Cymbidium Ringspot Tombusvirus Research Articles

A simplified method for cloning of short interfering RNAs from Brassica juncea infected with Turnip mosaic potyvirus and Turnip crinkle carmovirus

RNA silencing is a plant defense mechanism in which virus infected plants produce short interfering RNAs (siRNAs) derived from viral RNA, that attack the virus at the post-transcriptional level. In a previous study on Cymbidium ringspot tombusvirus (CymRSV) infection in Nicotiana benthamiana, siRNAs (determined by cloning and sequencing) predominantly originated from the sense (+) strand of the viral RNA, suggesting that the majority of siRNAs are produced through the direct cleavage of the virus single strand (ss) RNA by the plant Dicer-like enzyme. To test whether this asymmetry in strand polarity is a generic rule for all plant viruses, siRNAs from Brassica juncea, either singly infected by Turnip mosaic potyvirus (TuMV, the family Potyviridae), or doubly infected with TuMV and Turnip crinkle carmovirus (TCV, the family Tombusviridae) were investigated. A simplified siRNA cloning method was developed, using a single ligation reaction to attach both 5′ and 3′ adapters to the target short RNAs followed by one-step RT-PCR amplification. In the TCV infection, as for the CymRSV infection, siRNAs were produced predominantly (97.6%) from the +ss RNA. However, for TuMV infections, siRNAs were derived from both strands (+/−, 58.1–41.9%), indicating the presence of alternative siRNA production mechanisms.

Heterologous movement protein strongly modifies the infection phenotype of cucumber mosaic virus.

A hybrid virus (CMVcymMP) constructed by replacing the movement protein (MP) of cucumber mosaic cucumovirus (CMV) with that of cymbidium ringspot tombusvirus (CymRSV) was viable and could efficiently spread both cell to cell and long distance in host plants. The hybrid virus was able to move cell to cell in the absence of functional CP, whereas CP-deficient CMV was restricted to single inoculated cells. In several Chenopodium and Nicotiana species, the symptom phenotype of the hybrid virus infection was clearly determined by the foreign MP gene. In Nicotiana debneyi and Nicotiana tabacum cv. Xanthi, the hybrid virus could move systemically, contrary to CymRSV.

Cymbidium ringspot tombusvirus coat protein coding sequence acts as an avirulent RNA.

Avirulent genes either directly or indirectly produce elicitors that are recognized by specific receptors of plant resistance genes, leading to the induction of host defense responses such as hypersensitive reaction (HR). HR is characterized by the development of a necrotic lesion at the site of infection which results in confinement of the invader to this area. Artificial chimeras and mutants of cymbidium ringspot (CymRSV) and the pepper isolate of tomato bushy stunt (TBSV-P) tombusviruses were used to determine viral factors involved in the HR resistance phenotype of Datura stramonium upon infection with CymRSV. A series of constructs carrying deletions and frameshifts of the CymRSV coat protein (CP) undoubtedly clarified that an 860-nucleotide (nt)-long RNA sequence in the CymRSV CP coding region (between nt 2666 and 3526) is the elicitor of a very rapid HR-like response of D. stramonium which limits the virus spread. This finding provides the first evidence that an untranslatable RNA can trigger an HR-like resistance response in virus-infected plants. The effectiveness of the resistance response might indicate that other nonhost resistance could also be due to RNA-mediated HR. It is an appealing explanation that RNA-mediated HR has evolved as an alternative defense strategy against RNA viruses.

