Systemic silencing signal(s)

Grafting experiments have revealed that transgenic plants that undergo co-suppression of homologous transgenes and endogenous genes or PTGS of exogenous transgenes produce a sequence-specific systemic silencing signal that is able to propagate from cell to cell and at long distance. Similarly, infection of transgenic plants by viruses that carry (part of) a transgene sequence results in global silencing (VIGS) of the integrated transgenes although viral infection is localized. Systemic PTGS and VIGS strongly resemble recovery from virus infection in non-transgenic plants, leading to protection against secondary infection in newly emerging leaves and PTGS of transiently expressed homologous transgenes. The sequence-specific PTGS signal is probably a transgene product (for example, aberrant RNA) or a secondary product (for example, RNA molecules produced by an RNA-dependent RNA polymerase with transgene RNA as a matrix) that mimics the type of viral RNA that is targeted for degradation by cellular defence. Whether some particular cases of transgene TGS could also rely on the production of such a mobile molecule is discussed.     

Replicating satellite RNA induces sequence-specific DNA methylation and truncated transcripts in plants

Tobacco plants were transformed with a chimeric transgene comprising sequences encoding beta-glucuronidase (GUS) and the satellite RNA (satRNA) of cereal yellow dwarf luteovirus. When transgenic plants were infected with potato leafroll luteovirus (PLRV), which replicated the transgene-derived satRNA to a high level, the satellite sequence of the GUS:Sat transgene became densely methylated. Within the satellite region, all 86 cytosines in the upper strand and 73 of the 75 cytosines in the lower strand were either partially or fully methylated. In contrast, very low levels of DNA methylation were detected in the satellite sequence of the transgene in uninfected plants and in the flanking nonsatellite sequences in both infected and uninfected plants. Substantial amounts of truncated GUS:Sat RNA accumulated in the satRNA-replicating plants, and most of the molecules terminated at nucleotides within the first 60 bp of the satellite sequence. Whereas this RNA truncation was associated with high levels of satRNA replication, it appeared to be independent of the levels of DNA methylation in the satellite sequence, suggesting that it is not caused by methylation. All the sequenced GUS:Sat DNA molecules were hypermethylated in plants with replicating satRNA despite the phloem restriction of the helper PLRV. Also, small, sense and antisense approximately 22 nt RNAs, derived from the satRNA, were associated with the replicating satellite. These results suggest that the sequence-specific DNA methylation spread into cells in which no satRNA replication occurred and that this was mediated by the spread of unamplified satRNA and/or its associated 22 nt RNA molecules.     

Map-based cloning in crop plants: tomato as a model system II. Isolation and characterization of a set of overlapping yeast artificial chromosomes encompassing the jointless locus

Constructs carrying the entire or part of the tobacco nitrate reductase cDNA (NIA) cloned between the promoter and terminator sequences of the 35S RNA of the cauliflower mosaic virus were introduced into tobacco, in an attempt to improve nitrate assimilation. Several transgenic plants that had elevated NIA mRNA and nitrate reductase (NR) activity were obtained. In addition, a few plants that exhibited a chlorotic phenotype characteristic of NR-deficient mutants were also obtained. One of these plants contained no NIA mRNA, no NR activity and accumulated nitrate. This phenotype was therefore assumed to result from co-suppression of 35S-NIA transgenes and host NIA genes. NR-deficient plants were also found among the progeny of transformants overexpressing NIA mRNA. Genetic analyses indicated that these NR-deficient plants were homozygous for the 35S-NIA transgene, although not all homozygous plants were deficient for NR. The ratio of normal to NR-deficient plants in the progeny of homozygous plants remained constant at each generation, irrespective of the state of expression of the NIA genes (active or inactive) in the previous generation. This ratio also remained unchanged when field trials were performed in two areas of France: Versailles and Bergerac. The analysis of homozygous plants revealed that co-suppression was reversible at some stage of sexual reproduction. Indeed, host genes and transgenes reactivated at each generation, and co-suppression always appeared after a lag period of normal growth, suggesting that the phenomenon is developmentaly regulated. We observed that the triggering of cosuppression was delayed when plants were initially grown under limited light and/or watered with limited nitrate supply (light and nitrate both being required for the expression of the host NIA genes). However, this delay did not affect the final ratio between normal and NR-deficient plants after transfer to nitrate-fertilized fields. Independent transformants exhibited either different co-suppression ratios or no co-suppression at all, irrespective of the transgene copy number, suggesting that genomic sequences surrounding the transgene might play a role in determining co-suppression.     

