Expression and sequence requirements for nitrite reductase co-suppression.
We have previously reported that the introduction of a full-length tobacco nitrite reductase Nii1 cDNA under the control of the 35S promoter triggers co-suppression of endogenous Nii genes in 25% of tobacco transformants. Here we show that introduction of chimeric Nii1-uidA, uidA-Nii1 and Nii1-uidA-Nii1 transgenes carrying 186 bp of the 5' end and/or 241 bp of the 3' end of the Nii1 cDNA do not trigger co-suppression of endogenous Nii genes. In addition, we show that when introduced by crossing or transformation into co-suppressed transgenic tobacco lines carrying full-length Nii1 transgenes, these chimeric transgenes are not silenced. These results therefore suggest that the 5' and 3' ends of the Nii1 cDNA are not sufficient to trigger co-suppression and are not targets for homology-dependent RNA degradation. Surprisingly, co-suppression was released in a double transformant obtained by introduction of one of these constructs into the co-suppressed transgenic tobacco line 461-2.1 homozygous for a full-length Nii1 transgene, and in one plant regenerated from untransformed leaf discs (plant 461-2.1*). The reappearance of co-suppression at very low frequency (less than 10(-3)) in the F2 progeny of plant 461-2.1* and the apparent absence of structural modification of the transgene locus suggest a metastable epigenetic modification. The steady-state level of Nii mRNAs in the plant 461-2-.1* was higher than in wild-type plants but lower than in hemizygous plants 461-2.1 which never trigger silencing. These results therefore confirm that transcription of the transgene above a particular threshold is required to trigger co-suppression.
Crété
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Laboratoire de Biologie Cellulaire, INRA, Versailles, France.
Vaucheret
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D019175:DNA Methylation; D018076:DNA, Complementary / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics / Q000378:metabolism; D004843:Epistasis, Genetic; D018506:Gene Expression Regulation, Plant; D005966:Glucuronidase / Q000235:genetics; D009572:Nitrite Reductases / Q000235:genetics*; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*; D014170:Transformation, Genetic
D018076:DNA, Complementary; D018744:DNA, Plant; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D009572:Nitrite Reductases; D005966:Glucuronidase