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.

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.

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.

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.

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.