Heterologous replicase driven 3' end repair of Cucumber mosaic virus satellite RNA.

To investigate the extent of the 3' end repair in a satellite RNA of Cucumber mosaic virus (CMV) strain Q (Q(sat)) by a heterologous Tomato aspermy virus (TAV), a set of biologically active agrotransformants corresponding to the three genomic RNAs of TAV was developed. Analysis of Nicotiana benthamiana plants agroinfiltrated with TAV and either wild type or each of the six 3' deletion mutants of Q(sat) revealed that (i) heterologous replicase failed to generate Q(sat) multimers, a hallmark feature of homologous replicase dependent replication of Qsat; (ii) manifestation of severe symptom phenotypes and progeny analysis suggested that heterologous replicase was competent to repair Q(sat) deletion mutants lacking up to 3'13 nucleotides (nt) but not beyond and (iii) comparative in silico analysis indicated that the 3' secondary structural features of the repaired Q(sat) progeny from heterologous vs homologous driven replicases are remarkably very similar. The significance of these observations is discussed.

Regulation of a virus-induced lethal disease in tomato revealed by LongSAGE analysis.

Infection of Cucumber mosaic virus (CMV) and D satellite RNA (satRNA) in tomato plants induces rapid plant death, which has caused catastrophic crop losses. We conducted long serial analysis of gene expression (LongSAGE) in control and virus-infected plants to identify the genes that may be involved in the development of this lethal tomato disease. The transcriptomes were compared between mock-inoculated plants and plants infected with CMV, CMV/D satRNA, or CMV/Dm satRNA (a nonnecrogenic mutant of D satRNA with three mutated nucleotides). The analysis revealed both general and specific changes in the tomato transcriptome after infection with these viruses. A massive transcriptional difference of approximately 400 genes was found between the transcriptomes of CMV/D and CMV/Dm satRNA-infected plants. Particularly, the Long-SAGE data indicated the activation of ethylene synthesis and signaling by CMV/D satRNA infection. Results from inoculation tests with an ethylene-insensitive mutant and treatments with an ethylene action inhibitor further confirmed the role of ethylene in mediating the epinastic leaf symptoms and the secondary cell death in the stem. Results from Northern blot analysis demonstrated the partial contribution of ethylene in the induced defense responses in CMV/D satRNA-infected plants.

Response of tomato and its wild relatives in the genus Solanum to cucumber mosaic virus and satellite RNA combinations.

The differential response of 29 genotypes of tomato and wild tomato relatives (Solanum section Lycopersicon species) to cucumber mosaic virus strain Fny (CMV-Fny), alone or in combination with three different satellite RNA (satRNA) variants, allowed the identification of four disease phenotype patterns, each including plants that developed very severe symptoms (leaf malformations, top stunting and lethal necrosis) and plants that remained asymptomatic. No resistance or tolerance to CMV-Fny was observed, whilst individual host genotypes displayed latent infection upon inoculation with one (CMV-Fny/Tfn-satRNA, phenotype patterns 1 and 4), two (CMV-Fny/Tfn-satRNA and CMV-Fny/TTS-satRNA, phenotype pattern 2) or all three (the former two plus CMV-Fny/77-satRNA, phenotype pattern 3) CMV/satRNA combinations. RNA gel-blot analyses showed that latent infection generally correlated with a strong downregulation of CMV RNA accumulation levels. Introgression lines derived from a cross between Solanum habrochaites LA1777, which displayed disease phenotype pattern 2, and Solanum lycopersicum were screened for tolerance to the stunting phenotype induced by CMV-Fny/TTS-satRNA, and only one line, carrying an introgression on chromosome 6, was identified as being partially tolerant. Solanum chilense LA1932xS. lycopersicum back-cross introgression lines were screened for tolerance to lethal necrosis induced by CMV-Fny/77-satRNA (phenotype pattern 3); the tolerant phenotype was observed in 33 % of plants of the BC(1)F(2) progeny and <1 % of plants of the BC(1)F(3) progeny. Thus, potentially useful sources of tolerance to CMV/satRNA-induced diseases were identified, although the tolerant phenotypes appeared to be controlled by complex quantitative trait loci.

[Studies on construction of artificial mutants of Cucumber mosaic virus satellite RNA and their biological activity].