NICOTIANA BENTHAMIANA PLANTS TRANSFORMED WITH THE COMPLETE PRE-READTHROUGH DOMAIN OR THE N-PROXIMAL REGION OF THE REPLICASE GENE FROM CYMBIDIUM RINGSPOT VIRUS RNA ARE RESISTANT TO VIRUS INFECTION

Nicotiana benthamiana plants were transformed with sequences derived from the 5’-terminal open reading frames (ORF 1 and 2) of the cymbidium ringspot tombusvirus (CymRSV). These sequences included the pre-readthrough region of the replicase or its N-terminal region, the readthrough domain, and the full-length replicase gene in which the stop codon of ORF 1 was abolished. Some lines transformed with the first two sequences showed complete resistance when challenged with CymRSV. Experiments using protoplasts from resistant lines showed that the resistance operates at the single cell level by abolishing viral replication. Lines resistant to CymRSV were susceptible to two other tombusviruses, artichoke mottled crinkle and carnation Italian ringspot. These data and the lack of correlation between transgene expression and resistance would suggest this to be a form of RNA-mediated resistance. Since resistant lines were only found among those transformed with sequences involved in the build-up of the virus replication complex, it is suggested that resistance may be due to the interference of these sequences with those of the infecting virus.

FULL-LENGTH OR TRUNCATED MOVEMENT PROTEINS OF CYMBIDIUM RINGSPOT TOMBUSVIRUS DO NOT CONFER RESISTANCE ON TRANSGENIC NICOTIANA PLANTS

The full-length and truncated versions of the cymbidium ringspot tombusvirus (CymRSV) movement protein gene under the control of the cauliflower mosaic virus 35S promoter and terminator were used to transform Nicotiana benthamiana plants. Integration of the viral sequence in the plant genome was confirmed by PCR amplification of DNA extracts from transformed plants and by detection of virus-related transcripts in RNA extracts. Western blot analysis failed to show the presence of the transgene-encoded protein in transgenic plants. None of these plants was protected from infection when challenged with CymRSV, regardless of the type of transgene, whether full-length or truncated. It is suggested that RNA-mediated resistance was not acting in these plants and that failure to obtain resistance was due to lack of accumulation of the transgenic protein.

Molecular analysis of the pothos latent virus genome.

Pothos latent virus (PoLV) is an isometric virus with a positive-sense, single-stranded RNA genome of 4415 nt. The genome contains five open reading frames (ORF), coding for five proteins with approximate molecular masses of 25, 84, 40, 27 and 14 kDa, respectively. In vitro synthesized PoLV RNA was infectious to Nicotiana benthamiana plants and protoplasts, but could not support replication of the defective interfering (DI) and satellite RNAs associated with Cymbidium ringspot tombusvirus. No DI RNA related to PoLV was generated after repeated passaging with infected sap. Mutagenesis studies defined the role of the ORF 4 product (27 kDa) as a movement protein, and the ORF 5 product (14 kDa) as being responsible for symptom severity. Moreover, it was shown that the coat protein (CP) is important in regulating synthesis of the 14 kDa protein, excess production of which is lethal to infected plants. CP mutants defective in capsid formation infected plants systemically and induced severe necrotic symptoms. Conversely, CP mutants able to form apparently normal virus particles induced symptoms indistinguishable from those elicited by wild-type virus.

3′ Terminal Putative Stem–Loop Structure Required for the Accumulation of Cymbidium Ringspot Viral RNA

Defective interfering (DI) RNA of cymbidium ringspot tombusvirus (CymRSV) was used to identify thecis-acting nature of the last 77-nt sequence of the viral genome which is required for DI RNA accumulation. The 3′-terminalcis-essential domain of both genomic and DI RNAs can be folded into a stable stem–loop structure composed of three hairpins and two short non-base-paired regions. None of the three conserved stem–loops can be deleted without abolishing the infectivity of DI RNA. Similarly, those mutants in which base-paired stem regions were disrupted by single-, double-, or triple-base substitutions were unable to replicate. However, when the original structures were reconstructed by compensatory mutations the viability of the molecules was also restored. Limited mutation (1 or 2 nt) in the non-base-paired region did not show any significant effect on viral replication. Our results strongly suggest that the proposed structure for the 3′ terminus of the viral genome is very important for viral RNA replication. It is very likely that the function of this structure is to promote the minus-strand synthesis of CymRSV DI RNA. Evidence is provided that the proposed 3′-terminal structure is relevant not only for CymRSV DI but for genomic RNA as well.

