A Resource of Mapped Dissociation Launch Pads for Targeted Insertional Mutagenesis in the Arabidopsis Genome

Ds Launch Pad T-DNA Vector

A schematic representation of the Ds launch pad T-DNA vector is shown in Figure 1. The Ds element contains a nos::BAR transcriptional fusion, conferring resistance to the herbicide phosphinothricin (Ppt, or gluphosinate ammonium), and a gene trap. The gene trap comprises a promoterless β-glucuronidase (uidA or GUS) reporter gene with a GPA1 intron sequence and splice acceptor sites in all three frames fused upstream of the GUS ATG codon (Sundaresan et al., 1995; engineered to remove an upstream ATG codon). Activation of the GUS reporter gene requires Ds insertion within a gene. When Ds inserts in the correct orientation into an intron, splicing occurs between the 5′ splice donor site of the gene and the 3′ splice acceptor site in the GUS fusion resulting in a GUS translational fusion. Insertion into an exon can also lead to a translational fusion because sequences at the 3′ end of the Ds element can act as splice donor sites. GUS expression, which is expected to mirror the expression of the native gene, can be simply visualized by histochemical staining (Jefferson et al., 1987). The presence of the gene trap extends the utility of the insertion element by enabling the expression pattern of the flanking gene to be characterized.

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Figure 1.

Ds launch pad T-DNA vector. Schematic representation of Ds launch pad T-DNA vector. Arrows represent the direction of open reading frames. Restriction enzyme sites relevant to vector construction and DNA gel-blot analysis are shown (for details, see “Materials and Methods”). The Ds element was cloned into the XhoI site (bold) within the 5′-untranslated leader (UTL) of the ALS gene.

The Ds element was inserted into the 5′-UTL of a chimeric tobacco (Nicotiana tabacum) ACETOLACTATE SYNTHASE (ALS) gene (Fig. 1), encoding a mutant version of ALS, which confers resistance to the herbicide chlorsulfuron (Cs, or sulfonylurea). The chimeric ALS gene is a fusion of two genes, SuRA/Hra and SuRB, from Cs-resistant mutants of tobacco (Mazur et al., 1987; Lee et al., 1988). The presence of the Ds element within the 5′-UTL inactivates expression of the ALS gene, and its activity is restored upon Ds excision, resulting in a Cs-resistant phenotype. Ds excision and re-insertion events (following introduction of a transposase source) can therefore be selected by treatment with Cs and Ppt.

Transposase Source T-DNA Vectors

For transactivation of Ds elements from the Ds launch pads, two new transposase source vectors (Fig. 2A) were constructed that incorporated the phenotypic marker Lc (Ludwig et al., 1989) and either the 35S::Ac transposase fusion (Scofield et al., 1992; Swinburne et al., 1992) or an sΔNaeIAc element, where the 3′ end of the element has been removed (from position 4,385 to the end) and a 537-bp deletion has been created within the 5′-UTL of Ac (Bancroft et al., 1992), the latter causing an increase in excision frequency. The two transposase fusions have previously been demonstrated to provide different levels of Ds transactivation and generate transposition events at different times during plant development. The 35S::Ac fusion has been shown to give rise to high levels of Ds excision (typically >30% germinal excision frequency) but a low and variable re-insertion frequency of Ds (Swinburne et al., 1992; Long et al., 1993). Also, Ds excision events occur early during vegetative growth of the F₁ plant, leading to sectors encompassing many flowers, resulting in many of the F₂ progeny carrying the same transposition event (Long et al., 1993). Therefore, when using the 35S::Ac transposase source many F₁ hybrid and F₂ families need to be generated to produce large numbers of independent transposition events. In contrast, the sΔNaeIAc transposase source results in a lower germinal excision frequency (approximately 5%; Bancroft et al., 1992), but a higher proportion of reinsertion events (approximately 50%; Bancroft and Dean, 1993a). The sΔNaeIAc transposase generally results in transposition very late in plant development (after the divergence of cell lines leading to pollen and ova), so Ds transposition events carried in F₂ progeny are generally independent (Bancroft and Dean, 1993b). However, a significant proportion (approximately one-third) of the F₂ progeny selected to be doubly resistant for Ds excision and integration markers do not carry transposed Ds elements but contain a combination of nonexcised Ds alleles and empty donor sites (Bancroft and Dean, 1993b). The two transposase sources thus provide different strategies to generate lines carrying new insertions, the choice depending on the difficulty of the mutant screens to be employed.

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Figure 2.

sAc-Lc T-DNA vector. A, Schematic representation of sAc-Lc T-DNA vector. Arrows represent the direction of open reading frames. Restriction enzyme sites relevant to vector construction are shown (for details see “Materials and Methods”). B, Hirsute phenotype of sAc-Lc line.

