Petunia plants escape from negative selection against a transgene by silencing the foreign DNA via methylation.
Transgenic Petunia hybrida clones harbouring the T-DNA gene 2 of Agrobacterium tumefaciens were used to test a strategy for the trapping of plant transposable elements. In the Petunia line used, floral variegation is due to the presence of the non-autonomous transposable element dTph1 at the An1 locus. The gene 2 product converts the auxin precursor indole-3-acetamide and its analogue 1-naphthalene acetamide into the active auxins indole-3-acetic acid and 1-naphthalene acetic acid. Plant cells that express gene 2 can use a low concentration of the precursors as auxins and become sensitive to the toxicity of high concentrations of these compounds. By selecting protoplast-derived microcalli or seedlings able to grow on medium with high precursor concentrations, variant plants were obtained in which gene 2 was no longer expressed. Southern analysis, using gene 2-specific probes, revealed that in one variant the T-DNA was deleted. For 30 other variants no alteration in gene 2 structure was observed, indicating that transposable element insertion was not responsible for the inactivation of gene 2. Analysis with restriction enzymes allowing discrimination between methylated or non-methylated DNA sequences showed that the inactivated gene 2 sequences were methylated. Addition of the in vivo methylation inhibitor 5-azacytidine to the medium led to reactivation of gene 2 expression in some of the variants. These observations demonstrated that reversible DNA methylation was the main cause of silencing of gene 2 in this system.
Renckens
S
S
Laboratorium voor Genetische Virologie, Vrije Universiteit Brussel, St-Genesius-Rode, Belgium.
De Greve
H
H
Van Montagu
M
M
Hernalsteens
J P
JP
D016960:Agrobacterium tumefaciens / Q000235:genetics; D000581:Amidohydrolases / Q000235:genetics; D001374:Azacitidine / Q000494:pharmacology; D015139:Blotting, Southern; D002460:Cell Line; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D008745:Methylation; D009693:Nucleic Acid Hybridization; D010641:Phenotype; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D012641:Selection, Genetic; D014170:Transformation, Genetic
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; C036483:T-DNA; D000581:Amidohydrolases; C058772:indole-3-acetamide hydrolase; D001374:Azacitidine