Localization of cis-acting sequences essential for cymbidium ringspot tombusvirus defective interfering RNA replication

The smallest defective interfering RNA (DI-2) of cymbidium ringspot tombusvirus (CyRSV) was used to identify the cis-acting sequences necessary for its replication by making a series of deletions throughout the 404 nt long molecule and testing the biological activity of mutants. Deletion or substitution of the conserved sequence blocks (A, B and C) always yielded inactive molecules. The deletion of only a few nucleotides could be tolerated beyond the natural deletion sites in blocks A and B. However, either half of block C1 (34 nt) and the first 25 nt of C2 (102 nt) could be deleted without loss of infectivity. It was also demonstrated that either one of the two halves of block C1 was specifically required for replication. We suggest that the last 77 nt of the viral genome and either half of block C1 represent the complementary strand promoter sequence recognized by the viral replicase.

Characterization of Resistance to Cymbidium Ringspot Virus in Transgenic Plants Expressing a Full-Length Viral Replicase Gene

Transgenic Nicotiana benthamiana plants expressing the replicase gene of cymbidium ringspot tombusvirus (CymRSV) were tested for the biological activity of the transgene. Protoplasts from one resistant and three susceptible transgenic lines were transfected with in vitro synthesized defective interfering (DI) RNA, a small subviral molecule which requires only the virus replicase for replication. Only protoplasts of susceptible transgenic lines were able to sustain the replication of DI RNA, whereas protoplasts of the resistant line were not. The transgene of the resistant line was recovered from transgenic cells, cloned, and shown to be intact and functional, which suggests that this is not a case of dysfunctional replicase conferring resistance to transgenic plants as a "dominant negative mutant." Plants of the transgenic line resistant to CymRSV were shown to be susceptible to two other tombusviruses, artichoke mottled crinkle and carnation Italian ringspot virus. Since the resistance is specific and inversely correlated to the level of expression of the transgene, it appears to be a form of RNA-mediated resistance.

Generation of Defective Interfering RNA Dimers of Cymbidium Ringspot Tombusvirus

Inoculation of Nicotiana clevelandii and N. benthamiana plants with in vitro transcripts of both genomic and defective interfering (DI) RNAs of cymbidium ringspot tombusvirus resulted in a rapid accumulation of new DI-like RNA species which were demonstrated by cloning and sequencing to be head-to-tail dimers of unit length DI RNAs. The junction regions of dimers were represented by sequences derived precisely from the 5′ and 3′ termini of DI RNAs. Only infection with DI RNAs of smaller size (DI-2 and DI-3, 402 and 482 nt, respectively) produced detectable amount of dimers; in contrast, infection with the largest DI RNA (DI-13, 679 nt) was unable to accumulate dimers during viral infection. Analysis of mutant DI RNAs containing deletions or insertions revealed that the size of the monomer molecule is a major factor in the accumulation of dimers. Monomeric DI RNAs were formed in both plants and protoplasts inoculated with in vitro-transcribed directs. No heterodimers were found in plants inoculated simultaneously with DI-2 and DI-3 RNA molecules, which may indicate that replicase is not released from the template during synthesis of dimer molecules. However, the occurrence of a recombinant DI RNA dimer molecule derived from the two DI RNAs suggests that simultaneous infection of the same cells with two DI RNAs did indeed take place and that absence of heterodimers did not depend on compartmentalization.

Replication of Cymbidium Ringspot Virus Satellite RNA Mutants

Cymbidium ringspot tombusvirus (CyRSV) supports the replication of an RNA molecule known to be a satellite (sat-RNA). Sequence analysis of wild-type sat-RNA showed that the 3′ terminus is heterogeneous and terminates with one, two, or three C residues. Transcripts from mutant clones which lacked up to eight nucleotides at the 3′ end were biologically active and yielded progeny RNA that had the 3′ end restored as in wild-type RNA. Deletions or substitutions at the 5′ end produced nonviable molecules. Studies with mutants containing deletions in internal regions showed that replication was affected essentially by the location of deletions; viable mutants were much less encapsidated than the wild type, showing that size also may be important in the encapsidation and survival of CyRSV sat-RNA.

