Transformation of statice (Limonium sinuatum Mill.) by Agrobacterium tumefaciens-mediated gene trans

Transformation of statice (Limonium sinuatum Mill.) by Agrobacterium tumefaciens-mediated gene trans

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Unformatted text preview: The Plant Journal (1998) 13(5), 707–716 TECHNICAL ADVANCE Gene identification with sequenced T-DNA tags generated by transformation of Arabidopsis cell suspension Jaideep Mathur1, Laszlo Szabados2, Sabine Schaefer1, ´´ Britta Grunenberg1, Andrea Lossow1, Esther Jonas-Straube1, Jeff Schell1, Csaba Koncz1,2 and Zsuzsanna Koncz-Kalman1,* ´´ 1Max-Planck Institut fur Zuchtungsforschung, D-50829 ¨ ¨ Koln, Carl-von-Linne-Weg 10, Germany, and ´ ¨ 2Institute of Plant Biology, Biological Research Center of Hungarian Academy of Sciences, H-6701 Szeged, PO Box 521, Temesvari krt. 62, Hungary ´ Summary A protocol for establishment and high-frequency Agrobacterium-mediated transformation of morphogenic Arabidopsis cell suspensions was developed to facilitate saturation mutagenesis and identification of plant genes by sequenced T-DNA tags. Thirty-two self-circularized TDNA tagged chromosomal loci were isolated from 21 transgenic plants by plasmid rescue and long-range inverse polymerase chain reaction (LR-iPCR). By bidirectional sequencing of the ends of T-DNA-linked plant DNA segments, nine T-DNA inserts were thus localized in genes coding for the Arabidopsis ASK1 kinase, cyclin 3b, Jdomain protein, farnesyl diphosphate synthase, ORF02, an unknown EST, and homologues of a copper amine oxidase, a peripheral Golgi protein and a maize pollenspecific transcript. In addition, 16 genes were identified in the vicinity of sequenced T-DNA tags illustrating the efficiency of genome analysis by insertional mutagenesis. Introduction Following large-scale sequencing of expressed sequence tags (ESTs) and construction of physical maps, genome projects in Arabidopsis and other plants are advancing towards sequencing of whole chromosomes in conjunction with functional analysis of the sequenced genes (Cooke et al., 1996; Hofte et al., 1993; Huang and Miao, 1997; ¨ Newman et al., 1994; Sasaki et al., 1994; Schmidt et al.,1995; Zachgo et al., 1996). As gene replacement is not yet routine in higher plants, the T-DNA of Agrobacterium and transposons are used for saturating the genome Received 8 July 1997; revised 30 October 1997; accepted 7 November 1997. *For correspondence (fax 49 221 5062 213; e-mail © 1998 Blackwell Science Ltd with insertional mutations in order to establish correlations between sequence data, mutant phenotypes and gene functions by reverse genetics (for reviews see Feldmann et al., 1994; Long et al., 1993; Miao and Lam, 1995; Osborn et al., 1991; Souer et al., 1995). Application of reporter gene fusion, enhancer trap, suicide gene and activator tagging technologies facilitate both targeted tagging of single genes and mutagenesis of particular gene classes characterized by well-defined regulatory functions and/or expression patterns (Franzmann et al., 1995; Hayashi et al., 1992; Kertbundit et al., 1991; Knapp et al., 1994; Koncz et al., 1989, 1994; Sunderesan et al., 1995; Topping et al., 1991). In addition, PCR screening techniques adopted from Drosophila and Caenorhabditis genome research are used in the sequence-based identification of transposon and T-DNA insertions in known genes (Ballinger and Benzer, 1989; Kaiser and Goodwin, 1990; Koes et al., 1995; Krysan et al., 1996; McKinney et al., 1995; Rushforth et al., 1993; Souer et al., 1995; Zwaal et al., 1993). Thus far, partial sequencing of 30 000 cDNAs in Arabidopsis has identified about 10 000 genes, corresponding to 40–65% of the total gene number, as calculated on the basis of genome size, mRNA complexity and mutation ´ rates (Koncz and Redei, 1994; Meyerowitz, 1994; Miklos and Rubin, 1996). Although the construction of equalized cDNA libraries may further increase this number (Kohchi et al., 1995), the identification of all Arabidopsis genes now requires a link between