Low frequency of T-DNA based activation tagging in Arabidopsis is correlated with methylation of CaMV 35S enhancer sequences.
A powerful system to create gain-of-function mutants in plants is activation tagging using T-DNA based vehicles to introduce transcriptional enhancer sequences. Large Arabidopsis populations of individual plants carrying a quadruple cauliflower mosaic virus (CaMV) 35S enhancer are frequently used for mutant screenings, however the frequency of morphological mutants remains very low. To clarify this low frequency we analyzed a subset of lines generated by this method. The correlation between the number of T-DNA insertion sites, the methylation status of the 35S enhancer sequence and 35S enhancer activity was determined. All plants containing more than a single T-DNA insertion showed methylation of the 35S enhancer and revealed a dramatic decrease in 35S enhancer activity. The results support the notion that in a large proportion of the T-DNA based activation tagged lines the 35S transcriptional enhancer is silenced due to methylation, which is induced by multiple T-DNA integrations.
Chalfun-Junior
Antonio
A
Business Unit Bioscience, Plant Research International, Bornsesteeg 65, 6708 PD, Wageningen, The Netherlands. gerco.angenent@wur.nl
Mes
Jurriaan J
JJ
Mlynárová
Ludmila
L
Aarts
Mark G M
MG
Angenent
Gerco C
GC
D017360:Arabidopsis / Q000235:genetics* / Q000821:virology; D001483:Base Sequence; D015139:Blotting, Southern; D017796:Caulimovirus / Q000235:genetics*; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D005966:Glucuronidase / Q000032:analysis / Q000378:metabolism; D016254:Mutagenesis, Insertional; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D014170:Transformation, Genetic / Q000235:genetics*
D004269:DNA, Bacterial; C036483:T-DNA; D005966:Glucuronidase