To phenotypically mark plants carrying a transposase source, a 35S::Lc transcriptional fusion was cloned into the transposase T-DNA vector (Fig. 2A). Lc is a member of the maize R gene family and is involved in the regulation of anthocyanin pigmentation (Ludwig et al., 1989). In Arabidopsis, the expression of the 35S::Lc fusion increases the level of pigmentation and density of trichomes (Lloyd et al., 1992). The Lc fusion therefore provides a visual marker, and plants carrying transposase can be easily identified by their hirsute, darkly pigmented phenotype (Fig. 2B). This makes identification of plants lacking transposase very straightforward, simplifying stabilization of the Ds element. The 35S::Lc transcriptional fusion has previously been demonstrated to be a successful visual marker for sAc transposase T-DNAs and shown not to adversely affect Ds transposition (Osborne et al., 1995).

Mapping Ds Launch Pad T-DNA Vectors

To produce the required number of mapped Ds launch pads, a large population of transgenic Arabidopsis plants was generated. The Ds launch pad T-DNA vector was introduced into Arabidopsis genotype Landsberg erecta via Agrobacterium tumefaciens-mediated transformation (Valvekens et al., 1988). The NPTII gene (which confers resistance to the antibiotic kanamycin) contained within the T-DNA (Fig. 1) was used to select for transgenic plants. More than 300 independent Arabidopsis transformants were generated. It was important to identify transgenic lines that contained a single copy of the Ds launch pad T-DNA. The selection for Ds excision and re-insertion events requires a single Ds launch pad. In addition, analysis of lines containing transposed Ds elements is simplified if only one copy is present, i.e. an observed phenotype is solely due to one Ds insertion. Initially, lines containing single-locus Ds launch pad T-DNA inserts were identified by segregation of the NPTII gene. A segregation ratio of 3:1 (kanamycin resistant: kanamycin sensitive) in the T₂ generation was diagnostic of a single-locus insert. DNA gel-blot analysis was subsequently performed on these single-locus lines to identify those with single-copy Ds launch pad inserts. Probes specific for the left and right T-DNA borders were used to determine Ds launch pad copy number, and single hybridizing bands with both probes indicated a single insert. An internal probe (GUS gene fragment) was also used to demonstrate the Ds launch pad was intact. More than 150 lines were identified to contain single-copy Ds launch pad T-DNA inserts (approximately 50% of transgenic lines generated). This illustrated the efficiency of the binary vector pSLJ1711 in obtaining transformants with single, simple T-DNA inserts.

The first step toward mapping the Ds launch pads onto the Arabidopsis genome was to isolate DNA flanking the Ds launch pad T-DNA insertion. For the majority of lines this was achieved using thermal asymmetric interlaced (TAIL) PCR (Liu and Whitter, 1995; Liu et al., 1995), using either right border (RB) or left border (LB) specific primer sets (for details, see “Materials and Methods”). Generally, TAIL PCR was more successful when using the LB primers. In cases where T-DNA flanking fragments were not amplified by TAIL PCR, inverse PCR (IPCR) was used (Ochman et al., 1988; Triglia et al., 1990).

To map the Ds launch pad T-DNA insertion sites amplified by TAIL PCR or IPCR onto the Arabidopsis genome, two strategies were adopted. Initially, the Ds launch pad flanking fragments were used as probes to screen YAC and, to a lesser extent, bacterial artificial chromosome (BAC) library filters (for details, see “Materials and Methods”; Muskett et al., 2002). This approach was extremely useful before the availability of the entire Arabidopsis genome sequence. However, when the genome sequence became available, the second approach was to sequence the TAIL PCR or IPCR products and map them simply by using the BLAST alignment program (Altschul et al., 1990). Sequencing of the PCR products also confirmed the presence of expected T-DNA border sequences, eliminating the need to perform DNA gel-blot analysis to confirm that the amplified products truly flanked the Ds launch pads in each line. By combining the two mapping strategies, 89 Ds launch pads were accurately positioned onto the Arabidopsis genome (Fig. 3). Mapping revealed the distribution of Ds launch pads to be fairly even over the five Arabidopsis chromosomes. With the exception of centromeric and nucleolar organizer regions (insertions into these repetitive DNA regions could not be mapped by the techniques employed), few chromosomal regions contained no Ds launch pads. The two most notable regions lacking mapped Ds launch pads included an approximately 24-cM region between m421 and B68 on chromosome 2 and an approximately 22-cM region between g4108 and g13838 on chromosome 4. Conversely, there were no obvious “hotspots” of Ds launch pad insertions (the most clustered region being between FLS and mi322 on chromosome 5). Therefore, the 89 mapped Ds launch pads provide starting locations to target genes throughout the majority of the genome. Details of all 89 lines, including line number, amplification of Ds launch pad flanking fragment, hybridizing YAC or BAC clones, GenBank accession number of Ds launch pad insertion site sequence, and BLAST hits are shown in Table I.

