The Plant Journal (2000) 22(3), 265±274 TECHNICAL ADVANCE A high throughput system for transposon tagging and promoter trapping in tomato Rafael Meissner, Veronique Chague, Qianho Zhu, Eyal Emmanuel, Yonatan Elkind and Avraham A. Levy* Plant Sciences Department, The Weizmann Institute of Science, Rehovot, 76100 Israel Received 21 October 1999; revised 22 February 2000; accepted 23 February 2000. *For correspondence (fax +972 8934 4181; e-mail [email protected]). Summary We describe new tools for functional analysis of the tomato genome based on insertional mutagenesis with the maize Ac/Ds transposable elements in the background of the miniature cultivar Micro-Tom. 2932 F3 families, in which Ds elements transposed and were stabilized, were screened for phenotypic mutations. Out of 10 families that had a clear mutant phenotype, only one mutant was Ds-tagged. In addition, we developed promoter trapping using the ®re¯y luciferase reporter gene and enhancer trapping, using b-glucuronidase (GUS). We show that luciferase can be used as a non-invasive reporter to identify, isolate and regenerate somatic sectors, to study the time course of mutant expression, and to identify inducible genes. Out of 108 families screened for luciferase activity 55% showed expression in the ¯ower, 11% in the fruit and 4% in seedlings, suggesting a high rate of Ds insertion into genes. Preferential insertion into genes was supported by the analysis of Ds ¯anking sequences: 28 out of 50 sequenced Ds insertion sites were similar to known genes or to ESTs. In summary, the 2932 lines described here contain 2±3 Ds inserts per line, representing a collection of approximately 7500 Ds insertions. This collection has potential for use in high-throughput functional analysis of genes and promoter isolation in tomato. Introduction Ef®cient tools for forward and reverse genetics are 1991; Rommens et al., 1992; Yoder, 1990). A number of invaluable to determine gene function. Such tools are tomato genes have been isolated with Ac/Ds, such as the available in maize and petunia, using insertional muta- Cf-9 (Jones et al., 1994) and Cf-4 loci (Takken et al., 1998), genesis with native tranposons (Koes et al., 1995; Mena et controlling resistance to various races of Cladosporium al., 1996; Walbot, 1992). In Arabidopsis, forward and fulvum; Dwarf, a gene encoding a cytochrome P450 reverse genetics have been implemented with the T-DNA homologue (Bishop et al., 1996), DCL, which controls of Agrobacterium tumefaciens (Feldman et al., 1989; chloroplast development (Keddie et al., 1996) and the Gaymard et al., 1998; Krysan et al., 1996) and with feebly gene which is involved in metabolism and develop- transposons from heterologous species such as the maize ment (Van der Biezen et al., 1996). In all cases genes were Ac/Ds (Fedoroff and Smith, 1993; Sundaresan et al., 1995) tagged by targeted tagging, by taking advantage of the and En/Spm transposons (Aarts et al., 1993; Wisman et al., preferential transposition of Ac/Ds to sites close by (Carroll 1998). Recently, databases of transposon insertion sites et al., 1995; Healy et al, 1993) and of the linkage of the have been produced in Arabidopsis that enable rapid target to the previously mapped Ds elements (Knapp et al., identi®cation of knockouts (Parinov et al., 1999; Speulman 1994; Thomas et al., 1994). Despite these successes in et al., 1999; Tissier et al., 1999). In tomato, insertional transposon tagging in tomato, there is still a need for more mutagenesis has been performed mostly with the Ac/Ds ef®cient forward and reverse genetics tools, especially in elements. These elements were shown to be active (Yoder light of the present release of tomato ESTs into sequence et al., 1988), and patterns of Ac/Ds transposition in this databases (www.tigr.org/tdb/lgi/index/html). It has been species were described (Carroll et al., 1995; Osborne et al., proposed that the miniature cultivar Micro-Tom is well ã 2000 Blackwell Science Ltd 265 266 Rafael Meissner et al. suited for large-scale mutagenesis in tomato owing to its tain the NPTII gene as a transformation marker and/or as a small size, rapid life cycle, easy transformability, and re-insertion marker. The indole acetamide hydrolase (iaaH) ef®cient activity of the Ac/Ds elements (Meissner et al., gene confers sensitivity to NAM and was used as a 1997). negative selection marker to select against Bam35s±Ac Another application of insertional mutagenesis is to and thus obtain stable transposition events. The ALS gene combine a reporter gene within the non-autonomous confers resistance to 100 p.p.b. chlorosulfuron in plants mobile element (T-DNA or transposon) as a tool for carrying an unexcised Ds element and confers resistance discovering genes and/or transcriptional regulators, such to 3 p.p.m. chlorosulfuron in plants where the Ds element as enhancers and promoters. Enhancers