genome sequencing and reverse genetics. Sequencing of plant DNA junctions of transposon and T-DNA tags may in fact provide such a link, assuming that saturation mutagenesis can be achieved with these insertional mutagens. Currently, one- and two-element systems based on the maize transposons Ac/Ds and En/I/ dSpm (Bancroft et al., 1993; Cardon et al., 1993) are used to build up mutagenized populations, and already two large collections of T-DNA tagged lines, consisting of about 55 000 plants, are available in Arabidopsis (Bechtold et al., 1993; Forsthoefel et al., 1992). Genome saturation with TDNA inserts is a realistic goal, because within a pool of 75 000–100 000 transgenic plants, a T-DNA tag is expected to occur in each Arabidopsis gene at 95–97.5% probability when the average copy number of inserts is between 1 and 3 (Bechtold et al., 1993; Feldmann et al., 1994). Saturation mutagenesis with T-DNA inserts can be reproducibly achieved by Agrobacterium transformation of Arabidopsis cell suspensions as described below. In addition to promoting the development of targeted gene tagging 707 708 Jaideep Mathur et al. approaches (e.g. activator T-DNA tagging; Kakimoto, 1996), this technique is applicable for mass regeneration of transgenic plants to generate mutant pools for identification of T-DNA-tagged Arabidopsis chromosomal loci by genome sequencing. Plasmid rescue and long-range inverse PCR offer two simple methods for isolation by self-circularization and bi-directional sequencing of T-DNA-tagged chromosomal DNA segments, facilitating the analysis of the Arabidopsis genome by reverse genetics. Results and discussion For Agrobacterium-mediated transformation of Arabidopsis, a wide range of tissue culture and in planta transformation methods are available. From Agrobacterium-infected leaf discs, cotyledon, stem or root explants, transgenic calli can be obtained and regenerated to plants with varying efficiencies (for review see Koncz et al., 1994; Morris and Altmann, 1994). Because Agrobacterium is capable of systemically transforming diverse cell types when infiltrated into plants (e.g. as was demonstrated using a T-DNA with an intron-containing uidA reporter gene by Vancanneyt et al., 1990), the need for plant regeneration from tissue culture can be overcome by in planta transformation. Because the yield of current transformation methods is generally a few thousand transgenic plants per experiment, saturation of the Arabidopsis genome with TDNA tags is a cumulative process leading to the construction of a single representative mutant collection (for review see Bechtold et al., 1993; Feldmann et al., 1994). However, when mutant selection techniques are applicable at the cellular level using, e.g. activator mutagenesis (Hayashi et al., 1992), saturation T-DNA tagging can be achieved in a single experiment by co-cultivation of plant protoplasts with Agrobacterium (Horsch et al., 1987). In Arabidopsis, this technology is not yet efficient when applying the available leaf mesophyll- and root-derived protoplast systems (Damm et al., 1989; Mathur et al., 1995). However, similarly to tobacco and rice (An, 1985; Hiei et al., 1994), Agrobacterium infection of morphogenic Arabidopsis cell suspensions yields very high transformation rates. Initiation and maintenance of Arabidopsis cell suspensions A protocol for the establishment of cell suspensions from Arabidopsis ecotypes Col-1, Col-5, RLD1 and WS2 is described in Experimental procedures. Briefly, root cultures were initiated after germination of sterilized seeds for a week by growing 15–20 seedlings in Erlenmeyer flasks containing basal medium (BM: regular MS medium (Murashige and Skoog, 1962) with B5 vitamins, Gamborg et al., 1968) for 15–20 days. Roots were excised from seedlings, dissected and cultured in CM (callus medium, BM Figure 1. Establishment of cell suspension from root explants and seedlings of Arabidopsis thaliana for co-cultivation with Agrobacterium. (a) Proliferating cells on root explants after 7 days of culture in liquid CM. (b) Cell