Site-Selected Transposon Mutagenesis at the hcf106 Locus in Maize

Abstract

The High chlorophyll fluorescencel06 (HcflO6) gene in maize is required for chloroplast membrane biogenesis, and the hcf706muml allele is caused by the insertion of a Robertson's Mutator Mul element into the promoter of the gene. Seedlings homozygous for hcfl06-muml are pale green and die 3 weeks after germination, but only in the presence of Mutator activity conferred by active, autonomous Mu regulatory transposons elsewhere in the genome. When Mutator activity is lost, the mutant phenotype is suppressed, and homozygous plants have an almost wild-type phenotype. To isolate derivative alleles at the hcflO6 locus that no longer require Mutator activity for phenotypic expression, we have developed a method for site-selected transposon mutagenesis in maize. This procedure, first described for Caenorhabditis elegans and Drosophila, involves using polymerase chain reaction (PCR) to screen pools of individuals for insertions and deletions in genes of known sequence. Pools of seedlings segregating for the progenitor allele hcf708muml were screened by PCR for insertions and deletions associated with Robertson's Mutator. In a 360-bp target region, two new insertions and one deletion were identified in only 700 Mu-active gametes screened. One of the insertions was in the progenitor hcfl06-muml allele and the other was in the wild-type allele, but all three new alleles were found to have breakpoints at the Same nucleotide in the first intron. Unlike the hcfl06-muml progenitor allele, the deletion and one of the insertions conferred pale green seedling lethal phenotypes in the absence of Mutator activity. However, the second insertion had a weak, viable phenotype under these conditions. Although the sample size was small, our results suggest that this procedure can be used to rapidly identify transposon insertions into known genes in a single generation. Null derivative alleles,can then be isolated in a second generation using the insertion as a starting point. This two-step procedure represents a powerful and simple way to "knock out" maize genes identified by sequence alone by using only a few thousand progeny from a simple cross.

An essential role of a TatC homologue of a ΔpH- dependent protein transporter in thylakoid membrane formation during chloroplast development in Arabidopsis thaliana

“…Hcf106 and Tha4 are homologues of TatA, TatB, and TatE of E. coli. hcf106-mun1 mutant plants are pale green and die 3 weeks after germination in soil (15,36). tha4 mutants are subtly chlorophylldeficient and die after the development of three to four leaves (16).…”

A Two-Component Enhancer-Inhibitor Transposon Mutagenesis System for Functional Analysis of the Arabidopsis Genome

“…1854 The Plant Cell pooling strategies then can lead to identification of the desired mutant individual or individuals within the treated population. This target-selected mutational strategy has been applied successfully within studies of Drosophila, Caenorhabditis, maize, and petunia (Kaiser and Goodwin, 1990;Rushforth et al, 1993; Zwaal et al, 1993;Das and Martienssen, 1995;Koes et al, 1995;Mena et al, 1996).…”

“…1854 The Plant Cell pooling strategies then can lead to identification of the desired mutant individual or individuals within the treated population. This target-selected mutational strategy has been applied successfully within studies of Drosophila, Caenorhabditis, maize, and petunia (Kaiser and Goodwin, 1990;Rushforth et al, 1993; Zwaal et al, 1993;Das and Martienssen, 1995;Koes et al, 1995;Mena et al, 1996).In Arabidopsis, most reverse genetics screens have used T-DNA insertional mutagenesis (McKinney et al, 1995;Krysan et al, 1996;Azpiroz-Leehan and Feldmann, 1997;Bouchez and Höfte, 1998;Winkler et al, 1998) and have required the use of large populations so that only a few inserts per plant would result in genome saturation. To develop an effective reverse genetics strategy in Arabidopsis based on transposons, we have previously described a two-component maize Enhancer-Inhibitor system ( En-I ; Enhancer is sometimes designated as Suppressor-mutator…”

“…The three-dimensional pooling strategy that we used to find these mutations within our population of 2592 multiple-transposon lines was described and successfully used previously for petunia (Koes et al, 1995), maize (Das and Martienssen, 1995), and Caenorhabditis (Zwaal et al, 1993). We successfully identified two independent ap1::I alleles, ap1::I(XIX-6-E) with an insertion in an intron and an average excision rate of 12%, and ap1::I(V-11-G) with an insertion in the seventh exon and an average excision rate of 35%.…”

Function Search in a Large Transcription Factor Gene Family in Arabidopsis: Assessing the Potential of Reverse Genetics to Identify Insertional Mutations in R2R3 MYB Genes

“…Individual lines carrying insertions within a gene of interest can be identified by polymerase chain reaction (PCR) by using a gene-specific primer in combination with a primer complementary to border sequences of the insertion element. This method was first described for Drosophila (Ballinger and Benzer, 1989;Kaiser and Goodwin, 1990) but subsequently has been applied to several other organisms, including Caenorhabditis elegans , petunia, and maize (Zwaal et al, 1993;Das and Martienssen, 1995;Koes et al, 1995). In Arabidopsis, this process of "reverse" genetics (McKinney et al, 1995) was used to screen for T-DNA insertions in actin gene sequences.…”