Kulbir Singh Sandhu, Pushpa Sharma Koirala, Michael M Neff

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.