A High Throughput System for Transposon Tagging and Promoter Trapping in Tomato

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

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chlorophyll mutants (e.g. Figure 2c,d); two mimicked the symptoms of TYLCV (tomato yellow leaf curl virus)- infected plants (e.g. Figure 2b); one mimicked hypersensi- tive response (not shown); and one showed narrow cotyledons and was almost lea¯ess at the mature plant stage (e.g. Figure 2e±g). The ¯ower of the lea¯ess mutant had four fused ovary styles and stigma (data not shown). Eight out of 10 mutants showed 3 : 1 segregation, indicating that the mutation was caused by a single recessive allele. Southern blot analysis was done to test whether the mutants were caused by Ds insertion: DNA was extracted from 13 individuals of each of the mutant families, digested, blotted and probed with a Ds probe. To distinguish homozygote- from heterozygote-dominant plants, seeds from each one of the 13 plants of the mutant family were collected separately and sown. Co-segregation of the Ds element with the mutant phenotype was found only for the chlorophyll mutant shown in (Figure 2h,i) (data not shown).

Non-invasive gene trapping with the luciferase reporter gene

One hundred and eight F3 families derived from the Ds- Figure 1. Schematic representation of constructs transformed into Micro- luciferase parents and corresponding to independent and Tom. Constructs Bam35S±Ac and Ds378±GUS were used for enhancer trapping stable transposition events were screened for luciferase (Fedoroff and Smith, 1993). Sequences similar to Ac are shown in grey, expression in various plant organs by imaging with with the terminal inverted repeats shown as grey arrows. Constructs are an ultra-low light-cooled charge coupled device (CCD) ¯anked by the right (RB) and left (LB) borders of their respective T-DNA. The b-glucuronidase gene (GUS) is fused to Ac weak promoter in Ds378± camera. The same plants were screened for luminescence GUS. The luciferase gene in Ds251±LUC is fused to the Ac promoterless at the seedling, ¯ower and young fruit stages. Different fragment from 1 to 251. Resistance to basta (Barr), kanamycin (Kanar)or expression patterns were obtained, for example in hypo- hygromycin (Hygror) is conferred by the phosphinothricin, neomycin phosphotransferase or aminocyclitol phosphotransferase genes, cotyls, anthers, sepals or fruits (Figure 3). The frequency of respectively. Chlorosulfuron resistance (Chlorosulfuronr) is obtained occurrence of light signals was 4, 11 and 55% in the upon excision of the Ds element from the Ds378±GUS, Ds251±LUC seedlings, fruits and ¯owers, respectively (Table 1). Organ- constructs and activation of a mutated acetolactate synthase gene from Arabidopsis (Fedoroff and Smith, 1993). Sensitivity to naphtalene speci®c signals, those only in ¯owers, only in fruits or only acetamide (NAMs) is conferred by the indole acetic hydrolase gene. in seedlings, were found at a frequency of 48, 2 or 1%, respectively (Table 1). Unlike GUS, which is usually destructive, luciferase is a

2932 F2 plants that were selected for stable transposition non-destructive and probably non-invasive reporter gene. were collected for each plant separately, and F3 seeds This opens a number of possibilities, such as regenerating sown for each family and screened for phenotypic a somatic sector, following gene expression in real time, mutations. Approximately 13 plants were grown for each and analysing inducible genes. Examples of such applic- of the 2932 families: In total the plants grown were: 18 863 ations are given below. Imaging of F1 plants derived from

F3 plants derived from 1451 different F2 plants of cross crosses between Ds-luciferase and transposase, enabled to

Ds378±GUS 3 Bam35S±Ac, and 19 253 F3 plants derived identify luminescent sectors, which correspond to inser- from 1481 different F2 plants for crosses of Ds251± tions into genes. One such cotyledon sector, which LUC 3 Bam35S±Ac. emitted a particularly strong light signal, was cut under sterile conditions and was regenerated into a mature plant. All the organs of the regenerated plant were screened and Mutant screening found to express luciferase (Figure 4). The promoter that

The F3 families described above were screened for obvious was trapped in this plant seems to be constitutive, and was phenotypes as seen only by visual observation. A total of stronger than the 35S promoter when the expression of 10 mutants were found, ®ve with the Ds-GUS cross and the 35S-luciferase-positive control plants and that of the ®ve with the crosses with Ds-LUC (Figure 2). Six were regenerated plant were compared side by side (data not

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Figure 3. Gene-trapping expression patterns as detected with the luciferase reporter gene.

