A novel strategy for one-step cloning of full-length cDNA and its application to the genome of barley stripe mosaic virus.

We have devised a novel vector-primer strategy for cloning of full-length (FL) cDNA which can be applied to non-polyadenylated RNA species. Single-stranded plasmid DNA is used to prime first-strand synthesis by reverse transcriptase, and plasmids covalently linked to FL cDNA are then circularized by the annealing of a specific oligodeoxyribonucleotide (band-aid oligo). Only limited nucleotide sequence data are required from the termini of each RNA species to be cloned to design the plasmid primer and band-aid oligo. The band-aid strategy has been applied to the cloning of barley stripe mosaic virus genomic RNAs, and found to be both rapid and efficient. A strategy for the preparation of linear double-stranded plasmid DNA templates (suitable for run-off in vitro transcription) which is independent of restriction sites present within the cloned cDNA is also described.

Two forms of the major barley stripe mosaic virus nonstructural protein are synthesized in vivo from alternative initiation codons.

Barley stripe mosaic virus (BSMV) has a tripartite genome comprising RNAs designated alpha, beta, and gamma, which collectively encode seven polypeptides. We show here that an antiserum raised against an abundant disease-specific protein from BSMV-infected plants reacts specifically with the viral beta b gene product expressed as part of a beta-galactosidase fusion protein in Escherichia coli. Two predominant forms of the protein, beta b and beta b', are synthesized in vivo. Infectious in vitro transcripts derived from wild-type and mutant BSMV cDNA clones have been used to map the initiation site for translation of the beta b protein in vivo. The results of our mutagenesis experiments are consistent with a model in which translation of the beta b' protein is initiated by ribosomes that scan past the 5'-proximal beta b initiation site. A mutant which is able to synthesize only the shorter beta b' protein was indistinguishable from the wild-type with respect to all of the phenotypes tested. Thus, the beta b form of the protein is dispensable in planta, and whether the two forms of the protein have different functions in vivo is unclear at present.

Mutational analysis of barley stripe mosaic virus RNA beta.

Barley stripe mosaic virus (BSMV), the type member of the hordeivirus group, has a plus-stranded genome comprising RNA species designated alpha, beta, and gamma. Although RNA beta 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 beta from which infectious in vitro transcripts can be derived to construct a number of mutations in its four genes. Mutations introduced into the beta b, beta c, or beta d 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 (beta b), (2) stability of the genomic RNA during virus multiplication in planta, (3) the requirement for a trans-acting BSMV protein (gamma b), (4) symptomatology and disease development in infected barley plants, and (5) host range.

Genetic analysis of bipartite geminivirus tissue tropism.

The bipartite geminiviruses bean golden mosaic virus (BGMV), cabbage leaf curl virus (CabLCV), and tomato golden mosaic virus (TGMV) exhibit differential tissue tropism in Nicotiana benthamiana. In systemically infected leaves, BGMV remains largely confined to vascular-associated cells (phloem-limited), whereas CabLCV and TGMV can escape into the surrounding mesophyll. Previous work established that TGMV BRi, the noncoding region upstream from the BR1 open reading frame (ORF), is required for mesophyll invasion, but the virus must also contain the TGMV AL23 or BL1/BR1 ORFs. Here we show that, in a BGMV-based hybrid virus, CabLCV AL23 also directed efficient mesophyll invasion in conjunction with TGMV BRi, which suggests that host-adaptation of AL23 is important for the phenotype. Cis-acting elements required for mesophyll invasion were delineated by analyzing BGMV-based hybrid viruses in which various parts of BRi were exchanged with those of TGMV. Interestingly, mesophyll invasion efficiency of hybrid viruses was not correlated with the extent of viral DNA accumulation. In conjunction with TGMV AL23, a 52-bp region of TGMV BRi with sequence homology to DNA A was sufficient for mesophyll invasion. This 52-bp sequence also directed mesophyll invasion in combination with the TGMV BL1/BR1 ORFs. Overall, these results are consistent with a model for mesophyll invasion in which AL2 protein, in association with host factors, acts through the 52-bp region in TGMV BRi to affect expression of the BR1 gene.

Tissue specificity of geminivirus infection is genetically determined.