can be detected was excised. F1 seeds of transposase 3 Ds plants were by cloning a reporter gene in between the borders of a produced by crossing the transposase plants (Bam35S±Ac) mobile element and downstream of a weak constitutive with 12 independent Ds378±GUS-transformed T1 plants promoter (Fedoroff and Smith, 1993; Wilson et al., 1989), and 15 independent Ds251±LUC-transformed T1 plants. the reporter being activated upon insertion near an The number of Ds inserts in each one of the T1 Ds parents enhancer. Promoters can be detected by cloning a used in the crosses was determined by Southern blotting promoterless reporter in between the borders of the and was found to vary from one to seven in the different mobile element (Sundaresan et al., 1995), the reporter plants (data not shown). F1 seeds were obtained from all being activated upon insertion downstream of a promoter the crosses involving Ds251±LUC. However, for a reason and in the correct orientation, thus generating transcrip- which is unclear, only one of the Ds378±GUS plants gave tional or translational fusion. Both enhancer and gene- rise to fertile F1 seeds, while embryos were aborted in the trapping methods enable the presence of genes and their other crosses. Somatic activity of the Ds element could be patterns of transcriptional regulation to be detected, detected through increased resistance to chlorosulfuron in independently of a mutant phenotype. The b-glucuronid- F1 plants compared to the Ds parent, and thus selection for ase (GUS) reporter gene has been used in most works on somatic activity could be carried out in F1 seedlings by enhancer and gene trapping in plants (Maes et al., 1999; germinating and selecting the 100 p.p.b. chlorosulfuron- Sundaresan, 1996). One problem with GUS staining resistant F1 seedling. Another indication of transposition (Jefferson et al., 1987) is the destructive nature of the activity is that somatic GUS or luciferase sectors could be staining and destaining procedure. Non-invasive and non- detected in F1 plants but not in the parents (data not destructive reporter genes such as the luciferase or GFP shown). A total of 1768 F1 chlorosulfuron (100 p.p.b.)- genes have not yet been widely used for gene trapping in resistant plants from the cross with Ds-GUS, and 971 F1 plants. plants from the crosses with Ds-LUC, were grown and We report on the production and analysis of a collection seeds were harvested from each F1 plant individually. F2 of 2932 families of miniature tomatoes containing stabil- seedlings were selected for germinally stable transposi- ized insertions of Ds elements. This collection was tion: excision (ALSr), re-insertion (Hygror or Kanar), and screened for mutant phenotypes and for enhancer and stabilization (Namr). Following this selection, 1451 out of gene trapping with the GUS and luciferase reporter genes. 19 005 F2 seedlings were obtained from the cross with Ds- The high frequency of luciferase-trapped genes and the GUS (7.6%), and 1481 out of 20 619 F2 seedlings were sequencing of transposon-¯anking regions both indicate obtained from the crosses with Ds-Luc (7.2%). The majority that the Ds elements preferentially insert into genes. We of these plants correspond to independent transposition discuss the utilization of this system for high-throughput events as determined by the fact that in most cases they insertional mutagenesis and for non-invasive gene originated from different F1 plants. For F1 plants which trapping in tomato. gave rise to more than one stably transposed seedling, Southern blot analysis indicated that in about half of the cases siblings corresponded to independent transposition Results events. Considering that half of the excised Ds elements do not re-insert (Meissner et al., 1997) and that transposase Establishingthe Ds insertion collection was counter-selected (1/4 of the F2 progenies survived The constructs used for preparing the Ds insertion NAM selection), this means that excision rates were very collection in Micro-Tom are shown in Figure 1. Among high, in the 60% range (7.5 3 2 3 4). those, Ds378±GUS, an enhancer trap, and Bam35S±Ac, a Recessive mutations may be observed in F2 plants only if stable transposase source, were previously transformed an early transposition event occurred in a given F1 plant and shown to be active in Micro-Tom (Meissner et al., and was transmitted to both male and female gametes. 1997). The Ds251±LUC construct (Figure 1) was built for Although we have detected such case (one of the gene trapping with the luciferase reporter gene and was chlorophyll mutants described below), we have focused also transformed into Micro-Tom. These constructs con- on screening for mutants in F3 families. F3 seeds from the ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 265±274 Gene tagging and trapping in tomato 267 chlorophyll mutants (e.g.