Each row represents the imaging of a given organ from F3 plants of the crosses Ds251±LUC 3 Bam35S±Ac where selection for stabilized Ds Figure 2. Mutant phenotypes obtained by insertional mutagenesis with transposition was made, and after treatment with luciferin. Left column, Ac/Ds. imaging of the organ under light conditions; right, the same object as (a) Wild type; (b) mimicry of tomato yellow leaf curl virus symptoms; (c) seen after imaging in total darkness. Imaging of seedlings grown chlorophyll mutant with white leaves throughout the plant; (d) upper vertically in agar plates (a) led to the identi®cation of one individual with new leaves are yellow and turn green when mature; (e) mutant with a luminescent hypocotyl (b). Imaging of a ¯ower (c) showing strong abnormal growth showing thin, elongated cotyledons in young seedlings luciferase activity in the anthers, a weak activity in the petals and no compared to (f) wild-type seedlings. Later during development the plant activity in sepals (d). Imaging of a young fruit (e) where luciferase activity grown from (e) is almost lea¯ess with thin, simple, wiry leaves (g) and is restricted to the sepals (f). Imaging of the two halves of a young fruit ¯owers with fused ovaries (not shown). (h±i) Chlorotic throughout (g) where luciferase activity is stronger in the core of the fruit rather than development with stronger phenotype during fruit set; (h) chlorophyll in other parts (h). mutant showing white leaves in the absence of transposase and (i) sibling showing variegated leaves when transposase is present.

and gene activity could again be localized to the bending shown). In one F3 seedling luciferase activity was detected and elongating region (Figure 5c,d), showing that gene in the root. This type of activity was very speci®c, localized expression can be followed over time. in the elongating zone of the root just above the root tip In order to identify if an inducible promoter was trapped

(Figure 5a,b). The luminescent root was manually bent into among the 108 F3 families tested, we imaged the seedlings a horizontal position, and the petri dish with the seedlings under normal conditions, after 30 h at 4°C and 12 h was grown further, vertically, for 24 h and then imaged recovery, and after 20 h at 42°C with 12 h recovery. Four again. Gravitropism caused the root to bend downward positive (light-emitting) seedlings could be seen when

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Table 1. Organ-speci®c patterns of luminescence in 108 F3 Ds251±LUC families

Total Organ Organ-speci®c patterns of luminescence (% luminescent)

Flowers + ± ± + ± + + ± 55 Fruits ± + ± + + ± + ± 11 Seedlingsa ±±+±+++± 4 Total luminescent 52 2 1 7 2 0 1 43 60 % luminescent 48 2 1 6 2 0 1 40 aSeedlings were grown in normal conditions or tested after cold or heat shock as described in the text. Two seedlings were shut off after cold treatment and one was shut off after the heat treatment. + indicates that luminescence was observed; ± indicates a lack of luminescence.

grown under normal conditions (16 h day, 25°C), two were fragments derived from inverse PCR is summarized in shut off after the cold treatment and one more was shut off Table 2. Out of 50 Ds ¯anking sequences, 16 are similar to after the heat treatment. No cold or heat upregulated known genes, 12 are similar to ESTs with no known promoter was detected in this collection. function, six are in the T-DNA region and 16 are sequences with no signi®cant homology to sequences from the GenBank database. Only three sequences (#2, #5, #7) were Enhancer trapping almost identical (within range of sequencing error) to

Fruits from 70 different F2 plants, derived from cross known genes from tomato, indicating that sequencing Ds378±GUS 3 Bam35S±Ac and selected for stable trans- insertion sites enabled the discovery of new tomato genes. position events, were screened for GUS activity, that is, for insertions near enhancers. Four fruits showed positive Discussion GUS staining with various expression patterns: in the vascular tissue (Figure 6a); throughout the fruit (Figure 6b) Forward and reverse genetics with the Micro-Tom Ac/Ds or in the outer layers of the pericarp (Figure 6c,d). Progeny system of these plants were further tested to determine whether GUS activity is restricted to fruits or can also be detected in We have described a new system for high-throughput other tissues such as roots, leaves and stems. In two lines insertional mutagenesis in tomato. Previous efforts to (Figure 6a,b), GUS activity was restricted to fruits and in developinsertional mutagenesis in tomato have mostly the other two (Figure 6c,d) it was found to be also in the focused on targeted transposon tagging (Bishop et al., roots, stems and leaves (data not shown). Note that 1996; Jones et al., 1994). The system described here is well negative controls were not coloured under the staining suited for non-targeted tagging as large populations can conditions used (data not shown). Interestingly, a gradient be screened; however the rate of tagged mutants is quite of blue colour was observed in mutants (Figure 6c,d); this low (one in 10 in this work). The reason for the low tagging gradient was observed again when mutants were tested in rate might be the strong activity of Ds elements, with the next generation. excision rates in the 60% range.