proliferation in the hypocotyl of seedlings in CM. (c) Cell suspension obtained after four sub-cultures (35 days) and filtration through a sieve of 250 µm. (d) Scanning electromicrograph showing Agrobacterium attachment to the surface of cells in suspension 5 h after co-cultivation. Bars: (a) 0.25 mm; (b) 0.05 mm; (c) 25 µm; (d) 5 µm. supplemented with auxins and cytokinin to promote cell division) in the dark for 15–20 days. Cell proliferation was initiated at the sites of lateral root initials, and more extensively, in cortical tissues of roots (Figure 1a). When seeds were directly germinated in CM, cell proliferation was also inducible in the hypocotyls (Figure 1b). Proliferating cell clumps released in the medium were separated from explants (which were further cultured to raise more material) using a sieve with a pore size of 850 µm, concentrated, and propagated for 5 weeks by weekly sub-culturing. Finally, the cultures were filtrated through a sieve of 250 µm pore size to obtain quickly cycling fine cell suspensions (Figure 1c). Combined application of 2,4-D (2,4-dichlorophenoxyacetic acid) and IAA (indole-3-acetic acid) was found to promote faster cell division in most Arabidopsis ecotypes than 2,4-D alone. The size of cell clumps obtained with 2,4D and IAA ranged between 60 and 100 µm, whereas the diameter of cell clumps with 2,4-D alone reached only 15– 30 µm during the first 3 weeks of culture. Cell proliferation was also induced by treatment of root explants of RLD ecotype with NAA (3.0 mg l–1 α-naphthaleneacetic acid) and yielded higher regeneration rates in comparison with © Blackwell Science Ltd, The Plant Journal, (1998), 13, 707–716 Gene identification with sequenced T-DNA tags 709 Figure 2. Transformation of Arabidopsis cell suspension with Agrobacterium. (A) Histochemical GUS staining of Arabidopsis Col-1 cell suspension 3 days after co-cultivation with Agrobacterium carrying the vector pPCV6NFGUS. (B) Counting GUS-stained cell clumps on a grid 21 days after co-cultivation with Agrobacterium carrying pPCV6NFGUS. (C) Plate (a): Arabidopsis cell suspension co-cultivated with Agrobacterium GV3101 (pMP90RK) on CM containing hygromycin; plate (b): Arabidopsis cell suspension co-cultivated with Agrobacterium GV3101 (pMP90RK) carrying pPCV6NFGUS on CM with hygromycin; plate (c): induction of greening and shoot regeneration from transformed cell suspensions 18 days after plating on RM containing hygromycin. The size of bar corresponds to 0.5 mm in (A) and (B), and to 2.0 cm in (C). application of 2,4-D as sole auxin. Following an amplification phase of 5 weeks, all cell cultures initiated from Arabidopsis ecotypes Col-1, Col-5, RLD1 and WS2 were stabilized and showed a regular threefold increase in biomass during the subculture periods. Maintenance of a constant cell density (i.e. logarithmic growth) by regular sub-culturing was found to be essential, because high density (i.e. stationary) cultures formed larger cell clumps. The average size of microcalli was therefore controlled by sieving the cell cultures through a 250 µm mesh after every second subculture. Experimental procedures). Bacterial attachment to plant cells (monitored by scanning electron microscopy, Figure 1d) occurred within 5–7 h and led to formation of long cellulose fibrils and pronounced clumping during coculture for 48 h. Bacterial growth was arrested by claforan and tricarcillin-clavunic acid (both at 150 mg l–1) to allow the plant cell cultures to proliferate without observable growth retardation until the end of the sub-culture period of 7 days. The efficiency of Agrobacterium transformation was determined using the binary T-DNA vector pPCV6NFGUS, which was constructed by inserting a CaMV 35S promoterdriven uidA reporter gene, containing an intron (Vancanneyt et al., 1990), as a HindIII fragment into the XbaI site of gene fusion vector pPCV6NFHyg (Figure 3, Koncz et al., 1989). In addition to histological detection of the T-DNA-encoded β-glucuronidase (GUS) reporter exclusively in plant cells, T-DNA tags in