Abstract: 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.
Abstract: Transgenic tomato (Lycopersicon esculentum Mill. cv. UC82) plants expressing a benign variant of Cucumber mosaic virus satellite RNA (CMV Tfn-satRNA) were generated. The transformed plants did not produce symptoms when challenged with a satRNA-free strain of CMV (CMV-FL). The same plant lines initially were susceptible to necrosis elicited by a CMV strain supporting a necrogenic variant of satRNA (CMV-77), but a phenotype of total recovery from the necrosis was observed in the newly developing leaves. The features of the observed resistance were analyzed and are consistent with two different mechanisms of resistance. In transgenic plants inoculated with CMV-FL strain, the symptomless phenotype was correlated to the down-regulation of CMV by Tfn-satRNA, amplified from the transgene transcripts, as the first resistance mechanism. On the other hand, the delayed resistance to CW-77 in transgenic tomato lines was mediated by a degradation process that targets satRNAs in a sequence-specific manner. Evidence is provided for a correlation between a reduced accumulation level of transgenic messenger Tfn-satRNA, the accumulation of small (approximately 23 nucleotides) RNAs with sequence homology to satRNAs, the progressively reduced accumulation of 77-satRNA in infected tissues, and the transition in infected plants from diseased to healthy. Thus, events leading to the degradation of satRNA sequences indicate a role for RNA silencing as the second mechanism determining resistance of transgenic tomato lines.
Abstract: Delivery into plants of T-DNAs containing promoter, terminator, or coding sequences generated small interfering RNAs (siRNAs) specific to each type of sequence. When both promoter and transcribed sequences were simultaneously present in the T-DNA, accumulation of siRNAs to transcribed sequences was favored over accumulation of siRNAs to the nontranscribed upstream promoter sequences. The generation of specific siRNA sequences occurred even in the absence of T-DNA homology to sequences in the plant. Delivery of T-DNA, with homology to the transgene limited to the nontranscribed cauliflower mosaic virus 35S promoter (35SP) and the transcribed nopaline synthase transcription termination (NosT)signal sequences, into transgenic plants expressing the green fluorescent protein (GFP), generated siRNAs in infiltrated tissues to both 35SP (35SsiRNAs) and NosT (NosTsiRNAs), but not to the GFP sequence (GFPsiRNAs). In infiltrated tissues, the 35SsiRNAs failed to trigger the transcriptional silencing of the transgene, accumulation of 35SsiRNAs could be prevented by the potyviral HC-Pro, and the NosTsiRNAs required an initial amplification to trigger efficient transgene silencing, which is mediated by transcripts from the exogenous T-DNA, and not from the transgene. In upper leaves, silencing correlated with the presence of GFPsiRNAs and the absence of 35SsiRNAs, confirming that its spread was posttranscriptionally mediated by the transgene mRNA.
Abstract: The replication-associated proteins encoded by Cucumber mosaic virus (CMV), the 1a and 2a proteins, were detected by immunogold labeling in two host species of this virus, tobacco (Nicotiana tabacum) and cucumber (Cucumis sativus). In both hosts, the 1a and 2a proteins colocalized predominantly to the vacuolar membranes, the tonoplast. While plus-strand CMV RNAs were found distributed throughout the cytoplasm by in situ hybridization, minus-strand CMV RNAs were barely detectable but were found associated with the tonoplast. In both cucumber and tobacco, 2a protein was detected at higher densities than la protein. The 1a and 2a proteins also showed quantitative differences with regard to tissue distributions in tobacco and cucumber. About three times as much 2a protein was detected in CMV-infected cucumber tissues as in CMV-infected tobacco tissues. In tobacco, high densities of these proteins were observed only in vascular bundle cells of minor veins. In contrast, in cucumber, high densities of 1a and 2a proteins were observed in mesophyll cells, followed by epidermis cells, with only low levels being observed in vascular bundle cells. Differences were also observed in the distributions of 2a protein and capsid protein in vascular bundle cells of the two host species. These observations may represent differences in the relative rates of tissue infection in different hosts or differences in the extent of virus replication in vascular tissues of different hosts.
Abstract: In the last few years, tomato crops of Southern coastal Croatia have been repetitively stricken by a devastating necrotic tomato disease on such a scale that it endangered tomato production in the area. Cucumber mosaic cucumovirus (CMV) was found in symptomatic plants by serology. Low-molecular weight RNA bands were detected in total nucleic acid extracts from naturally infected tomato fruits. The satellite nature of this RNA was proven by molecular hybridization with a digoxigenine-labelled satellite RNA probe. Molecular and biological characterization of the satRNA proved to be involved in codetermination of the lethal necrosis disease. This is the first report of CMV strain containing a satRNA in Croatia.(1)
Abstract: A cucumber mosaic virus (CMV) isolate supporting a natural 390-ribonucleotide satellite was used to reproduce under experimental conditions a disease of processing tomatoes called fruit necrosis. The virus induced incomplete differentiation of the vascular tissue of fruit stalks, which was the likely cause of the disease. On the other hand, the satellite RNA attenuated viral symptoms on tomato leaves reproducing the disease pattern typically observed in the field. The biological properties of this seemingly new variant of cucumoviral satellite RNAs were determined.