Evidence That ORF 1 and 2 Are the Only Virus-Encoded Replicase Genes of Cymbidium Ringspot Tombusvirus

The 33-kDa and its 92-kDa readthrough protein genes (open reading frames (ORFs) 1 and 2) of cymbidium ringspot tombusvirus (CyRSV) were introduced into Nicotiana benthamiana plants. Protoplasts derived from transgenic plants expressing the first two ORFs were inoculated with in vitro transcripts of defective interfering (DI) RNA. The efficient replication of input DI RNA, which is incapable of autonomous replication, confirmed the biological activity of the expressed viral replicase. In addition, in vitro transcripts of a genomic RNA deletion mutant clone encoding only ORFs 1 and 2 were able to replicate on their own and could support the replication of CyRSV DI RNAs in N. benthamiana protoplasts.

The nature of multimeric forms of cymbidium ringspot tombusvirus satellite RNA

The multimeric forms of cymbidium ringspot tombusvirus (CyRSV) satellite (sat) RNA were analysed. Attempts to amplify the putative junction region of oligomers using the polymerase chain reaction (PCR) failed, indicating the absence of such structures. SatRNA-related species having the size double than the unit length were shown to be double-stranded monomers and not single-stranded dimers. Similarly, satRNA species of a size four times the unit length were shown to be constituted by aggregates of double-stranded monomers. The absence of single-stranded CyRSV satRNA oligomers indicates that the formation of multimers is not a step in the replication of this RNA molecule.

Immunodetection of the 33 K/92 K polymerase proteins in cymbidium ringspot virus-infected and in transgenic plant tissue extracts.

An antiserum was raised against the 33 K protein encoded by the 5' proximal gene of cymbidium ringspot tombusvirus RNA. This antiserum reacts specifically with the 33 K and 92 K proteins, which constitute the viral replicase, in CyRSV-infected Nicotiana benthamiana plants and in transgenic plants transformed with the full-length replicase gene. In inoculated leaves of infected plants, synthesis of the 33 K/92 K proteins stops ten days after inoculation, whereas in newly produced systemically infected leaves there was continuous production of these proteins. In transgenic plants, both proteins were detected showing that readthrough of the termination codon of the 33 K protein does not depend on the presence of the replicating virus. The subcellular localization of the 33 K/92 K proteins is similar in infected and transgenic plants. No correlation was found between the level of expression of integrated virus gene and level of resistance to the challenging virus.

Expression of homologous and heterologous viral coat protein-encoding genes using recombinant DI RNA from cymbidium ringspot tombusvirus

Defective interfering (DI) RNA of cymbidium ringspot tombusvirus (CyRSV) was tested as a potential RNA vector. The coat protein-encoding gene ( CP) of the same virus or of the unrelated tomato aspermy cucumovirus (TAV) was inserted in a biologically active clone of CyRSV DI-3 RNA. Both homologous and heterologous CP genes were inserted into DI-3 cDNA clone in two different positions. The CyRSV CP was expressed only in the leaves which were inoculated with DI-3CPWt plus CP-less mutant helper virus (ΔABgII). Chimaeric DI RNA carrying a heterologous CP gene (DI-3HCPtav) was able to replicate and express the inserted TAV CP in the presence of a wild-type (wt) helper genome. Both constructs, which were stable and active for gene expression, carried the inserted CP genes in the same position, between the A and B blocks of DI-3 RNA. Other constructs in which the CP were cloned between the B and C blocks of DI-3 RNA were not able to direct the translation of the encoded CP. The expression level of CP derived from recombinant DI RNA was lower relative to expression of CP from wt virus infection.

Consequences of gene transfer between distantly related tombusviruses

Hybrid cDNA clones were constructed by fusing the coat protein-encoding gene and/or the 3'-terminal region (including the 22- and 19-kDa protein-encoding genes) derived from a clone of artichoke mottled crinkle tombusvirus to the 5'-terminal region of a full-length clone of cymbidium ringspot tombusvirus. In vitro transcripts from recombinant clones were infectious when inoculated into Nicotiana clevelandii plants. Inoculated plants showed symptoms different from those induced by parent viruses. In particular, systemic invasion depended very much, although not exclusively, on the type of protein that coated progeny viral RNA, suggesting a role of the capsid protein in the long-distance movement of tombusvirus infections.

