Developmentally regulated silencing and reactivation of tobacco chitinase transgene expression

The tobacco class I chitinase gene CHN48 driven by CaMV 35S RNA expression signals introduced into Nicotiana sylvestris can inactivate its own expression as well as expression of homologous host genes at the mRNA level. Comparison of the silencing of chitinase and beta-1,3-glucanase transgenes with the same expression signals and breeding experiments with transgenes inserted in different chromosomes showed that chitinase-gene silencing depends on transcribed sequence homology and transgene dose. The CHN48 transgene silenced host class I but not class II chitinases suggesting that greater than about 60% identity of sequences encoding the catalytic domain is required for silencing. Although steady-state mRNA contents were greatly reduced in silent plants, the levels of nascent chitinase transcripts detected in nuclear run-on experiments were the same in nuclei from high-expressing and silent plants. This strongly suggests that chitinase-gene silencing is a post-transcriptional phenomenon. Silencing and resetting to high-level expression of chitinase genes is developmentally regulated. Silencing occurs stochastically in 25-100% of seedlings at the 6-10 leaf stage of development and is preceded by a marked, transient increase in transgene-encoded chitinase. Resetting of silent genes to the high-expressing state is a non-stochastic event that occurs in developing seeds 8-11 days after pollination. Experiments with lateral buds and plants regenerated from cultured leaf disks show that once established, competence for silencing can persist in dormant, actively growing, and de novo established shoot meristems. Some plants show a variegated pattern of silencing. Analyses of these patterns indicate that chitinase-gene silencing is not a clonal event. Thus, it is likely that communication between cells rather than the lineage of the cells is important in stabilizing the silent state. Possible mechanisms for developmentally regulated post-transcriptional silencing are discussed.