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Employing libraries of zinc finger artificial transcription factors to screen for homologous recombination mutants in Arabidopsis
Author(s) -
Lindhout Beatrice I.,
Pinas Johan E.,
Hooykaas Paul J.J.,
Van Der Zaal Bert J.
Publication year - 2006
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2006.02877.x
Subject(s) - arabidopsis , homologous recombination , zinc finger , mutant , biology , genetics , gene , arabidopsis thaliana , dna , fusion protein , transcription factor , microbiology and biotechnology , recombinant dna
Summary A library of genes for zinc finger artificial transcription factors (ZF‐ATF) was generated by fusion of DNA sequences encoding three‐finger Cys 2 His 2 ZF domains to the VP16 activation domain under the control of the promoter of the ribosomal protein gene RPS5A from Arabidopsis thaliana . After introduction of this library into an Arabidopsis homologous recombination (HR) indicator line, we selected primary transformants exhibiting multiple somatic recombination events. After PCR‐mediated rescue of ZF sequences, reconstituted ZF‐ATFs were re‐introduced in the target line. In this manner, a ZF‐ATF was identified that led to a 200–1000‐fold increase in somatic HR (replicated in an independent second target line). A mutant plant line expressing the HR‐inducing ZF‐ATF exhibited increased resistance to the DNA‐damaging agent bleomycin and was more sensitive to methyl methanesulfonate (MMS), a combination of traits not described previously. Our results demonstrate that the use of ZF‐ATF pools is highly rewarding when screening for novel dominant phenotypes in Arabidopsis.