The types of cells and tissues infected by a virus define its tissue tropism. Determinants of tissue tropism in animal-infecting viruses have been extensively investigated, but little is known about plant viruses in this regard. Some geminiviruses in the genus Begomovirus exhibit phloem limitation and are restricted to cells of the vascular system, whereas others can invade mesophyll tissue. To identify viral genetic determinants of tissue tropism, we established a model system using two begomoviruses and their common host plant, Nicotiana benthamiana. Analysis by DNA in situ hybridization confirmed that tomato golden mosaic virus invades mesophyll tissues in systemically infected leaves, whereas bean golden mosaic virus remains phloem limited. Through genetic complementation and analysis of recombinant hybrid viruses, we demonstrated that three genetic elements of tomato golden mosaic virus determine its mesophyll tissue tropism. A noncoding region of the viral genome is essential for the phenotype, but it must be accompanied by one of two different coding regions. To our knowledge, this is the first example documented in a plant virus of noncoding DNA sequences that determine tissue tropism.

Tomato golden mosaic virus open reading frame AL4 is genetically distinct from its C4 analogue in monopartite geminiviruses.

Tomato golden mosaic virus (TGMV) is a bipartite geminivirus with six well-characterized genes. An additional open reading frame (ORF), AL4, lies within the essential AL1 gene. Recent studies of monopartite, dicot-infecting geminiviruses have revealed that mutations in their analogous C4 ORFs have host-specific effects on infectivity, symptomatology, virus movement and/or viral DNA accumulation. We have investigated whether TGMV has a similar host-specific requirement for AL4. The phenotypes of three TGMV al4 mutants were determined in a range of hosts, which included species that revealed c4 mutant phenotypes for monopartite geminiviruses. Each TGMV al4 mutant was indistinguishable from wild-type TGMV in all hosts tested. Additional analyses of double mutants revealed no evidence for functional redundancy between AL4 and the AL3, or AR1 genes. In contrast to the putative C4 proteins of monpartite geminiviruses, TGMV AL4, if it is expressed, is either non-functional, or functionally redundant with an essential TGMV gene product.

Systemic movement of an RNA plant virus determined by a point substitution in a 5' leader sequence.

The ability of viruses to move through infected plants is an important determinant of host range and pathogenicity. We have investigated the genetic basis for the inability of the Type strain of barley stripe mosaic hordeivirus to undergo long-range systemic movement in the tobacco Nicotiana benthamiana. We show that, in this model system, a short open reading frame in the 5' leader of the smallest viral genomic RNA prevents long-range vascular movement. As predicted by the ribosome scanning model, the leader open reading frame decreases the efficiency with which the 5'-proximal gene is translated in vitro. Thus, systemic pathogenicity in this system may be determined by the efficiency of translation of a viral gene in vivo and is not determined by the primary sequence of the encoded protein.

RNA recombination in the genome of barley stripe mosaic virus.

Barley stripe mosaic Hordeivirus (BSMV) is a positive-strand RNA virus requiring three single-stranded RNAs (alpha, beta, and gamma) for infectivity. A terminal-sequence-dependent cloning strategy was used to clone the entire genome of the CV17 strain. Full-length gamma cDNA clones were obtained when oligonucleotides specific for the 5'-terminal sequence of RNA alpha were used in the cloning procedure, but not when RNA gamma-specific oligonucleotides were used. Sequence analysis of six putative gamma cDNA clones revealed that nucleotides 1-70 possess 89% homology with the first 70 nucleotides of RNA alpha. This leader region is separated from the gamma-specific coding region by an eight-base intervening sequence common to both CV17 RNAs alpha and gamma. Northern and Southern hybridization with oligonucleotide probes specific for either alpha or gamma leader sequences indicated that CV17 gamma cDNA clones are representative of native CV17 gamma RNAs. Furthermore, bioassays indicated that in vitro transcripts derived from these gamma cDNA clones were infectious when coinoculated with in vitro transcripts of full-length alpha and beta cDNA clones. Thus, the evidence suggests that RNA gamma of BSMV strain CV17 is a recombinant molecule which may have arisen as a result of natural recombination between RNAs alpha and gamma.

Genetic requirements for local and systemic movement of tomato golden mosaic virus in infected plants.

Tomato golden mosaic geminivirus (TGMV) has two DNA components, A and B. Replication of DNA A can be detected in inoculated leaves, but DNA B is additionally required for virus movement in planta. Using viral DNA accumulation as an indication of the number of infected cells, we show here that both the BL1 and BR1 genes are necessary for local TGMV movement. We also demonstrate that transient expression of BL1 and BR1 together allows wild-type TGMV DNA A to move systemically. When the transient movement assay was used to analyze various A component mutants, all were found to move locally in inoculated leaves, and only an ar1 (coat protein) mutant was unable to move systemically. In addition, we confirm that a TGMV al2 (AR1 and BR1 trans-activator) mutant has a defect in local movement which can be rescued by provision of exogenous BR1, but not BL1. Finally, we show that the ability of TGMV coat protein mutants to accumulate single-stranded (ss) DNA is dependent on BR1. These results provide experimental evidence obtained in planta which supports three predictions of published models for bipartite geminivirus movement: (i) BL1 and BR1 have distinct and essential roles in cell-to-cell movement as well as systemic movement; (ii) BR1 may interact with viral ssDNA in vivo; and (iii) AL2 is indirectly required for movement through its effect on BR1 expression. In addition, our data suggest that specific models of bipartite geminivirus systemic movement should accommodate a role for the coat protein.

