Molecular and genetic analysis of nitrite reductase co-suppression in transgenic tobacco plants

Silencing of Nia host genes and transgenes (encoding nitrate reductase) was previously achieved by introducing into tobacco plants the tobacco Nia2 cDNA cloned downstream of the cauliflower mosaic virus (CaMV) 35S promoter. To check whether Nii host genes and transgenes (encoding nitrite reductase, the second enzyme of the nitrate assimilation pathway) were also susceptible to silencing, a transgene consisting of the tobacco Nii1 gene with two copies of the enhancer of the 35S promoter cloned 1 kb upstream of the Nii promoter region was introduced into tobacco plants. Among nine independent transformants analysed, two showed silencing of Nii host genes and transgenes in some descendants after selfing, but never after back-crossing with wild-type plants, suggesting that silencing depends on the number of transgene loci and/or on certain allelic or ectopic combinations of transgene loci. In one transformant carrying a single transgene locus in a homozygous state, silencing was triggered in all progeny plants of each generation, 20 to 50 days after germination. Field trial analysis confirmed that silencing was not triggered when the transgene locus of this latter line was present in a hemizygous state. In addition, it was revealed that silencing can be triggered, albeit at low frequency and later during the development, when this transgene locus is brought into the presence of a non-allelic transgene locus by crossing, suggesting that a homozygous state is not absolutely required.