dramatic changes in symptomatology and seed transmission, depending on the parental source of RNAs alpha and beta. These results suggest that a complex interaction of the RNAgamma leader, the gammab gene, and RNAs alpha and beta are involved in BSMV pathogenesis. Considering the putative regulatory role of the gamma gene (Donald and Jackson 1994, Plant Cell 6:1593-1606) and the trans effects that alterations in the gammab gene have on RNAbeta gene expression (Petty et al., 1990, EMBO J. 9:3453-3457), phenotypic effects attributed to elements of RNAgamma could result from cis or trans interactions involving the RNAgamma leader, the gammab gene, and RNAs alpha and beta. Clearly, virus replication and movement play pivotal roles in the seed transmission of BSMV.

Evidence That the αa Gene of Barley Stripe Mosaic Virus Encodes Determinants of Pathogenicity to Oat (Avena sativa)

Complementary DNA clones from which infectious RNA can be transcribed were used to map the genetic determinants for oat (Avena sativa L.) pathogenicity of barley stripe mosaic virus (BSMV). Pseudorecombinant analysis of BSMV strains NDI8 (nonpathogenic to oat) and CV42 (pathogenic to oat) indicated that the ability to systemically infect oat mapped to RNA α. A homologous recombinant of ND18 (18αTKTKIN), possessing nucleotides 2218 to 2454 from CV42 RNA α, induced symptoms on oat similar to those generated by wild-type CV42. Six amino acids encoded by the αa gene differ between ND18 (PQSQTK) and CV42 (TKTKIN) in this region. Fine structure recombinants that encoded subsets of the six amino acid changes either were slow in their infection phenotype (18αTKTK and 18αTQSQIN) relative to recombinant 18αTKTKIN or were not infectious (18αKTKIN and 18αKTK) to inoculated oat plants. Neither coat protein antigen nor viral RNA was detected in inoculated plants that did not display symptoms. All recombinants infected isolated oat protoplasts, however, as determined by Northern and Western blots of extracts from inoculated oat protoplasts. The accumulation of viral genomic RNAs in protoplasts among the recombinants tested was similar, except for that of recombinant 18αTKTK which was reduced by 30%. The data support an earlier suggestion (Y. Zheng. and M. C. Edwards, 1990. J. Gen. Virol. 71. 1865-1868) that resistance of oat to BSMV strain ND18 is due to restricted virus movement in planta and provide evidence that the αa protein itself may be involved in the movement of BSMV in the infected plant.

Expression of Resistance to Barley Stripe Mosaic Virus in Barley and Oat Protoplasts

Mesophyll protoplasts from both susceptible and resistant hosts were inoculated with RNA purified from barley stripe mosaic virus (BSMV) strains CV52 and CV42 using the polyethylene glycol (PEG) method. Protoplasts derived from the susceptible Hordeum vulgare L. cv. Black Hulless were susceptible to both BSMV strains, as indicated by fluorescein isothiocyanate staining and ELISA. More than 80% of protoplasts derived from an oat cultivar resistant to CV52, but not to CV42, were readily infected by either CV52 or CV42. Protoplasts from 10 barley lines resistant to CV42 remained resistant to CV42, although a limited number of protoplasts were infected. Functional resistance in the cultured barley protoplasts, but not in those from oat plants, suggests that resistance in these barley lines may be the result of restriction of replication, whereas resistance in oat plants is more likely due to restriction of cell-to-cell movement.

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.

A comparative study on the in vitro translation products of individual RNAs from two-, three-, and four-component strains of barley stripe mosaic virus

Passaging through plants of the three-component barley stripe mosaic virus (BSMV) strain Norwich (Norwich III) yielded a two-component isolate (Norwich II). A study was made of the sets of polypeptides translated in a rabbit reticulocyte and wheat embryo cell-free systems from individual RNAs of (1) the natural three-component strain Norwich III, (2) a two-component isolate derived from the former (Norwich II), and (3) an isolate intermediate between the three- and two-component ones, Norwich;. Translation of RNA 1 from all three variants of the Norwich strain in vitro yields a single major product with a molecular weight (M r) of 120,000 (p120). RNA 2 from Norwich III in vitro produces two polypeptides: the viral coat protein (p23) and, in certain ionic conditions, a polypeptide of 25,000 M r (p25). RNA 3 from Norwich III is translated into a protein with M r of 75,000 (p75). Conversion of Norwich III into Norwich II is accompanied by the changes in the coding specificity of RNA 2; beside the coat protein and p25, a protein of 85,000 M r (p85) is formed upon its translation—a feature characteristic of RNA 2 of the naturally occurring bipartite BSMV strains (Russian, type). With the Norwich i variety, which is marked by a significantly reduced portion of RNA 3 in the total virion RNA preparation, RNA 2 in addition to the coat protein, p25, and p85 directs the synthesis of another product with M r of about 78,000. Thus, in conversion of the three-component BSMV into a two-component one, the loss of RNA 3 is concomitant with the actuation in RNA 2 of a sequence coding for p85. RNAs 1–3 of the quadripartite Argentina Mild (AM) BSMV strain code in vitro for the same polypeptides as RNAs 1–3 of Norwich III. AM RNA 4 is translated as a monocistronic template into a polypeptide with M r of 55,000 (p55). Amino acid sequences of p85, p75, and p55 are shown to overlap among these polypeptides but not to appreciably overlap with p120 sequences. Data presented here allowed a tentative structure to be proposed for genome of two-, three-, and four-component BSMV strains.

Evidence of multiple virion components in leaf-dip preparations of barley stripe mosaic virus

Virion length distributions of each of four strains of barley stripe mosaic virus (BSMV) were obtained from metal-shadowed, leaf-dip preparations of infected barley (Hordeum vulgare L. emend. Lam. cv. ‘Black Hulless’) before and after three successive transfers of each strain. Three major virion components with normal lengths of 108, 125, and 148 nm were recognized. All three virion components were consistently resolved in preparations of one strain, while in preparations of two strains, only the latter two components were resolved. The 125-nm component was most common in all preparations of these strains. For a fourth strain, the 108-nm component was most common and although the three components were resolved in the initial preparation, after three successive transfers only the 108- and 148-nm components were resolved.Sample size and the method of grouping virions according to length were both important factors in resolving virion components of BSMV. The failure of previous workers to resolve either the 108- or 148-nm components in metal-shadowed preparations can probably be attributed mainly to the latter factor.It is suggested that the number, normal lengths, and ratio of virion components are useful criteria for differentiating and characterizing BSMV strains.

Comparisons of poa semilatent and barley stripe mosaic viruses

Poa semilatent virus (PSLV), which caused chlorosis and rapid death of inoculated wheat, infected a number of grasses susceptible to barley stripe mosaic virus (BSMV); but it also infected Agropyron trachycaulum and Poa palustris which were not susceptible to a barley strain or two oat-infecting strains of BSMV. Conversely BSMV caused local lesions on several species of Chenopodium that did not become infected with PSLV. BSMV protected wheat from infection by PSLV, but PSLV caused only partial protection from BSMV.The normal length and thickness of particles in leaf-dip preparations was 161 × 26 mμ for PSLV and 133 × 25 mμ for BSMV.Serological relationship was not indicated by the Ouchterlony agar double-diffusion test or leaf-dip serology. A distant relationship was shown with the microprecipitin test and the ring interface precipitin test. In cross absorption tests the titers of the antisera to the homologous viruses were not reduced by absorption with the heterologous viruses.PSLV and BSMV appear to be distantly related serotypes.