Development of a virus induced gene silencing vector from a legumes infecting tobamovirus.

Medicago truncatula, the model plant of legumes, is well characterized, but there is only a little knowledge about it as a viral host. Viral vectors can be used for expressing foreign genes or for virus-induced gene silencing (VIGS), what is a fast and powerful tool to determine gene functions in plants. Viral vectors effective on Nicotiana benthamiana have been constructed from a number of viruses, however, only few of them were effective in other plants. A Tobamovirus, Sunnhemp mosaic virus (SHMV) systemically infects Medicago truncatula without causing severe symptoms. To set up a viral vector for Medicago truncatula, we prepared an infectious cDNA clone of SHMV. We constructed two VIGS vectors differing in the promoter element to drive foreign gene expression. The vectors were effective both in the expression and in the silencing of a transgene Green Fluorescent Protein (GFP) and in silencing of an endogenous gene Phytoene desaturase (PDS) on N. benthamiana. Still only one of the vectors was able to successfully silence the endogenous Chlorata 42 gene in M. truncatula.

RNA silencing: an antiviral mechanism.

RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as an antiviral mechanism in higher plants and insects. To overcome antiviral RNA silencing, viruses express silencing-suppressor proteins which can counteract the host silencing-based antiviral process. After the discovery of virus-encoded silencing suppressors, it was shown that these viral proteins can target one or more key points in the silencing machinery. Here we review recent progress in our understanding of the mechanism and function of antiviral RNA silencing in plants, and on the virus's counterattack by expression of silencing-suppressor proteins. We also discuss emerging evidence that RNA silencing and expression of viral silencing-suppressor proteins are tools forged as a consequence of virus-host coevolution for fine-tuning host-pathogen coexistence.

Identification of sequence elements of tombusvirus-associated defective interfering RNAs required for symptom modulation.

Defective interfering (DI) RNAs of tombusviruses are short, non-coding, symptom-modulating RNAs originating from the viral genome. The presence of homologous DI RNA in virus infection attenuates the otherwise lethal viral symptoms. Nicotiana benthamiana plants infected with tomato bushy stunt tombusvirus pepper isolate (TBSV-P) show severe symptoms, which culminate in the death of the plant. In contrast, plants co-inoculated with TBSV-P and TBSV-P-derived DI RNA display attenuated symptoms. However, co-inoculation of TBSV-P with heterologous DI RNA, originating from Carnation Italian ringspot tombusvirus results in development of apical necrotic symptoms. To localize the symptom-determining factors on DI RNA genome, chimeras of protective and non-protective DI RNAs have been constructed. All chimeras were biologically active and accumulated to a high level in the presence of helper virus. We identified a 5' proximal sequence element of the DI RNA as the most important symptom determinant region. However, our results demonstrated that the symptom modulating ability of this region is also influenced by the sequence composition of whole DI RNAs.

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.

The ORF1 products of tombusviruses play a crucial role in lethal necrosis of virus-infected plants.

Hybrids of cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) tombusviruses were used to identify viral symptom determinants responsible for the generalized necrosis in tombusvirus-infected plants. Surprisingly, symptoms of Nicotiana benthamiana infected with CymRSV/CIRV hybrids were distinctly different. It was demonstrated that not all chimeras expressing wild-type (wt) levels of p19 protein caused systemic necrosis as both parents CymRSV and CIRV did. We showed here that hybrids containing chimeric ORF1 were not able to induce lethal necrosis even if the viral replication of these constructs was not altered significantly. However, if a wt p33 (product of ORF1) of CymRSV was provided in trans in transgenic plants expressing p33 and its readthrough product p92, the lethal necrosis characteristic to tombusvirus infection was restored. In addition, the expression of p33 by a potato virus X viral vector in N. benthamiana caused severe chlorosis and occasionally necrosis, indicating the importance of p33 in wt symptoms of tombusviruses. Thus, our results provide evidence that elicitation of the necrotic phenotype requires the presence of the wt p33 in addition to the p19 protein of tombusviruses.

The complete nucleotide sequence and synthesis of infectious RNA of genomic and defective interfering RNAs of TBSV-P.