Multiple genetic determinants of barley stripe mosaic virus influence lesion phenotype on Chenopodium amaranticolor.

The ND18 and Type strains of barley stripe mosaic hordeivirus (BSMV) differ in the local lesion phenotypes they elicit on Chenopodium amaranticolor. The ND18 strain produces large necrotic lesions on this host by 3 to 4 days postinoculation, whereas the Type strain is less virulent and elicits chlorotic local lesions which appear about 2 weeks after inoculation. We have used infectious in vitro transcripts derived from full-length cDNA clones of these two BSMV strains to investigate the genetic basis for their differential virulence on C. amaranticolor. Pseudorecombination of the wild-type alpha, beta, and gamma genomic RNAs of each strain revealed that the lesion forming phenotype segregated with RNA gamma. Fine mapping of the phenotypic determinants on RNA gamma was carried out by constructing deletion mutants, chimeric recombinants, and point mutants. These experiments showed that three different genetic elements in the Type strain RNA gamma contribute significantly to its attenuated virulence on C. amaranticolor. In addition, pseudorecombination experiments using mutant Type strain gamma RNAs that were more virulent than native Type RNA gamma indicated that the clean segregation of the lesion forming phenotype observed with wild-type RNA gamma is fortuitous. This lesion phenotype is dependent on both the multiple attenuating determinants in the wild-type Type strain RNA gamma and the source of genomic RNAs alpha and beta in the inoculum. The complexity of these virulence determinants clearly illustrates the limitations of classical pseudorecombination as a tool for the genetic analysis of plant viruses.

Virus and host-specific adaptations in the BL1 and BR1 genes of bipartite geminiviruses.

The host range of individual geminiviruses may be quite narrow, and closely related viruses can exhibit distinct host adaptations. Two such bipartite geminiviruses are bean golden mosaic virus (GBMV) and tomato golden mosaic virus (TGMV). In both, the BL1 and BR1 genes are required for the spread of virus infection in plants. We have investigated the contributions of BL1 and BR1 to host-specific phenotypes of BGMV and TGMV by constructing hybrid viruses in which these coding regions were exchanged. Hybrids were assayed on bean, a good host for BGMV, and Nicotiana benthamiana, a good host for TGMV. A BGMV hybrid having TGMV BL1 and BR1 efficiently infected beans, but elicited attenuated symptoms. In N. benthamiana, this hybrid had slightly increased virulence and DNA accumulation relative to wild-type BGMV. A TGMV hybrid having BGMV BL1 and BR1 was virulent in N. benthamiana, but elicited attenuated symptoms. However, this hybrid exhibited no gain of function in beans relative to wild-type TGMV. Hybrid viruses with TGMV BL1 and BGMV BR1 had severely defective phenotypes in either viral or host background. Although exchanging BL1 and BR1 between BGMV and TGMV did not change host range, some host adaptation of these genes is suggested. However, virus-specific compatibility between BL1 and BR1 is of more importance for viability. Thus, these gene products may act in concert to potentiate virus movement.

Identification of barley stripe mosaic virus genes involved in viral RNA replication and systemic movement.

Barley stripe mosaic hordeivirus (BSMV) has a tripartite positive-sense RNA genome which encodes seven major polypeptides. Infectious in vitro transcripts derived from full-length wild-type and mutant cDNA clones have been used to investigate the contribution made by various BSMV gene products to viral RNA replication and systemic movement. We show that whereas all three of the BSMV RNA components are required for plant infection, RNAs alpha and gamma can replicate together in barley protoplasts, and therefore RNA beta must encode functions required for systemic invasion of plants. The alpha a and gamma a proteins, which contain helicase and RNA polymerase sequence motifs, together comprise the essential virus-encoded components of BSMV RNA replicase. A second BSMV protein (beta b) which contains a helicase motif is not required for RNA replication. A small cysteine-rich protein (gamma b) is dispensable for infection of plants, but in its absence the accumulation of viral coat (beta a) and beta b proteins is significantly reduced. In addition, mutations in both the gamma b and gamma a (replicase) proteins can affect the systemic movement phenotype.