Transgene Induced Co-Suppression during Vegetative Growth in Cryptococcus neoformans

Introduction of DNA sequences into the genome often results in homology-dependent gene silencing in organisms as diverse as plants, fungi, flies, nematodes, and mammals. We previously showed in Cryptococcus neoformans that a repeat transgene array can induce gene silencing at a high frequency during mating (～50%), but at a much lower frequency during vegetative growth (～0.2%). Here we report a robust asexual co-suppression phenomenon triggered by the introduction of a cpa1::ADE2 transgene. Multiple copies of the cpa1::ADE2 transgene were ectopically integrated into the genome, leading to silencing of the endogenous CPA1 and CPA2 genes encoding the cyclosporine A target protein cyclophilin A. Given that CPA1-derived antisense siRNAs were detected in the silenced isolates, and that RNAi components (Rdp1, Ago1, and Dcr2) are required for silencing, we hypothesize that an RNAi pathway is involved, in which siRNAs function as trans factors to silence both the CPA1 and the CPA2 genes. The silencing efficiency of the CPA1 and CPA2 genes is correlated with the transgene copy number and reached ～90% in the presence of >25 copies of the transgene. We term this transgene silencing phenomenon asexual co-suppression to distinguish it from the related sex-induced silencing (SIS) process. We further show that replication protein A (RPA), a single-stranded DNA binding complex, is required for transgene silencing, suggesting that RPA might play a similar role in aberrant RNA production as observed for quelling in Neurospora crassa. Interestingly, we also observed that silencing of the ADE2 gene occurred at a much lower frequency than the CPA1/2 genes even though it is present in the same transgene array, suggesting that factors in addition to copy number influence silencing. Taken together, our results illustrate that a transgene induced co-suppression process operates during C. neoformans vegetative growth that shares mechanistic features with quelling.     

Virus‐induced gene silencing in transgenic plants: transgene silencing and reactivation associate with two patterns of transgene body methylation

We used bisulfite sequencing to study the methylation of a viral transgene whose expression was silenced upon plum pox virus infection of the transgenic plant and its subsequent recovery as a consequence of so‐called virus‐induced gene silencing (VIGS). VIGS was associated with a general increase in the accumulation of small RNAs corresponding to the coding region of the viral transgene. After VIGS, the transgene promoter was not methylated and the coding region showed uneven methylation, with the 5′ end being mostly unmethylated in the recovered tissue or mainly methylated at CG sites in regenerated silenced plants. The methylation increased towards the 3′ end, which showed dense methylation in all three contexts (CG, CHG and CHH). This methylation pattern and the corresponding silenced status were maintained after plant regeneration from recovered silenced tissue and did not spread into the promoter region, but were not inherited in the sexual offspring. Instead, a new pattern of methylation was observed in the progeny plants consisting of disappearance of the CHH methylation, similar CHG methylation at the 3′ end, and an overall increase in CG methylation in the 5′ end. The latter epigenetic state was inherited over several generations and did not correlate with transgene silencing and hence virus resistance. These results suggest that the widespread CG methylation pattern found in body gene bodies located in euchromatic regions of plant genomes may reflect an older silencing event, and most likely these genes are no longer silenced.     

Homology-dependent resistance: transgenic virus resistance in plants related to homology-dependent gene silencing

Tobacco plants transformed with the RNA polymerase (RdRp) gene of potato virus X (PVX) that are extremely resistant to infection by potato virus X have previously been described. The PVX-resistant plants accumulated the RdRp protein at a lower level than fully susceptible plants transformed with the same RdRp construct. In this paper the difference between the PVX-resistant and susceptible transformed plants is investigated and it is demonstrated that there are three associated phenotypes of the RdRp transgene that vary in parallel between transformed lines. These phenotypes are: accumulation of the transgenic RdRp RNA at a low level; strain-specific resistance to PVX; and the ability of the transgene to trans -inactivate homologous transgenes. This gene-silencing potential of the transgenes conferring PVX resistance was illustrated by analysis of progeny from a cross between a transformant that was extremely resistant to PVX and a second PVX-susceptible transformant. In other transformants, in which the resistance was less extreme, the same three phenotypes were associated but in a transgene dosage-dependent manner. The same association of strain-specific resistance and low-level accumulation of the transgenic RdRp RNA was observed with plants that were transformed with mutant or wild-type versions of the RdRp gene. Strain-specific resistance was also produced in plants transformed with untranslatable versions of the RdRp transgene. Based on these data it is proposed that homology-dependent gene silencing and transgenic resistance to PVX may be due to the same RNA-based mechanism. An undefined genomic feature is proposed to account for the variation in the resistance and trans -inactivation phenotypes of different transformants. It is further proposed that this genome feature influences a cytoplasmic mechanism that degrades RNA with sequence homology to the silencing transgene.