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Figure 3.

Positions of mapped Ds launch pad T-DNA inserts on the Arabidopsis genome. Genomic locations of Ds launch pad T-DNA inserts in 89 independent lines (one insert per line) are shown. Ds launch pads are represented by triangles. Circles represent centromeric regions, gray boxes represent nucleolar organizer regions. Each chromosome is labeled with a number of genetic markers to give an approximate reference position.

Ds Excision and Re-Insertion

To demonstrate excision and re-insertion of the Ds element from the Ds launch pads, DNA gel-blot analysis was performed on F₂ progeny (generated from a cross to a line containing the 35S::Ac fusion) selected for double herbicide resistance, and the Ds integration site was analyzed using the GUS gene or BAR gene as a hybridization probe. This analysis detects both intact Ds launch pads (i.e. with a nonexcised Ds element) and re-inserted Ds elements (Fig. 4). DNA gel-blot analysis revealed hybridizing fragments, corresponding to re-inserted Ds elements, of various sizes (Fig. 4, asterisks). This demonstrated the ability of Ds to excise and re-insert into different genomic locations in the independent lines. Single hybridizing fragments corresponding to re-inserted Ds elements also demonstrated germinal excision events (i.e. transposition events inherited from the previous generation). In lines that did not contain a re-inserted Ds element (Fig. 4, lanes 4 and 5), Ppt resistance was conferred by an intact Ds launch pad revealed by a hybridizing fragment of expected size, 2.8 kb. Further analysis of the double herbicide-resistant seedlings also showed that a small proportion did not contain an empty donor site, suggesting some leakiness of the Cs selection.

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Figure 4.

Analysis of Ds excision and reinsertion. DNA gel-blot analysis to detect Ds excision and re-insertion. Genomic DNA from 10 independent F₂ families was digested with HindIII. A GUS probe was used to detect Ds excision and re-insertion. A Ds launch pad with a nonexcised Ds element resulted in a hybridizing band of expected size, 2.8 kb. Excised and re-inserted Ds elements gave rise to hybridizing bands of variable sizes, marked by asterisks. The GUS probe also hybridized to an unknown fragment, resulting in a background band of 8 kb in all lanes.

DNA Constructs

The Ds launching pad T-DNA vector (Fig. 1) was constructed as follows: A XhoI site was introduced into the 5′-untranslated region of the tobacco, chimeric ALS gene by site-specific mutagenesis, as described by Kunkel (1985; shown in bold in Fig. 1). The ALS gene was excised from pSBAD (kindly supplied by Barbara Mazur [DuPont, Wilmington, DE]) as a BamHI/PstI fragment and ligated into the same restriction sites in pBluescript (pKSII). The ALS gene was then cloned as a SalI fragment from pKSII into the XhoI site of the pdBS polylinker in the binary vector pSLJ1711 (Jones et al., 1992). Finally, the “gene trap” Ds element (from PS116; P.S. Springer and R. Martienssen, unpublished data) was cloned into the introduced XhoI site in the 5′-untranslated region of the tobacco, chimeric ALS gene.

The transposase source T-DNA vectors (Fig. 2A) were constructed in the following way: Three modifications were made to the double-35S::Lc::nos3′ transcriptional fusion (Osborne et al., 1995). Plasmid BIO167 (kindly supplied by Brian Osborne, Cognia, New York, and Barbara Baker, Plant Gene Expression Center, University of California, Berkely, and US Department of Agriculture, Albany, CA) was cut with NotI and treated with the Klenow fragment of DNA polymerase I to result in a blunt-ended linear plasmid. ClaI linkers (Promega, Madison, WI) were ligated to the blunt ends, and the plasmid was recircularized. Plasmid BIO167 was then cut with BamHI, treated with the Klenow fragment, and recircularized, therefore removing the BamHI site. The double-35S::Lc::nos3′ transcriptional fusion was cloned as a ClaI fragment into the ClaI site of the binary vector SLJ3869 (Jones et al., 1992). Each one of the two transposase sources was then cloned into the BamHI and HpaI sites of SLJ3869. The 35S::Ac transposase fusion (Scofield et al., 1992; Swinburne et al., 1992) was excised as a BamHI/SmaI fragment from SLJ1101 (kindly supplied by Jonathan Jones [Sainsbury Laboratory, Norwich, UK]) and ligated into SLJ3869. The sΔNaeIAc transposase source (Bancroft et al., 1992) was excised from SLJ1941 (kindly supplied by Jonathan Jones) in two stages. Plasmid SLJ1941 was cut with SstI, treated with the Klenow fragment (to result in a blunt end) and then cut with BglII. This fragment was then ligated into SLJ3869.