Turnip crinkle virus genes required for RNA replication and virus movement.

We have used infectious in vitro transcripts from mutagenized turnip crinkle virus (TCV) cDNA clones to identify the gene products required for viral RNA replication, virion assembly, and intercellular movement. Previous sequence analysis of the TCV genome revealed the presence of five open reading frames which had the potential to encode gene products of 88, 38, 28, 9, and 8 kDa. Inoculation of protoplasts with infectious RNA revealed that only the p28 and p88 gene products are required for viral RNA synthesis. Although the p8 and p9 gene products were dispensable for RNA replication and virion assembly in protoplasts, mutations in the p8 and p9 genes prevented the production of systemic infections in plants. No viral RNA or protein was observed in the inoculated or systemic leaves of plants inoculated with transcripts synthesized from p8 or p9 mutant cDNAs. In contrast to these results, viral RNA was recovered from the inoculated, but not the systemic leaves, of plants inoculated with an RNA lacking the coat protein (CP) gene. With the CP mutant, no symptoms were observed on normally systemic hosts, but small local lesions were induced on Chenopodium amaranticolor. These results indicate that p8, p9, and CP are required for viral 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 alpha, beta, and gamma 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 gamma-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.

Complementable and noncomplementable host adaptation defects in bipartite geminiviruses.

Members of the geminvirus group of plant viruses collectively infect a broad spectrum of species. Individual viruses which are genetically very similar may nevertheless have distinct host ranges. Two such geminiviruses are tomato golden mosaic virus (TGMV) and been golden mosaic virus (BGMV), for which common hosts have not previously been reported. Each virus has two genome components, designated A and B. The A component is capable of autonomous replication and encapsidation, whereas the B component provides viral functions required for the spread of infection in plants. To investigate the basis for the distinctive host ranges of BGMV and TGMV, we have introduced plasmids containing cloned viral genome components into Nicotiana benthamiana, a good host for TGMV, and bean, Phaseolus vulgaris, a good host for BGMV. We found that TGMV has a low specific infectivity for bean and is virulent, whereas BGMV has a high specific infectivity for N. benthamiana, but infections are asymptomatic and viral DNA accumulation is low. To investigate which viral functions were defective in the poor host in each case, we attempted to complement them by co-inoculation with the well-adapted virus. After inoculation of beans with both viruses, only BGMV was detected. Thus, TGMV exhibits a noncomplementable host adaptation defect in beans. This suggests that the defect has a cis-acting or virus-specific trans-acting genetic basis. In contrast, the BGMV phenotype of low DNA accumulation in N. benthamiana was partially complemented by TGMV A alone and complemented further by the complete TGMV genome. This suggests that a virus nonspecific, trans-acting factor encoded by the BGMV A component is poorly adapted to N. benthamiana. The results of this study indicate that bipartite geminivirus host range may be limited by defective virus-host interactions of more than one kind.

Host and viral factors determine the dispensability of coat protein for bipartite geminivirus systemic movement.

Geminiviruses have unique, twinned icosahedral particles which encapsidate circular single-stranded DNA. Their genomes are composed of either one or two DNA segments. Monopartite geminiviruses absolutely require a functional coat protein (CP) for infectivity, whereas bipartite geminivirus CP null mutants can infect plants systemically. However, we show here that a CP mutant of the bipartite tomato golden mosaic virus (TGMV), which can infect Nicotiana benthamiana systemically, is confined to the inoculated leaves of Nicotiana tabacum or Datura stramonium. We also show that a CP mutant of the related bean golden mosaic virus (BGMV), which can infect beans systemically, is confined to the inoculated leaves of N. benthamiana. In each case, the extent of viral DNA accumulation in inoculated leaves was unaffected by the absence of CP, which suggests that CP is required specifically for systemic movement. The dispensability of CP is correlated with the degree of virus-host adaptation. TGMV is well adapted to N. benthamiana and does not require CP to infect this host systemically, whereas BGMV is poorly adapted to N. benthamiana and requires CP. Analysis of TGMV-BGMV hybrid viruses revealed that the viral genetic background can also affect the dispensability of CP for systemic movement in N. benthamiana. Thus, bipartite geminivirus movement in planta can be resolved genetically into three components: (i) local, cell-to-cell movement, which does not require CP; (ii) CP-dependent systemic movement, which occurs in all hosts tested; and (iii) CP-independent systemic movement, which occurs in hosts to which a given virus is well adapted.

Genetic determinants of host-specificity in bipartite geminivirus DNA A components.