The complete nucleotide sequences of the genome of the pepper isolate of tomato bushy stunt Tombusvirus (TBSV-P), and its defective interfering (DI) RNAs were determined. The genome of TBSV-P is a linear single-stranded monopartite RNA molecule of positive polarity, 4776 nucleotides long and has an organisation identical to that reported for other tombusviruses. In vitro transcripts of the genome were highly infectious, and it could support replication of the DI RNAs associated with the wild type virus. Two DI RNAs were found in the infected leaves of Nicotiana clevelandii, whose sequences were completely derived from the genomic RNA. The longest DI RNA (DI-5) has 550 nucleotides (nt), while the shorter DI RNA (DI-4) composed of 463 nt, both of them were formed by essentially the same genomic sequence blocks. Since host specificity of TBSV-P and other tombusviruses with available infectious cDNA clones is different, it is feasible to carry out gene exchange studies to determine viral host specificity factors for tombusviruses.

Interactions between tombusviruses and satellite RNAs of tomato bushy stunt virus: a defect in sat RNA B1 replication maps to ORF1 of a helper virus.

The biological properties of two recently described satellite RNAs of tomato bushy stunt virus (TBSV) were analyzed in natural and experimental hosts. Full-length cDNA clones were constructed for sat RNAs B1(822 nt) and B10 (612 nt) and used in inoculations with satellite-free transcripts of different tombusviruses. In all virus-host combinations tested, TBSV sat RNA B10 drastically reduced the accumulation of viral genomic RNA and attenuated symptoms. In contrast, sat RNA B1 caused a less marked reduction of viral RNA level and did not have any effect on symptoms. Experiments with Nicotiana benthamiana protoplasts showed that the differential effects of sat RNAs B1 and B10 on TBSV titer were related to differential abilities to interfere with virus replication. Three tombusviruses tested were able to maintain both sat RNAs in N. benthamiana plants, although carnation Italian ringspot virus (CIRV) was a poor helper for sat RNA B1. Using chimeric viruses, a strong determinant for low sat RNA B1 accumulation was mapped to the 5'-terminal part of the genome of CIRV. The poor helper activity of CIRV was shown to be due to low sat RNA B1 replication. A single-nucleotide mutation in the start codon of CIRV ORF1 restored the ability to replicate sat RNA B1 to high levels. This mutant encodes an ORF1 that is 22 amino acids shorter at the N-terminus than the wild-type virus.

Characterization of the molecular mechanism of defective interfering RNA-mediated symptom attenuation in tombusvirus-infected plants.

Different tombusviruses were able to support the replication of either homologous or heterologous defective interfering (DI) RNAs, and those infected plants usually developed typical attenuated symptoms. However, in some helper virus-DI RNA combinations the inoculated plants were necrotized, although they contained a high level of DI RNA, suggesting that the accumulation of DI RNA and the resulting suppression of genomic RNA replication were not directly responsible for the symptom attenuation. Moreover, the 19-kDa protein product of ORF 5, which is known to play a crucial role in necrotic symptom development, accumulated at the same level in the infected plants in the presence of protective homologous DI RNA and in the presence of nonprotective heterologous DI RNA. It was also demonstrated, by chimeric helper viruses, that the ability of heterologous DI RNA to protect the virus-infected plants against systemic necrosis is determined by the 5'-proximal region of the helper virus genome. The results presented suggest that DI RNA-mediated protection did not operate via the specific inhibition of 19-kDa protein expression but, more likely, DI RNAs in protective DI-helper virus combinations specifically interacted with viral products, preventing the induction of necrotic symptoms.

Template-independent repair of the 3' end of cucumber mosaic virus satellite RNA controlled by RNAs 1 and 2 of helper virus.

RNA viruses which do not have a poly(A) tail or a tRNA-like structure for the protection of their vulnerable 3' termini may have developed a different strategy to maintain their genome integrity. We provide evidence that deletions of up to 7 nucleotides from the 3' terminus of cucumber mosaic cucumovirus (CMV) satellite RNA (satRNA) were repaired in planta in the presence of the helper virus (HV) CMV. Sequence comparison of 3'-end-repaired satRNA progenies, and of satRNA and HV RNA, suggested that the repair was not dependent on a viral template. The 3' end of CMV satRNA lacking the last three cytosines was not repaired in planta in the presence of tomato aspermy cucumovirus (TAV), although TAV is an efficient helper for the replication of CMV satRNA. With use of pseudorecombinants constructed by the interchange of RNAs 1 and 2 of TAV and CMV, evidence was provided that the 3'-end repair was controlled by RNAs 1 and 2 of CMV, which encode subunits of the viral RNA replicase. These results, and the observation of short repeated sequences close to the 3' terminus of repaired molecules, suggest that the HV replicase maintains the integrity of the satRNA genome, playing a role analogous to that of cellular telomerases.