plant genes were also identified by transformation with pPCV6NFGUS. This vector (as well as its precursor plasmid pPCV6NFHyg) carried a promoterless neomycin phosphotransferase (aph(3 )II) reporter gene linked to the right T-DNA border. Translational aph(3 )II gene fusions resulting from T-DNA integration into plant genes could thus be scored by selecting for kanamycin-resistant transformed calli. After Agrobacterium co-cultivation, aliquots of infected cell suspensions were plated on grids and stained with X-gluc (Figure 2A,B; Jefferson, 1987) to determine the frequency of cells displaying GUS activity. As summarized for the Arabidopsis ecotypes Col-1 and RLD1 in Table 1, the frequency of GUSexpressing cells decreased gradually from 93–95% to a stable value of about 50% between days 2 and 14 after cocultivation, indicating that about half of the infected cells were stably transformed by T-DNA. Because not all cells showed GUS activity within the cell clumps, selection was applied against the untransformed cells using the T-DNAencoded hygromycin resistance marker. As expected, about 50% of microcalli formed hygromycin-resistant colonies, whereas control cells co-cultivated with an Agrobacterium strain (GV3101 pMP90RK) lacking the vector pPCV6NFGUS turned brown and did not yield any viable colonies on hygromycin-containing CM (Figure 2C). Selection for kanamycin (50 or 100 mg l–1) resistance after transformation with pPCV6NFGUS or pPCV6NFHyg yielded about 4–5 times fewer colonies in comparison with hygromycin selection (data not shown). This was in accordance with previous results showing that the average frequency of translational aph(3 )II gene fusions induced by T-DNA integration into plant genes ranges between 15 and 20% in Arabidopsis (Koncz et al., 1989). Agrobacterium transformation of cell suspensions Plant regeneration To transform the cells, Agrobacterium was added to the suspension cultures immediately after sub-culturing (see For high-frequency plant regeneration, only cultures that had undergone no more than 10–15 sub-culture periods © Blackwell Science Ltd, The Plant Journal, (1998), 13, 707–716 710 Jaideep Mathur et al. Table 1. Agrobacterium transformation and regeneration of Arabidopsis cell cultures Days after cocultivation GUS-stained microcalli (%) Arabidopsis ecotypes Col-1 RLD 2 3 4 5 6 7 14 21 98.38 84.14 69.56 66.81 68.56 68.76 52.10 52.98 0.90 5.08 3.28 5.58 8.87 4.34 5.67 10.43 Diameter of microcalli (µm) 100 250 500 850 850 Transformed colonies (%) 56.00 0.70 85.95 1.76 75.45 0.91 45.55 0.49 33.45 3.18 95.46 94.72 87.54 87.23 80.86 75.94 51.10 57.00 0.41 1.96 9.53 5.19 13.08 12.57 1.74 9.94 Regenerating colonies (%) 11.19 0.39 15.92 1.79 51.15 0.96 81.30 4.30 93.17 2.37 Upper section: Frequency of GUS-expressing cell clumps following co-cultivation with Agrobacterium GV3101 (pMP90RK) carrying pPCV6NFGUS. Lower section: Correlation between the size of microcalli and frequencies of Agrobacterium transformation and plant regeneration. Frequency of GUS-stained cell clumps was scored 5 days after co-cultivation with Agrobacterium, whereas regeneration rate was observed 35 days after plating the microcalli on RM containing hygromycin. Standard deviation of the data ( ) resulted from three independent experiments. proved to be suitable. The age of suspension cultures grown in the presence of 2,4-D dramatically affected both ploidy level and fertility of regenerated plants. From cell suspensions passing more than 40–50 sub-cultures, practically no fertile plants could be regenerated. Therefore, a careful standardization of the transformation protocol proved to be essential for obtaining continuously regenerating transformed callus and shoot cultures. Plant regeneration was induced 7–14 days after cocultivation with Agrobacterium by sub-culturing the cells in RM (regeneration medium, i.e. BM with 2.0 mg l–1 isopentenyl adenosine riboside (IPAR) and 0.05 mg l–1 NAA). Some ecotypes, such as RLD1 and Col-5, also showed regeneration in liquid RM. For clonal propagation, the cells were