Developmentally regulated silencing and reactivation of tobacco chitinase transgene expression

Abstract

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 β‐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 hornology and trans‐gene 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.

Resistance in plants transformed with the P1 or P3 gene of tobacco vein mottling potyvirus.

“…4), where the silencing is triggered simultaneously both in inoculated and non-inoculated plants, and seems to occur at a certain developmental stage. A similar example of development-associated silencing has been reported recently for a viral transgene, the tospoviral N gene (Pang et al, 1996) and there are also examples of this phenomenon for non-viral transgenes (Elmayan & Vaucheret, 1996 ;Kunz et al, 1996). TVMV infection of our P3 lines may have an overlapping role resulting in a final degree of gene silencing that is more complete in inoculated than uninoculated plants (Fig.…”

Sense- and antisense-mediated gene silencing in tobacco is inhibited by the same viral suppressors and is associated with accumulation of small RNAs

“…Earlier we showed that all members of the CHN and GLU gene families can be posttranscriptionally silenced in sense Nicotiana sylvestris transformants (25)(26)(27). Here we report that posttranscriptional sense-and antisense-silencing of these genes in tobacco is suppressed by plant viruses known to suppress PTGS and that smRNAs diagnostic for PTGS (7) are associated with both forms of silencing.…”

“…Plants at the six-leaf stage were assayed for expression of the silent and high-expressing phenotypes as described (25). A fully expanded leaf of plants with ca.…”

“…Posttranscriptional inactivation was detected by comparing the content of unspliced, primary transcripts, which are not decreased in PTGS (37), with that of the mature mRNAs in sibling silent and high-expressing plants. Leaves of transformants were judged to express the silent phenotype if the protein and mRNA contents after ethylene treatment were appreciably lower than that of wild-type plants under comparable conditions in the same experiment (25).…”

Expression Pattern of the Carrot EP3Endochitinase Genes in Suspension Cultures and in Developing Seeds1

“…Another, indirect indication for the importance of chitinases during seed development comes from a recent study of tobacco plants with low chitinase levels because of silencing caused by transgene expression of a tobacco class I chitinase. The silenced chitinases were transiently reset to a high-expressing state 8 to 11 DAP (Kunz et al, 1996). An explanation for the resetting of the silenced chitinases was not given, but it cannot be excluded that the requirement for chitinases during this period in seed development is a driving force for the resetting.…”