The 2932 F3 stable insertion lines (38 000 plants) described here contain approximately 7500 Ds insertions Sequencing Ds insertion sites (two to three copies per line on average) and might The high frequency of luciferase-trapped genes (Table 1) therefore be useful for reverse genetics. Assuming that suggests either a high copy number of Ds insertions and/ about half the transposition events land in genes, as or preferential insertion into genes. Copy number was discussed below, we roughly estimate that the 2932 F3 determined by Southern blot analysis for 30 out of the 108 lines contain around 3000 mutated genes. While only 10

F3 Ds-LUC families that were used in the trapping experi- families with a clear mutant phenotype were found, careful ments. On average, the copy number of new Ds insertions analysis, including measurement of quantitative traits, was 2±3 (ranging from 1±4) in F3 plants derived from F2 suggests that many subtle alterations in plant growth can plants where Ds had been stabilized (data not shown). be detected in this population (unpublished data) and

Taken together, the 108 F3 families constitute a collection therefore that it might be useful for forward genetics. of approximately 250 Ds insertions. Preferential insertion Sequencing of insertion sites also suggests that Ac/Ds into genes was tested by sequencing Ds ¯anking regions insertional mutagenesis can be useful for reverse genetics in this collection. Sequence analysis of the 50 DNA in tomato because of a preferential insertion into genes:

ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 265±274 270 Rafael Meissner et al.

Figure 5. Trapping genes and following expression in real time. (a) Tomato seedlings were grown vertically and screened for luminescence. (b) After imaging in complete darkness, one seedling was identi®ed as luciferase positive in the root, in the elongation zone, just above the root cap. Arrows indicate the position of the signal. In order to test whether expression was limited to the elongation zone, the positive root was lifted, laid horizontally and imaged again after 24 h. Cell elongation could be observed in the kink of the root formed as a result of gravitropism (c). The luminescent signal was again restricted to the newly formed elongation zone (d).

Figure 4. Regeneration of luminescent sectors. The luciferase assay being non-destructive, it is possible to isolate luminescent sectors from within a chimeric tissue and to regenerate whole plants from these sectors. This was done with a luminescent sector from the cotyledons of an F1 seedling of the cross Ds251± LUC 3 Bam35S±Ac (a,b). Staining of several organs was done after regeneration and activity was discovered in roots (c,d); ¯owers (e,f); young fruits (g,h) and mature fruit (i,j).

considering that the tomato genome is 10 times bigger Figure 6. X-Gluc staining of fruits from F2 plants derived from the cross Ds378±GUS 3 Bam35S±Ac. A variety of staining patterns are found; in than that of Arabidopsis, and assuming that the number of one line staining was localized only to the vascular cells of fruits from genes is in the same range as in Arabidopsis, the average the breaker stage (a); in other lines staining was observed throughout the gene density in tomato is much lower than that in pericarp (b) or only in the outer layer of the pericarp (c,d). Arabidopsis. Nevertheless the majority of the insertion sites (28 out of 50) corresponded to coding regions (known genes or ESTs), indicating a bias for insertion into coding are virtually identical to known tomato genes (the feebly regions. Out of the 28 (16 + 12) sequences that had a gene, the rDNA, and the proteinase inhibitor). Other signi®cant similarity to known genes or ESTs, only three sequences were similar, but not identical, to other genes

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Table 2. Analysis of Ds ¯anking sequences