Secondary structure-dependent evolution of Cymbidium ringspot virus defective interfering RNA.

Mutational analysis of defective interfering (DI) RNAs of Cymbidium ringspot virus (CymRSV) was used to study the mechanism of DI RNA evolution. It was shown that a highly base-paired structure in the 3' region of the longer DI RNA directed the formation of smaller DI RNA molecules. Mutations which increased the stability of the computer-predicted, highly structured 3' region of the longest DI RNA of CymRSV significantly enhanced the generation and accumulation of the smaller derivatives. Sequence analysis of smaller progeny molecules revealed that the highly base-paired region was deleted from the precursor DI RNA. Moreover, sites of recombination were found in other regions of the DI RNA progenies due to transposition of the highly base-paired structure. It is likely that the deletion event was structure- and not sequence-specific, and operated when a foreign sequence containing a 37-nt-long base-paired stem was inserted at the appropriate position of DI RNA.

Complete nucleotide sequence of tobacco necrosis virus strain DH and genes required for RNA replication and virus movement.

The complete genome sequence of tobacco necrosis virus strain D (Hungarian isolate, TNV-DH) was determined. The genome (3762 nt) has an organization identical to that reported for TNV-D. Highly infectious synthetic transcripts from a full-length TNV-DH cDNA clone were prepared, the first infectious necrovirus transcript reported. This clone was used for reverse genetic studies to map the viral genes required for replication and movement. Protoplast inoculation with delta 22 and delta 82 mutants revealed that both the 22 kDa and 82 kDa gene products are required for RNA replication. Although the products of three small central genes (p7(1), p7a and p7b) were not essential for RNA replication in protoplasts, mutations in these ORFs prevented infection of plants. In contrast, viral RNA accumulation and cell-to-cell movement were observed in the inoculated, but not the systemically infected, leaves of Nicotiana benthamiana challenged with RNA lacking the intact coat protein (CP) gene. These results strongly suggest that p7(1), p7a, p7b and CP are involved in TNV-DH cell-to-cell and long-distance movement, respectively.

The 5'-terminal region of a tombusvirus genome determines the origin of multivesicular bodies.

Multivesicular bodies (MVB) are membranous cytoplasmic inclusions that are invariably associated with tombusvirus infections regardless of the virus species, the host, or the tissue examined. MVB are virus-induced structures since they are absent from tissues of healthy plants and are always present both in infected plants and protoplasts. MVB derive from peroxisomes in cells infected by a number of tombusviruses including cymbidium ringspot virus (CymRSV) and from mitochondria in cells infected by another tombusvirus, carnation Italian ringspot virus (CIRV). By using common restriction sites in full-length infectious clones, hybrid clones of these two viruses were constructed. In addition, a mutant of CIRV was prepared in which the protein encoded by the first open reading frame was shortened by 22 amino acids. All mutant transcripts were viable and infected Nicotiana benthamiana plants. Infected leaf tissue samples were collected, processed for thin sectioning, and observed in the electron microscope. The origin of MVB was shown to be under the control of the 5' region of the viral genome. A sequence as short as about 600 nucleotides in ORF 1 contained the determinants for formation of MVB from peroxisomes or mitochondria.

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 the cis-acting nature of the last 77-nt sequence of the viral genome which is required for DI RNA accumulation. The 3'-terminal cis-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.

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 dimers. 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.

Molecular cloning and complete nucleotide sequence of carnation Italian ringspot tombusvirus genomic and defective interfering RNAs.