Plant Transformation

The T-DNA constructs were introduced into Arabidopsis ecotype Landsberg erecta by Agrobacterium tumefaciens-mediated transformation (Valvekens et al., 1988) using the strain A. tumefaciens C58C1 pGV2260. Transgenic plants were selected by resistance to the antibiotic kanamycin (conferred by the NPTII gene contained in the T-DNA). Antibiotic resistance analysis was performed by growing plants under sterile conditions on growth medium supplemented with appropriate antibiotics as described by Bancroft et al. (1992).

Preparation of Plant Genomic DNA

Small-scale preparation of Arabidopsis genomic DNA was performed using a modification of the method described by Carroll et al. (1995), using a sap extractor (Erich Pollarne, Hannover, Germany).

DNA Gel-Blot Analysis

DNA gel-blot analysis was carried out as described by Jarvis et al. (1997). Probes used to detect the T-DNA borders and internal fragment were: RB probe, HindIII/EcoRI fragment from pCL0622 comprised of two RB sequences; LB probe, SphI/XbaI fragment from pCL0481 comprising 2′1′ promoter and NPTII coding region sequences; and GUS probe, XbaI/SacI fragment from pGUS (kindly supplied by Keith Lindsey, Department of Biological Sciences, University of Durham, UK) comprising the GUS coding region. T-DNA copy number was ascertained by hybridizing HindIII digested genomic DNA from each line with probes specific for LB and RB of T-DNA (for HindIII restriction sites within T-DNA, see Fig. 1). Lines that exhibited single hybridizing bands with LB and RB probes contained single T-DNA inserts. The completeness of the T-DNA was also tested by hybridizing the same blots with an internal fragment (GUS) probe. If the T-DNA copy was whole, the hybridization would result in a single, predicted size fragment (2.8 kb).

Amplification of T-DNA Insert Sites

TAIL PCR procedure was carried out as described by Liu et al. (1995), the only exception being that a 1-μL aliquot of the secondary reaction was diluted in 20 μL of water, and the tertiary reaction was performed in a 50-μL volume. Two sets of three nested specific primers were synthesized that corresponded to T-DNA LB and RB sequences. The LB set consisted of LB1 (5′-TGG GTA TCT GGG AAT GGC GAA ATA-3′; average T_m = 61.0°C), LB2 (5′-CAA GGC ATC GAT CGT GAA GTT T-3′; average T_m = 58.4°C), and LB3 (5′-AAT GTA GAC ACG TCG AAA TAA AGA-3′; average T_m = 55.9°C). The RB set consisted of RB1 (5′-GGG GCA TCG CAC CGG TGA GTA AT-3′; average T_m = 66.0°C), RB2 (5′-AGC GAA TTT GGC CTG TAG ACC TCA-3′; average T_m = 62.7°C), and RB3 (5′-TAT TCG GGC CTA ACT TTT GGT GTG-3′; average T_m = 61.0°C). LB1 and RB1 primers were used for the primary reactions, LB2 and RB2 for the secondary reactions, and LB3 and RB3 for the tertiary reactions. Two arbitrary degenerate primers were used, AD2 (Liu et al., 1995) and AD5 (Tsugeki et al., 1996).

IPCR procedure was carried out as described by Tissier et al. (1999). The primers used for amplification from the LB were LB1 and inverse (5′-CCT CAC ATA ATT CAC TCA AAT GCT A-3′; average T_m = 58.1°C).

YAC and BAC Library Filter Hybridizations

Gel-purified TAIL PCR or IPCR products were used as probes. CIC YAC library filters were prepared and hybridizations performed as described by Schmidt and Dean (1996). BAC filter hybridizations were performed as described by Bent et al. (1998), using the gel-purified T-DNA flanking fragments in place of YAC DNA. Institut für Genbiologische Forschung, Berlin and Texas A&M University BAC library filter sets were a kind gift of Ian Bancroft (John Innes Centre).

DNA Sequencing

Gel-purified TAIL PCR or IPCR products were sequenced using the ABI BigDye terminator reaction kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. To sequence LB-generated TAIL PCR or IPCR products, a primer positioned closer to the LB repeat was used, LB4 (5′-CGC TGC GGA CAT CTA CAT TTT TGA-3′; average T_m = 61.0°C).

Herbicide Selection

Selection of Ppt and Cs doubly resistant seedlings was performed by germinating seeds on GM supplemented with 20 ng mL^–1 Cs (kindly supplied by DuPont) and 10 μg mL^–1 Ppt (kindly supplied by Aventis UK, West Malling, Kent, UK). Only plants that had true leaves and well-developed roots were selected for further analysis.