Transgenes are dispensable for the RNA degradation step of cosuppression

Abstract

Cosuppression results in the degradation of RNA from host genes and homologous transgenes after transcription in the nucleus. By using grafting experiments, we have shown previously that a systemic signal mediates the propagation of cosuppression of Nia host genes and 35S-Nia2 transgenes from silenced 35S-Nia2 transgenic stocks to nonsilenced 35S-Nia2 transgenic scions but not to wild-type scions. Here, we examined the requirements for triggering and maintenance of cosuppression in various types of scions. Grafting-induced silencing occurred in 35S-Nia2 transgenic lines over-accumulating Nia mRNA whether they are able to spontaneously trigger cosuppression or not and in 35S-Nia2 transgene-free plants over-accumulating host Nia mRNA caused by metabolic derepression. When grafting-induced silenced scions were removed from the silenced stocks and regrafted onto wild-type plants, silencing was not maintained in the 35S-Nia2 transgene-free plants and in the 35S-Nia2 transgenic lines that are not able to trigger cosuppression spontaneously. Conversely, silencing was maintained in the 35S-Nia2 transgenic lines that are able to trigger cosuppression spontaneously. Our results indicate that the presence of a 35S-Nia2 transgene is dispensable for the RNA degradation step of posttranscriptional silencing when host Nia mRNA over-accumulate above the level of wild-type plants. They also suggest that grafting-induced RNA degradation does not result in the production of the systemic silencing signal required for spontaneous triggering and maintenance.

Figure 1

Nia mRNA accumulation in the different nonsilenced scions (a) wild-type plants (WT) and homozygous transgenic plants over-accumulating Nia mRNA caused by the presence of the 35S-Nia2 transgene (classes I and II). (b) Wild-type plants (WT) and NR- or NiR-deficient plants over-accumulating host Nia mRNA caused by metabolic derepression (class III). Ten micrograms of total RNA extracted from leaves were probed with the tobacco Nia2 cDNA. Photographs of the 25S rRNA band in the ethidium-stained gel indicate the amounts of RNA loaded in each lane.

Figure 2

Triggering and maintenance of Nia mRNA degradation. (a) Homozygous transgenic 35S-Nia2 scions of class I (34–19.4) or class II (30–46.7) were grafted onto wild-type (/WT) or spontaneously silenced 35S-Nia2 transgenic stocks of class II (/S). Six weeks later, the terminal apices of grafting-induced silenced scions were removed and regrafted onto wild-type stocks (/S → WT). (b) Scions of class III that do not contain the 35S-Nia2 transgene were grafted as described in a. Two plants of each genotype were analyzed for each conditions. In lane 10 (NIA30 scion grafted onto silenced 35S-Nia2 lines), the spot on the membrane is an artifact, which does not correspond to the Nia mRNA. Its position is above that of the Nia mRNA, and the migration of RNA on the gel was correct as indicated by the picture of the 25S rRNA, which comigrates with the Nia mRNA. Ten micrograms of total RNA extracted from apical leaves were probed with the tobacco Nia2 cDNA. Photographs of the 25S rRNA band in the ethidium-stained gel indicate the amounts of RNA loaded in each lane.

Figure 3

Absence of maintenance of Nia cosuppression homozygous transgenic 35S-Nia2 scions of class I (34–19.4) were grafted onto spontaneously silenced 35S-Nia2 transgenic stocks of class II. Six weeks later, the terminal apices of grafting-induced silenced scions were removed and regrafted onto wild-type stocks. The first developed leaf was completely chlorotic (a). Then, the upper leaves showed vein-localized chlorosis (b). Finally, chlorosis was absent from the uppermost leaves of the scion (c).