Geminiviruses are small, ssDNA-containing plant viruses. Bean golden mosaic virus (BGMV) and tomato golden mosaic virus (TGMV) have bipartite genomes, the components of which are designated A and B. Although they are closely related, BGMV and TGMV nevertheless exhibit distinct host-specific phenotypes, with BGMV being well adapted to beans and TGMV being well adapted to Nicotiana benthamiana. A previous study showed that the two open reading frames (ORFs) of DNA B only partially determine the host-adapted phenotypes of BGMV and TGMV. We have now investigated the contributions of A component ORFs to host adaptation. Co-inoculated TGMV DNA A enhances the accumulation of BGMV in N. benthamiana. Using mutant and hybrid TGMV A components, the determinant of this phenotype was mapped to a region encompassing the overlapping AL2 and AL3 ORFs (AL23). BGMV- and TGMV-based hybrid A components containing the heterologous AL23 region each displayed host-specific gain-of-function phenotypes, which indicates that these sequences contribute to host adaptation in both viruses. In N. benthamiana, al2 and al3 mutants of either virus can be complemented in trans by the heterologous A component, so adaptation of the AL23 region to this host is likely mediated through a virus nonspecific, trans-acting factor. In beans, however, co-inoculated BGMV A does not affect the accumulation of TGMV, and TGMV did not complement BGMV al2 or al3 mutants. Thus host-adaptation of the AL23 region may have a different mechanistic basis in beans than it does in N. benthamiana. Although our experiments did not reveal significant host adaptation of the coat protein, which is encoded by the AR1 ORF, a virus-specific effect on viral ssDNA accumulation was observed.

Bipartite geminivirus host adaptation determined cooperatively by coding and noncoding sequences of the genome.

Bipartite geminiviruses are small, plant-infecting viruses with genomes composed of circular, single-stranded DNA molecules, designated A and B. Although they are closely related genetically, individual bipartite geminiviruses frequently exhibit host-specific adaptation. Two such viruses are bean golden mosaic virus (BGMV) and tomato golden mosaic virus (TGMV), which are well adapted to common bean (Phaseolus vulgaris) and Nicotiana benthamiana, respectively. In previous studies, partial host adaptation was conferred on BGMV-based or TGMV-based hybrid viruses by separately exchanging open reading frames (ORFs) on DNA A or DNA B. Here we analyzed hybrid viruses in which all of the ORFs on both DNAs were exchanged except for AL1, which encodes a protein with strictly virus-specific activity. These hybrid viruses exhibited partial transfer of host-adapted phenotypes. In contrast, exchange of noncoding regions (NCRs) upstream from the AR1 and BR1 ORFs did not confer any host-specific gain of function on hybrid viruses. However, when the exchangeable ORFs and NCRs from TGMV were combined in a single BGMV-based hybrid virus, complete transfer of TGMV-like adaptation to N. benthamiana was achieved. Interestingly, the reciprocal TGMV-based hybrid virus displayed only partial gain of function in bean. This may be, in part, the result of defective virus-specific interactions between TGMV and BGMV sequences present in the hybrid, although a potential role in adaptation to bean for additional regions of the BGMV genome cannot be ruled out.

Geminivirus replication origins have a modular organization.

Tomato golden mosaic virus (TGMV) and bean golden mosaic virus (BGMV) are closely related geminiviruses with bipartite genomes. The A and B DNA components of each virus have cis-acting sequences necessary for replication, and their A components encode trans-acting factors are required for this process. We showed that virus-specific interactions between the cis- and trans-acting functions are required for TGMV and BGMV replication in tobacco protoplasts. We also demonstrated that, similar to the essential TGMV AL1 replication protein, BGMV AL1 binds specifically to its origin in vitro and that neither TGMV nor BGMV AL1 proteins bind to the heterologous origin. The in vitro AL1 binding specificities of the B components were exchanged by site-directed mutagenesis, but the resulting mutants were not replicated by either A component. These results showed that the high-affinity AL1 binding site is necessary but not sufficient for virus-specific origin activity in vivo. Geminivirus genomes also contain a stem-loop sequence that is required for origin function. A BGMV B mutant with the TGMV stem-loop sequence was replicated by BGMV A, indicating that BGMV AL1 does not discriminate between the two sequences. A BGMV B double mutant, with the TGMV AL1 binding site and stem-loop sequences, was not replicated by either A component, indicating that an additional element in the TGMV origin is required for productive interaction with TGMV AL1. These results suggested that geminivirus replication origins are composed of at least three functional modules: (1) a putative stem-loop structure that is required for replication but does not contribute to virus-specific recognition of the origin, (2) a specific high-affinity binding site for the AL1 protein, and (3) at least one additional element that contributes to specific origin recognition by viral trans-acting factors.