title: The ben1-1 brassinosteroid-catabolism mutation is unstable due to epigenetic modifications of the intronic T-DNA insertion.
abstract: Loss-of-function genetic analysis plays a pivotal role in elucidating individual gene function as well as interactions among gene networks. The ease of gene tagging and cloning provided by transfer DNA (T-DNA) insertion mutants have led to their heavy use by the Arabidopsis research community. However, certain aspects of T-DNA alleles require caution, as highlighted in this study of an intronic insertion mutant (ben1-1) in the BEN1 (BRI1-5 ENHANCED 1) gene. As a part of our analysis of brassinosteroid catabolic enzymes, we generated a genetic triple-mutant from a cross between the bas1-2 sob7-1 double-null (T-DNA exonic insertion mutants of phyB-4 ACTIVATION TAGGED SUPPRESSOR 1 and SUPPRESSOR OF phyB-4 7) and ben1-1. As previously described, the single ben1-1 line behaves as a transcript null. However, in the triple-mutant background ben1-1 was reverted to a partial loss-of-function allele showing enhanced levels of the wild-type-spliced transcript. Interestingly, the enhanced expression of BEN1 remained stable when the ben1-1 single-mutant was reisolated from a cross with the wild type. In addition, the two genetically identical pretriple and posttriple ben1-1 mutants also differed phenotypically. The previously functional NPTII (NEOMYCIN PHOSPHOTRANSFERASE II) T-DNA marker gene (which encodes kanamycin resistance) was no longer functional in the recovered ben1-1 allele, though the length of the T-DNA insertion and the NPTII gene sequence did not change in the pretriple and posttriple ben1-1 mutants. Methylation analysis using both restriction endonuclease activity and bisulfite conversion followed by sequencing showed that the methylation status of the T-DNA is different between the original and the recovered ben1-1. These observations demonstrate that the recovered ben1-1 mutant is epigenetically different from the original ben1-1 allele.
authors.affiliation: Department of Crop and Soil Sciences, Washington State University, Pullman, Washington 99164.
authors.forename: Kulbir Singh
authors.initials: KS
authors.lastname: Sandhu
authors.forename: Pushpa Sharma
authors.initials: PS
authors.lastname: Koirala
authors.forename: Michael M
authors.initials: MM
authors.lastname: Neff
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000483:Alleles; D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D060406:Brassinosteroids / Q000494:pharmacology*; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D057890:Epigenomics; D017343:Genes, Plant; D007438:Introns; D019868:Kanamycin Kinase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic; D036226:Seedlings / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013260:Steroids, Heterocyclic / Q000494:pharmacology*
chemical_list: D029681:Arabidopsis Proteins; D060406:Brassinosteroids; D004269:DNA, Bacterial; D013260:Steroids, Heterocyclic; C036483:T-DNA; D000429:Alcohol Oxidoreductases; C523325:BEN1 protein, Arabidopsis; D000594:Amino Acid Oxidoreductases; C021883:nopaline synthase; D019868:Kanamycin Kinase; C023623:brassinolide
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