Based on the full length cDNA clone of a Cucumber mosaic virus satellite RNA, which was 369nt in size, artificial mutants were developed by the method of error-prone PCR and DNA shuffling. The new satellite cDNAs were transcribed in vitro into ssRNA and pseudo-recombined with a helper Cucumber mosaic virus, which contains no satellite RNA. Sequence analysis showed that A to T/G or G to A replacement all the four mutants, named MS1, MS5, MS6 and MS11 respectively, and there is no C to G or G to C replacement, but amongst, only the mutants MS11 could replicated when recombined with the helper virus strain. No satellite RNA could be detected by RT-PCR amplification and double-stranded RNA analysis for those pseudo-recombination constitution of Cucumber mosaic virus strain with mutants MS1, MS5 and MS6.Sequence homological comparison showed that the single replacement of mutants MS1, MS5 and MS6 occurred in the highly conservative regions and the T to A replacement of mutant MS11 was located in the normal-variation region. This is the first artificial mutation of satellite RNA of plant RNA viruses. The results indicated that single base in the region of satellite RNA maybe important to maintaining the biological activity of satellite RNA for its replication and stability. The variation and evolution of satellite RNA could be hopefully studied through combination directed evolution by DNA shuffling with pseudo-recombination in vitro.

Cucumber mosaic virus from Ixora: infectious RNA transcripts confirm deficiency in satellite support and unusual symptomatology.

A cucumber mosaic virus isolate from an Ixora plant originating in the Philippines and first described 20 years ago (CMV-Ix) by Waterworth and Povish differs from other characterized CMV strains in its anomalous satellite support and in several biological and molecular properties. We describe the preparation of infectious transcripts from cloned complementary DNA and the characterization of progeny virus. The results confirm that CMV-Ix has a more limited host range than most CMV isolates. Virions of CMV-Ix are smaller than those of the control CMV-S, independent of the type of negative stain used. Furthermore, CMV-Ix from transcripts supports the replication of the satellite T-CARNA 5 but not of D-CARNA 5.

Differential effects of satellite RNA on the accumulation of cucumber mosaic virus RNAs and their encoded proteins in tobacco vs zucchini squash with two strains of CMV helper virus.

The presence of cucumber mosaic virus (CMV) satellite RNA usually reduces the yield of accumulated helper virus, although more so in solanaceous than in cucurbit hosts. The accumulation of viral RNA and viral-encoded proteins of two strains of CMV (Fny- and Sny-) known to differ in their ability to support satellite RNA in zucchini squash was examined in squash and tobacco to determine the effect of satellite RNA on the accumulation of viral-associated components. In the absence of satellite RNA, Fny- and Sny-CMV showed similar levels of accumulation of RNA at 7 days postinoculation (p.i.), but by 14 days p.i. the Fny-CMV RNAs accumulated to lower levels than did both strains at 7 days p.i., in either host. The levels of accumulated Sny-CMV-encoded proteins were higher than those encoded by Fny-CMV in tobacco, but not squash plants, at 7 days p.i. At 14 days p.i., for Fny-CMV vs Sny-CMV, there were differences in the levels of accumulation of most CMV-encoded proteins in both hosts, more exacerbated in tobacco vs squash. The effect of satellite RNA was to intensify these differences; that is, by 7 days p.i., satellite RNA reduced the accumulation of Fny-CMV RNAs 1 and 2 and their encoded proteins in both tobacco and squash but had little or no effect on the accumulation of Sny-CMV RNAs or encoded proteins. By 14 days p.i., the levels of accumulation of all Fny-CMV RNAs and encoded proteins were severely reduced in both hosts, and the levels of accumulation of Sny-CMV RNAs 1 and 2 and their encoded proteins were also reduced in tobacco, but not squash. Sny-CMV did not support satellite RNA accumulation in squash plants or protoplasts. Satellite RNA did not appear to have a direct effect on the movement of either CMV strain. Rather, accumulation studies in tobacco protoplasts indicated that the difference in response of Fny-CMV vs Sny-CMV to satellite RNA in tobacco was due to the extent to which satellite RNA affected the levels of RNA 1, and to a lesser extent RNA 2, and their encoded proteins, 1a and 2a, both components of the CMV replicase.