plated on solid RM containing hygromycin and antibiotics to control Agrobacterium. When kanamycin selection was applied, kanamycin-resistant calli were transferred after 3–4 sub-cultures to kanamycin-free medium to ensure high-frequency shoot regeneration. Transformed calli started to green (Figure 2C) within 15–20 days and yielded a mass of regenerating shoots after 30–40 days. Shoot explants were transferred to 0.5 BM with 0.5% sucrose in test tubes and produced a seed set, even without rooting, within 3–4 weeks. To optimize the plant regeneration protocol, the transformed cell suspensions were either directly plated on RM, or embedded in gelrite layered on the surface of RM plates. Both plating techniques resulted in similar regeneration rates, but embedding in gelrite caused a delay of 7–14 days in shoot formation. Optimal (over 90%) regeneration frequencies were achieved when microcalli with an average size of 850 µm were plated on RM. In contrast, the highest transformation rates were obtained by Agrobacterium infection of microcalli with an average diameter of 250 µm. Therefore, to secure an optimal transformation efficiency, a size selection was carried out by sieving the suspensions through a mesh of 250 µm before co-cultivation with Agrobacterium, and the cultures were only plated on RM when the diameter of transformed microcalli reached about 850 µm (Table 1). This transformation protocol yielded a practically unlimited supply of transformed calli in each co-cultivation experiment. Because most transformed microcalli were capable of regenerating fertile plants, the yield of transgenic plants was only limited by the effort invested in plant propagation before the regeneration capability of cultures declined. A small proportion of plants obtained with pPCV6NFHyg transformation of Arabidopsis Col-1 was exploited to test the feasibility and efficiency of genome sequencing using random T-DNA tags. Gene identification with sequenced T-DNA tags To combine reverse genetics with genome sequencing, a protocol simplifying the rescue and sequencing of T-DNAtagged chromosomal DNA segments was developed (see Experimental procedures). To isolate both left and right insert junctions of a T-DNA tag, plant DNAs are usually digested with an enzyme which does not cleave within the T-DNA and self-ligated as described by Koncz et al., 1989, 1994). Plant DNA fragments entrapped by self-circularization between the T-DNA borders are isolated either by plasmid rescue (with the help of a plasmid replicon and a bacterial selectable marker carried by the T-DNA), or by PCR amplification and sequenced with primers facing the T-DNA ends. To double the output of genome sequencing, the plant DNA may also be digested with an enzyme which cleaves within the T-DNA tag. Following self-circularization, the plant DNA is thus ligated to known internal T-DNA sequences through an endonuclease cleavage site which may also serve as an RFLP marker. Following isolation by plasmid rescue or inverse PCR, the circularized plant DNA fragments can be bidirectionally sequenced using primers annealing to the left or right T-DNA arms. In addition to finding T-DNA-tagged genes, this sequencing method also allows the identification of plant genes that are located distantly from the ends of T-DNA tags. When two enzymes © Blackwell Science Ltd, The Plant Journal, (1998), 13, 707–716 Gene identification with sequenced T-DNA tags 711 Figure 3. Genome sequencing with T-DNA tags. A schematic map of pPCV6NFHyg T-DNA is shown at the top. To isolate the left T-DNA arm by plasmid rescue, the plant DNA is digested with EcoRI (R) or XbaI (X) and self-ligated before transformation into E. coli by electroporation. Plant DNA segments linked to the left T-DNA arm in the rescued plasmids are sequenced using primers lb2/lb4 at the left T-DNA 25 bp border (LB) and primers pBR and PC3 located upstream of the EcoRI and XbaI cleavage sites, respectively, within the T-DNA. The right T-DNA arm in junction with plant DNA sequences was similarly self-ligated after