Promotion of early Spm transposition and repression of Spm transcription by TnpA in transgenic tobacco

--Michael Schläppi and Nina Fedoroff

The maize suppressor-mutator (Spm) transposable element has been transformed into tobacco cells and a visual assay for Spm activity using a dSpm cloned between the CaMV 35S promoter and the b-glucuronidase (GUS) gene has been developed (Masson, P and Fedoroff, NV, PNAS 86:2219, 1989; Masson et al., Plant Cell 3:73, 1991). Callus lines exhibiting different frequencies of Spm excision were obtained from an initially highly active callus (Fedoroff, unpublished). This phenomenon resembles the epigenetic inactivation of Spm in maize, correlating with differences in methylation of sequences immediately upstream (a region termed the upstream control region or UCR) and downstream (a region termed the downstream control region or DCR) of the element's transcription start site (Banks et al., Genes Dev. 2:1364, 1988; Banks and Fedoroff, Dev. Genet. 10:425, 1989). By Southern blot analysis and copy number reconstitutions, we have selected a callus line, #8, containing 1-2 copies of Spm and 1 copy of the GUS excision assay sequence. Different plantlets regenerating from callus #8 that showed different Spm activity were analysed with the methylation sensitive restriction enzymes Eco109 (to detect UCR methylation) and SalI (to detect DCR methylation). Plant 8#47 exhibited very low Spm activity and revealed partial SalI methylation in the DCR whereas plant 8#49 showed high Spm activity and was fully cleavable at the SalI sites in the DCR (Schläppi, M, unpublished). DNA from both plants was fully cleavable and hence unmethylated at the Eco109 site in the UCR.

Different Spm element-encoded transcripts produced by alternative splicing have been cloned recently (Masson et al., Cell 58:755, 1989). Two transcripts, designated tnpA and tnpD, have been shown to be necessary and sufficient for the element's transposition in transgenic tobacco (Frey et al., EMBO J. 9:4037, 1990; Masson, P et al., Plant Cell 3:73, 1991). In order to investigate the effect of different element-encoded gene products on the activity of unmethylated and partially methylated Spm elements in transgenic tobacco, we retransformed the previously selected plants of callus line #8 with either tnpA, tnpC, or tnpD cDNA under the control of the CaMV 35S promoter and the nopaline synthase terminator by A. tumefaciens mediated gene trans-fer. Control plants were retransformed by A. tumefaciens without any Spm-specific sequences. Introduction of tnpA into plants with either methylated or unmethylated Spm sequences leads to a dramatic increase of early excision events whereas tnpC and tnpD have no effect as compared to the control (see Figure 1). Control and tnpC- or tnpD-retransformed plants of the partially methylated parent plant 8#47 had roughly 10X fewer GUS sectors and 10X less GUS activity than the equivalents of the unmethylated parent plant 8#49, but similarly large sectors and GUS activities when retransformed with tnpA (see Figure 2). The same phenomenon was observed when plants from the callus line #8 were crossed with tobacco plants containing only tnpA, tnpC, or tnpD cDNA. These results suggest that the production of a certain amount of TnpA protein might be the rate-limiting step for Spm transposition and that partial DCR methylation has no influence on the transposition process mediated by TnpA protein in trans.

The effect of the introduced cDNAs on transcription of Spm in transgenic tobacco was investigated by Northern blot analysis. Over-expression of tnpA from the CaMV 35S promoter leads to the reduction of Spm transcripts to undetectable levels (see Figure 3). This preliminary result is in agreement with the repression of transient expression of 5'-Spm-luciferase fusions in the presence of tnpA cDNA (Cook, D and Fedoroff, this volume). This observation suggests that Spm transcription is repressed upon formation of the pre-transposition complex due to saturation binding of TnpA to the subterminal repeats (see also Fedoroff, NV et al., this volume).

Figure 1. Histochemical visualization of dSpm excision events in Spm-containing transgenic tobacco plants retransformed with different element-encoded cDNAs driven by the CaMV 35S promoter. Excision sectors were visualized by staining with X-Gluc.

Figure 2. Quantification of b-glucuronidase (GUS) activity in Spm-containing transgenic tobacco plants retransformed with different Spm-encoded cDNAs. The Spm sequence of plant 8#47 was partially methylated in the DCR region, the Spm sequence of plant 8#49 was unmethylated. The GUS activity is given in arbitrary units per µg of measured protein.

Figure 3. Northern blot hybridization analysis of polyA+ RNA isolated from Spm-containing transgenic tobacco plant 8#49 retransformed with either tnpA, tnpC, or tnpD cDNA. The blot was probed with a DNA fragment homologous to the UCR and DCR region of Spm that was not present in the cDNA constructs.

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