No. Nucleotide similarity Ea Protein similarity Eb

Genes 1 Medicago trunculata Pi transporter, MTAF000355 2e-10 A. thaliana Pi transporter, U62331 1e-64 2 Solanum lycopersicum feebly gene, U35644 6e-29 S. lycopersicum feebly prot, S70648 6e-06 3 Tomato ovary EST 265819 2e-16 A. thaliana hypothetical. protein, AC002340 1e-04 4 Arabidopsis thaliana aap1 gene, X95622 6e-07 5 Lycopersicon esculentum 9.04 kb rDNA, X52215 0 6 Spirodela polyrrhiza PDR5-like ABC, Z70524 1e-20 7 L. esculentum auxin-induced proteinase 2e-25 inhibitor, L25128 8 D. radiodurans hypothetical. protein 8e-09 AE001961 9 L. esculentum polyprotein AF119040 6e-11 10 A. thaliana putative Na+/H+ exchanging 4e-10 protein, AL03539 11 Potato patatin pseudogene SB6B 5e-05 12 Solanum tuberosum mitoch trnC, X93575. 4e-09

13 Anthoceros punctatus photosystem I P700 9e-5 apoprotein A1, AB013664 14 Tomato EST 243864 4.6e-12 A. thaliana putative protein kinase, AC004260 9e-10 15 L. esculentum ripening-related mRNA, 0.004 X72734 16 Oryza sativa putative copia polyprotein, 1e-30 AC006248 EST 17 Tomato EST246491 0.0049 18 Tomato EST 265819 2.6e-17 19 Tomato EST 261212 6.3e-05 20 Tomato EST 248200 9.9e-05 21 A. thaliana EST P24675 9e-05 22 Tomato EST 261212 1.2e-11 23 Tomato EST TC3415 0.00057 24 Tomato EST 246491 4.9e-03 25 Tomato EST 266245 1.2e-05 26 Tomato EST TC5532 0.004 27 Tomato EST 261616 1.9e-05 28 Tomato EST249946 1.2e-05 In T-DNA 6 sequences Unknown 16 sequences aExpect value: statistical signi®cance threshold for reporting matches against database sequences obtained after BLASTN program search. bExpect value obtained after BLASTX program search.

or ESTs from tomato, Arabidopsis, potato, or other plant insertions are into genes, we roughly estimate that species. This indicates that sequencing Ds insertion sites 200 000±300 000 Ds insertions, derived from several un- is an effective tool for the discovery of new genes. linked T-DNAs, would be suf®cient to achieve a high Among interesting hits are sequences similar to a Pi (approximately 90%) chance of insertion into any speci®c transporter, an Na+/H+ antiporter, and an ABC transporter. target gene. Interestingly, feebly had been also isolated by Ac/Ds tagging (Van der Biezen et al., 1996), suggesting that it Probing the genome by gene trapping may be a hot-spot for Ac/Ds insertion. In our case the Ds insertion in feebly is in a different location. The proportion Enhancer and promoter trapping have not been described of insertions that were in the T-DNA and therefore are previously in tomato. This feature may be particularly not useful for reverse genetics was six out of 50. This useful for the isolation of transcriptional regulatory frequency (12%) is similar to that reported for Arabidopsis regions, particularly in fruit. Root or leaf promoters can (Parinov et al., 1999). Assuming that the tomato genome probably be borrowed from Arabidopsis trapping systems contains 20 000±30 000 genes, and that about half of the and retain their speci®city; however, ¯eshy berry fruit

ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 265±274 272 Rafael Meissner et al. types have a different structure, metabolism and develop- ideally suited to produce a large collection of insertions for ment compared to siliques and probably have some reverse genetics in tomato and for promoter isolation. unique sets of regulatory elements. Non-invasive gene Sequencing of insertion sites is an effective way to identify trapping has not been used extensively in plants. The new tomato genes. Subsequent analysis of homozygous ®re¯y luciferase gene used in this work proved to be a mutants for these genes should greatly facilitate their sensitive and non-invasive reporter, with many advant- functional analysis. ages compared to GUS: there is no endogenous background in the plant, and it is convenient to utilize ± 20 min Experimental procedures after the application of luciferin on the plant tissue it is possible to screen for luminescence. Furthermore, non- Constructs transformation invasive trapping opens up many prospects such as isolation of inducible promoters; follow-up of gene Constructs Ds378±GUS and Bam35S±Ac (Fedoroff and Smith, 1993) were kindly provided by Nina Fedoroff and Ds251±LUC was expression in mutants in real time; and regeneration of built as described below. These constructs were transformed in somatic trapping events. This latter feature can be useful tomato as previously described (Meissner et al., 1997). when dealing with species which cannot be propagated through seeds, or when searching for rare patterns of gene expression (each leaf represents a `®eld' of sectors). The Construction of plasmid Ds251±LUC major limitations of gene trapping with luciferase are the Ds251±LUC was built for promoter trapping with luciferase. For requirements of a dedicated imaging system, a dark room this purpose the 10 kb Asp718 fragment from plasmid 378 Ds± and the high cost of the substrate. GUS (Fedoroff and Smith, 1993) was cloned into the same site of pUC19, giving rise to plasmid pAA1±18. The internal MscI±PacI A surprising ®nding was the overall high rate of fragment within the Ds element of 378 Ds±GUS was digested from luciferase-positive plants: 65 out of the 108 families pAA1±18, removing the Ac promoter the GUS and HPT genes. screened (60%) (Table 1). To explain this, we assume that This fragment was replaced by the promoterless ®re¯y luciferase the promoterless system used here was ef®cient and gene linked to the NPTII gene giving rise to plasmid p22±8. The also that the Ds-luciferase preferentially inserts into luciferase gene was obtained from the SalI±BglII fragment of construct pJD301, kindly provided by Virginia Walbot and genes. Promoterless gene trapping was used previously described by Luehrsen et al. (1992). The NPTII gene was derived with T-DNA constructs, with the reporter cloned near the from the 2.3 kb SacII±ClaI fragment from plasmid pGA492 (An, T-DNA border (Maes et al., 1999). With Ac/Ds the presence 1986). In the new Ds in p22±8, the 5¢ of the promoterless luciferase of ATGs triplets at both termini of the element has caused gene was ligated downstream of nucleotide 251 from the 5¢ end of concern that the reporter gene would not be translated in- Ac. The Asp718 fragment of p22±8, containing the ALS excision frame, and thus that promoterless strategies may not cassette (Fedoroff and Smith, 1993) and the new Ds±LUC promoter trap, was cloned into a polylinker that was inserted in work. For this reason a gene-trapping system was the SLJ525 binary vector instead of the NPTII-containing HindIII± developed with an intron and a splice acceptor site ClaI fragment (details of the intermediate steps of the cloning are upstream of a GUS reporter (Sundaresan et al., 1995). available upon request). The resulting Ds251±LUC plasmid is The high frequency of luminescent traps in our experiment shown in Figure 1. indicates that the ATGs near the transposon 5¢ terminal region (nucleotides 1±251) did not prevent the ef®cient GUS staining recovery of traps. This may be caused by a favourable GUS activity in F plants was determined according to the nucleotide context that enables preferential initiation of 2 histochemical procedure described by Jefferson et al. (1987) with translation from the ATG of luciferase ORF, or by other some modi®cations. Fruits of the F2 plants were stained with X- unknown mechanisms. The high rate of luminescent Gluc (5-bromo-4-chloro-3-indolyl-b-D-glucuronic acid; Duchefa) at ±1 plants is consistent with the sequencing data showing a concentration of 0.5 mg ml , supplemented with in 100 mM that Ds preferentially inserts into genes (Table 2). We do NaH2PO4 pH 8.0 (phosphate buffer), 15 mM EDTA, 5 mM ferrocyanide, 5 mM ferricyanide, and 20% methanol. Seedlings were not understand the reason for the high frequency of stained with 100 mM NaH2PO4 pH 7.0 (phosphate buffer), 0.5 mM luminescence signals in ¯owers (Table 1); it may re¯ect the ferrocyanide, 0.5 mM ferricyanide, 0.1% Triton, and 10 mM EDTA. possibility that more genes are expressed in ¯owers than The clearing procedure was done as described (Beeckman and in other organs, or maybe gene expression is stronger in Engler, 1994). the ¯ower than in other organs where weak signals might have been undetected, or ¯owers might contain more ATP Luciferase screening than leaves fruits and roots (ATP is required for light emission and catalysis of luciferin) (A¯alo, 1991). The luciferase screening was done by imaging plant tissues in the dark chamber using a camera for ultra-low light imaging In summary, the utilization of a miniature tomato, (Princeton Instruments Inc., USA). Image acquisition and process- together with the Ac/Ds trapelements that insert prefer- ing was performed with IPLAB software (Signal Analysis Corp., entially into genes, make the Micro-Tom±Ac/Ds system USA).

ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 265±274 Gene tagging and trapping in tomato 273

The imaged tissues were supplemented with 1 mM beetle encodes the ®rst member of a new cytochrome P450 family. luciferin (Promega) and 0.01% Triton, and were kept for 20 min Plant Cell, 8, 959±969. in the dark after luciferin application to overcome the natural Carroll, B.J.V.I.K., Thomas, C.M., Bishop, G.J., Harrison, K., ¯uorescence emitted by the chlorophyll. The bioluminescence Sco®eld, S.R. and Jones, J.D.G. (1995) Germinal transposition was imaged during the ®rst 3 h of incubation with the substrate. of the maize element Dissociation from T-DNA loci in tomato. Genetics, 139, 407±420. Fedoroff, N.V. and Smith, D.L. (1993) A versatile system for Isolation of transposon-¯anking regions by long-range detecting transposition in Arabidopsis. Plant J. 3, 273±289. inverse PCR Feldman, K.A., Marks, M.D., Christianson, M.L. and Quatrano, R.S. (1989) A dwarf mutant of Arabidopsis generated by T-DNA Transposon-¯anking regions were isolated by long-range inverse insertion mutagenesis. Science, 243, 1351±1354. PCR adapted from Mathur et al. (1998). Genomic DNA was Gaymard, F., Pilot, G., Lacombe, B., Bouchez, D., Bruneau, D., extracted from pools of 5±13 Ds-insertion lines. Twenty DNA Boucherez, J., Michaux-Ferriere, N., Thibaud, J.B. and pools were prepared and 4 mg of each pool was digested Sentenac, H. (1998) Identi®cation and disruption of a plant overnight in 100 ml ®nal volume with 20±50 U of restriction shaker-like outward channel involved in K+ release into the endonucleases SpeI, XbaIorAsp718. Following phenol chloro- xylem sap. Cell, 94, 647±655. form extraction and ethanol precipitation, DNA was resuspended Gorbunova, V. and Levy, A.A. (1997) Circularized Ac/Ds in 100 ml, and 30 ml were run on a 0.8% agarose gel to check their transposons: formation, structure and fate. Genetics, 145, proper digestion. The other 70 ml were self-circularized in 500 ml 1161±1169. ®nal volume with 6 mlofT4 DNA ligase (Biolabs) overnight at 16°C. Healy, J., Corr, C., DeYoung, J. and Baker, B. (1993) Linked and After ethanol precipitation, half the resuspended DNA was unlinked transposition of a genetically marked Dissociation subjected to PCR ampli®cation using two sets of nested primers element in transgenic tomato. Genetics, 134, 571±584. speci®c of the Ds 5¢ and 3¢ termini, as previously described Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. (1987) GUS (Gorbunova and Levy, 1997). PCR reactions were performed in fusions: b-glucuronidase as a sensitive and versatile gene 50 ml using the Expand Long Template PCR system (Boehringer), fusion marker in higher plants. EMBO J. 6, 3901±3907. as recommended by the supplier, with buffer 3. The second Jones, D.A., Thomas, C.M., Hammond-Kosack, K.E., Balint-Kurti, nested ampli®cation was performed using 2 ml of a 500-fold P.J. and Jones, J.D.G. (1994) Isolation of the tomato Cf-9 gene diluted ®rst PCR mixture. The isolated PCR fragments were either for resistance to Cladosporium fulvum by transposon tagging. used directly as templates for sequencing with the internal Ds 5¢ and Ds 3¢ primers (Gorbunova and Levy, 1997), or cloned with the Science, 266, 789±792. PGEM-T system (Promega). Sequences were submitted to Keddie, J.S., Carroll, B., Jones, J.D.G. and Gruissem, W. (1996) databases at the National Center for Biotechnology Information The DCL gene of tomato is required for chloroplast (www.ncbi.nlm.nih.gov) and the Tigr Institute for Genomic development and palisade cell morphogenesis in leaves. research (www.tigr.org) for homology searching. EMBO J. 15, 4208±4217. Knapp, S., Larondelle, Y., Robberg, M., Furtek, D. and Theres, K. (1994) Transgenic tomato lines containing Ds elements at de®ned genomic positions as tools for targeted transposon Acknowledgements tagging. Mol. Gen. Genet. 243, 666±673. 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