Towards transposon tagging of the TMV resistance gene N using Activator in transgenic tobacco --Reinhard Hehl and Barbara Baker Utilization of the maize controlling element Activator

(Ac) as an insertional mutagen in heterologous plant species is facilitated by comprehensive understanding of its molecular genetic properties in novel genomic settings. Our results, summarized below, indicate that Ac can be effectively employed as an insertional mutagen in tobacco as well as other dicot species.

1) Ac maintains the genetic capacity to transpose and trans-activate Ds transposition at a high frequency for at least five tobacco generations. 2) Ac is transcribed and internal element sequences remain unmethylated. 3) The mechanism of Ac integration in maize and tobacco is similar, conserving Ac structure and function. 4) Ac transposes adjacent to unique and low copy genomic sequences and alters transcription of a unique target DNA, suggesting that it has transposed into a gene. 5) Increasing copies of Ac correlate with an increased frequency of Ds trans-activation.

We are currently employing Ac in an attempt to tag the dominant Tobacco Mosaic Virus (TMV) resistance gene N in transgenic tobacco. The N gene mediates a hypersensitive response (HSR) upon virus infection and restricts virus spread throughout the plant. We have introduced Ac into Nicotiana tabacum cv. Samsun NN, homozygous for N, and crossed NN plants bearing transposing Ac to N. tabacum cv. SRI (nn). Ac insertion into the single copy of N of the F1 population should lead to loss of HSR and to systemic TMV infection. Large F1 populations have been screened for loss of HSR as well as systemic virus infection by applying conditions lethal to seedlings expressing N. Screening of two F1 populations of 12,000 and 27,000, harboring transposing Ac, produced 2 and 28 systemically infected plants respectively. Surprisingly, several of these F1 plants display patches of necrotic lesions resembling HSR on mature leaves. This phenotype was not observed in the control population. The possibility that this phenotype correlates with transposon induced somatic instability of the N locus is under investigation.

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