The complete sequences of genomic and defective interfering (DI) RNAs of carnation Italian ringspot tombusvirus (CIRV) were determined. The genome (4760 nt) has an organization identical to that reported for other tombusviruses except that the pre-readthrough domain of the viral replicase encoded by the 5'-proximal open reading frame (ORF) is larger. In particular, the N-terminal region of this protein differs from the corresponding region of the other members of the genus Tombusvirus and Carmovirus. Two DI RNAs were found in infected tissues whose sequences were completely derived from genomic RNA. The smaller molecule (474 nt) is contained completely within the larger molecule (656 nt), which suggests that it is derived from the larger one.

Mapping helper virus functions for cucumber mosaic virus satellite RNA with pseudorecombinants derived from cucumber mosaic and tomato aspermy viruses.

P-TAV is a strain of tomato aspermy virus (TAV) able to efficiently support the systemic accumulation of some (i.e., B2-satRNA) but not of other (i.e., Ix-satRNA) strains of the satellite RNA (satRNA) of cucumber mosaic virus (CMV) in both tobacco and in tomato. As reported for V-TAV, the failure to support the systemic accumulation of Ix-satRNA seems to be due to an inefficient support of its systemic movement. Pseudorecombinants obtained by the exchange of RNAs 1 + 2 between P-TAV and Trk7-CMV, an efficient helper for the systemic accumulation of Ix-satRNA, were assayed for their ability to support the accumulation of CMV-satRNAs in tobacco plants and protoplasts. Pseudorecombinants having RNAs 1 + 2 from CMV supported the systemic movement and accumulation of CMV-satRNA as efficiently as CMV, whereas pseudorecombinants having RNAs 1 + 2 from TAV supported the CMV-satRNA very poorly. Thus, the ability to support the systemic movement and accumulation of CMV-satRNA is determined primarily by RNAs 1 + 2 and not by RNA 3, which is presumed to encode movement functions in the cucumoviruses and only has a minor, modulating effect on the systemic accumulation of satRNA. This suggests that for systemic movement CMV-satRNA has to interact with (the gene products of) RNAs 1 and/or 2 or that these viral RNAs compete with the satRNA for interaction with the coat or other movement proteins.

Sensitive non-radioactive nucleic acid hybridization assay for plum pox virus detection.

A new non-radioactive sandwich hybridization assay was designed to simplify the analysis of a large number of plant samples. Plant material was homogenized in 0.5% SDS and added directly to the hybridization reaction, in which a pair of identifying probes were used. One of the probes was biotinylated capture RNA specific for plum pox virus (PPV) strain SK-68; the other RNA probe was synthesized from a plasmid bearing the adjacent sequence of this strain and was labelled with digoxigenin (DIG). Both purified viral RNA and crude extracts from PPV-infected plants were used as target for sandwich hybridization. The hybridization reaction was carried out in a streptavidin-coated ELISA plate. After extensive washing, the viral RNA was detected by conventional colour reaction using anti-DIG/alkaline phosphatase conjugate. In comparative experiments, we have shown that this non-radioactive detection system is more sensitive than conventional ELISA techniques and we were able to detect virus-specific RNA in more than 50% of the ELISA-negative samples.

Increase in the relative fitness of a plant virus RNA associated with its recombinant nature.

A recombination event at the 3' end of RNA 3 in a pseudorecombinant virus (C1C2T3) having RNAs 1 and 2 from TrK7-cucumber mosaic virus (CMV) and RNA 3 from P-tomato aspermy virus (TAV) was detected by ribonuclease protection assay (RPA). Sequence analysis of the 3' end of RNA 3 of C1C2T3 and of its parental CMV and TAV strains showed that RNA 3 of C1C2T3 had a hybrid nature in which most of the 3' noncoding region (3' ncr), including the whole 3' terminal tRNA-like structure, was derived from CMV RNA 2 by recombination. Recombination may have occurred in two steps. The first one would have caused the duplication of a large region 5' to the 3' end tRNA-like structure, derived from TAV and from CMV. The second one would have eliminated the TAV-derived sequences in this duplicated region. Competition experiments in tobacco plants showed that in a context of RNA 1 + 2 from CMV, RNA 3 from TAV is outcompleted by RNA 3 of CMV, but the recombinant TAV RNA 3 with the 3' end of CMV outcompetes both TAV and CMV RNA 3. This shows an increase in relative fitness associated with the recombinant nature of RNA 3. Our results document the potential importance of RNA-RNA recombination in the determination of the genetic structure of RNA viral populations.