Functional Analysis of Cymbidium Ringspot Virus Genome

Genomic RNA of cymbidium ringspot tombusvirus (CyRSV) contains five large open reading frames (ORFs). The two 5′ proximal ORFs encode proteins of 33 and 92 kDa and the three 3′ proximal ORFs encode proteins of 41,22, and 19 kDa. In addition, a small reading frame encoding a protein of 4 kDa was recently observed by computer analysis of the 3′ nontranslated region of CyRSV and other tombusvirus RNAs (ORF 6). The function of putative gene products was investigated with the use of several mutants. Mutants in ORFs 1 and 2 were not viable indicating that both 33- and 92-kDa proteins are required for replication. Deletion of a large portion of the coat protein gene, encoding a 41-kDa protein, did not prevent replication of viral RNA and cell-to-cell movement, but interfered severely with long-distance translocation. No virus particles or viral RNA could be detected in inoculated or upper leaves of plants inoculated with transcripts obtained from mutants not expressing the 22-kDa protein. However, replication and encapsidation occurred normally in inoculated protoplasts indicating that this gene product is a transport protein. Conversely, no role could be assigned to putative gene products of ORF 5 (19-kDa protein) and QRF 6. It was also shown that factors other than gene expression can influence the replication of RNA mutants, probably due to instability of RNA molecules.

Defective Interfering RNA-Mediated Resistance against Cymbidium Ringspot Tombusvirus in Transgenic Plants

Defective interfering (DI) RNA of cymbidium ringspot tombusvirus was cloned downstream from the bacteriophage T7 RNA polymerase promoter. In vitro synthesized RNA was biologically active when coinoculated with parental genomic RNA onto Nicotiana benthamiana plants and prevented the occurrence of apical necrosis. N. benthamiana plants were transformed with the DI RNA sequences in both the positive and negative orientations relative to the cauliflower mosaic virus 35S promoter. Integration of DI RNA sequences in the plant genome was verified using PCR amplification of DNA extracts and Northern blot analysis of RNA extracts. DI RNA-related transcripts were detected in uninfected transgenic plants, but inoculation with the parental virus induced replication of the DI RNA only in transgenic plants expressing DI RNA in the positive orientation. Transgenic plants in which DI RNA accumulated were protected from apical necrosis and death.

Repair in Vivo of Altered 3′ Terminus of Cymbidium Ringspot Tombusvirus RNA

Progeny RNA of CyRSV RNA transcripts ending with -GGG, instead of -CCC as in wild-type RNA, was analyzed and shown to revert to wild-type. A new full-length cDNA clone of CyRSV was prepared, bearing the correct 3′ terminus, from which transcripts could be synthesized having very high infectivity. Mutants were prepared which lacked the three terminal Cs; transcripts were infectious and most cDNA clones recovered from progeny RNA had regained the wild-type sequence. Transcripts ending in -GGCCA n were also infectious, and the 3′ sequence of most cDNA clones recovered after inoculation was the same as the inoculum sequence. Deletion of G at position -4 completely abolished infectivity. The occurrence of a repair mechanism at the 3′ end of CyRSV similar to telomerase activity on chromosomes is suggested.

Resistance to cymbidium ringspot tombusvirus infection in transgenic Nicotiana benthamiana plants expressing the virus coat protein gene

Transgenic Nicotiana benthamiana plants expressing the coat protein gene of cymbidium ringspot virus (CyRSV) were tested for resistance against infection with CyRSV. Transgenic plants showed resistance to infection only when the purified virions concentration in the inoculum was as low as 0.05 micrograms/ml. No protection was observed in transgenic plants inoculated with virion concentrations of 0.5 and 5.0 micrograms/ml or when the inoculum was in vitro synthesized genomic RNA.