Regulated inactivation of homologous gene expression in transgenic Nicotiana sylvestris plants containing a defense-related tobacco chitinase gene

Abstract

The class I chitinases are vacuolar proteins implicated in the defense of plants against pathogens. Leaves of transgenic Nicotiana sylvestris plants homozygous for a chimeric tobacco (Nicotiana tabacum) chitinase gene with Cauliflower Mosaic Virus (CaMV) 35S RNA expression signals usually accumulate high levels of chitinase relative to comparable leaves of non-transformed plants. Unexpectedly, some transgenic plants accumulated lower levels of chitinase than nontransformed plants. We call this phenomenon silencing. The incidence of silencing depends on the early rearing conditions of the plants. When grown to maturity in a greenhouse, approximately 25% of plants raised as seedlings in closed culture vessels were of the silent type; none of the plants raised from seed in a greenhouse showed this phenotype. Silencing is also developmentally regulated. Plants showed three patterns of chitinase expression: uniformly high levels of expression in different leaves, uniformly low levels of expression in different leaves, and position-dependent silencing in which expression was uniform within individual leaves but varied in different leaves on the same plant. Heritability of the silent phenotype was examined in plants homozygous for the transgene. Some direct descendants exhibited a high-silent-high sequence of activity phenotypes in successive sexual generations, which cannot be explained by simple Mendelian inheritance. Taken together, the results indicate that silencing results from stable but potentially reversible states of gene expression that are not meiotically transmitted. Gene-specific measurements of chitinase and chitinase mRNA showed that silencing results from co-suppression, i.e. the inactivation of both host and transgene expression in trans. The silent state was not correlated with cytosine methylation of the transgene at the five restriction sites investigated.

Post‐transcriptional silencing of chalcone synthase in Petunia by inverted transgene repeats

“…A particular threshold level can also be reached by a high transgene copy number or by increasing the number of transgenes by crossings, by combining ectopic loci or by making plants homozygous (Angenent et al, 1993;De Carvalho et aL, 1992;De Carvalho Niebel et aL, 1995;Dorlhac de Borne et al, 1994;Hart et al, 1992;Palauqui and Vaucheret, 1995;Vaucheret et al, 1995). We also observed such gene dosage effects.…”

Posttranscriptional gene silencing of gn1 in tobacco triggers accumulation of truncated gn1-derived RNA species

“…In addition to the major products in regions A, B, C, and D, a significant number of less abundant PCR products were consistently present in samples of expressing and silenced T17 plants in independent experiments+ These products could reflect less abundant members of the normal gn1 RNA population present in the cell or they could result from inefficient PCR amplification+ Furthermore, a number of bands were observed that did not consistently occur in all experiments+ This probably reflects variations in the intensity of the gn1 gene silencing between different experiments, which in turn could be due to slight differences in environmental conditions between experiments+ It has been shown before that environmental conditions can affect the outcome of silencing (Napoli et al+, 1990;Hart et al+, 1992;Flavell et al+, 1998)+ A PCR reaction with an oligo located 205 bp upstream from the first gene-specific primer (oligo KL36) yielded products that were approximately 200 bp larger than the predominant species present in expressing and silenced T17 plants (data not shown)+ This shows that the RLM-RACE products truly represent gn1-derived RNA species and excludes the possibility that the predominant RLM-RACE products are due to technical PCR artefacts+ Taken together these results show that a changed pattern of gn1-derived RNA species coincides with b-1,3-glucanase gene silencing in T17 tobacco+…”

Transgene‐mediated suppression of chalcone synthase expression in Petunia hybrida results from an increase in RNA turnover

“…In the white parts, expression of the endogenous chs genes and that of the transgenes is suppressed. This phenomenon, called co-suppression, has now been reported for several different genes, in various organs and cell types (Angenent et al, 1993(Angenent et al, , 1994Brusslan et al, 1993;EIkind et al, 1990;Hart et al, 1992;Seymour et al, 1993;Temple et al, 1993;Van der Krol et al, 1993;Zhang et al, 1992), indicating that it is a general feature of sense transgene performance in plants.…”