digestion with EcoRI or XbaI. The ligated plant DNA was linearized by SmaI (Sm) or SphI (Sp) digestion, and used as template to amplify the plant DNA segment flanking the right T-DNA 25 bp border (RB) by long-range inverse PCR using primers at the right T-DNA border (Km1/Km2), as well as upstream of the EcoRI (EH1/EH2) and XbaI (XH1/XH2) sites within the T-DNA. The PCR products were gel-purified and sequenced with anchored primers Km1 and EH1 or XH1. Alternatively, the PCR fragments were digested with BamHI (B) and EcoRI (R) or XbaI (X) and subcloned in pBluescript for sequencing. The code of sequenced T-DNA tags (left corner, Table 2) identifies the transformed plant line (e.g. 612), the enzyme used for isolation of a particular T-DNA tag (e.g. X1 T-DNA insert no. 1 isolated with XbaI), and the primer used for sequencing (e.g. lb4, which also indicates whether the template was a plasmid or a PCR fragment). Abbreviations: LB and RB, left and right T-DNA 25 bp border repeats; pg5, promoter of T-DNA gene 5; oripBR, pBR322 replication origin; ApR, ampicillin resistance gene; pAg4, polyadenylation sequence of T-DNA gene 4; hpt, hygromycin phosphotransferase gene; pnos, promoter of the nopaline synthase gene; pAocs, polyadenylation sequence of the octopine synthase gene; aph(3 )II, neomycin/kanamycin phosphotransferase gene. are used for rescuing the T-DNA insert junctions, partial sequencing of up to six DNA segments (e.g. each with an average length of 450–650 bp, see below) may identify genes within a chromosomal locus of 4–10 kb. In addition, sequenced T-DNA tags provide RFLP/PCR and dominant selectable markers (e.g. hygromycin/kanamycin resistance) for genetic mapping as well as probes for physical mapping with yeast and bacterial artificial chromosome (YAC and BAC), or P1 phage clones. To test the efficiency of this technique, DNA was isolated from F2 progeny of 21 transgenic Arabidopsis (Col-1) plants carrying the T-DNA of pPCV6NFHyg gene fusion vector (Figure 3, the sequence of pPCV6NFHyg T-DNA is available from the Nottingham Arabidopsis Stock Center). Plant DNAs were digested independently with EcoRI or XbaI to © Blackwell Science Ltd, The Plant Journal, (1998), 13, 707–716 cleave the T-DNA into two parts, and with HindIII which had no cleavage site within the T-DNA. Aliquots (1.0 µg) of digested DNAs were self-circularized by ligation. Half of the DNA samples (0.5 µg) were used for transformation of E. coli by electroporation to isolate plant DNA fragments linked to the left T-DNA arm carrying a pBR322 replicon and an ampicillin resistance gene. The rescued plasmids were sequenced with primers hybrizing to the ends of the left T-DNA arm (Figure 3, primers lb2/4, pBR or PC3). The second half of DNA samples was digested with SmaI or SphI to cleave the right T-DNA arm and thereby linearize the self-circularized fragments. These DNAs served as templates for long-range PCR and sequencing with anchored T-DNA primers (Figure 3, primers Km1/2, EH1/2, Xh1/2) facing the termini of plant DNA fragments. Similarly, 712 Jaideep Mathur et al. Table 2. BLAST scores with sequenced T-DNA tags Table 2. Cont. T-DNA tagged sequence Genbank accession number Similarity to [accession number] T-DNA tagged sequence Genbank accession number Similarity to [accession number] 0612xa.lb4 AF005781 AF005782 2462eb.lb4 2462eb.pbr AF005822 AF005823 0612xa.pcr AF005783 0612xb.pcr 0759xa.lb4 0759xa.pcr AF005784 AF005785 AF005786 AF005787 2734ea.lb4 2734ea.pbr 2761xa.lb4 AF005824 AF005825 AF005826 AF005827 0864xc.lb2 AF005788 0864xc.pcr AF005789 0864xd.lb4 AF005790 None Amine oxidase (coppercontaining) [sp. P46881] Copper amine oxidase [gb D38508] None None None Arabidopsis ribosomal DNA spacer (#3) [emb X52636] Arabidopsis repeat region (clone 164 A) [emb X92080] Arabidopsis CER3-like gene [emb X95961] Human Golgi protein LDLC [pir A53542] None Yeast MSP1 protein (TAT-binding homolog) [sp. P28737] Arabidopsis cDNA [gb T45807], ASK1 kinase [sp. P43291] Arabidopsis cDNA, Fe (II) transporter [gb N37871] Zea mays pollen specific mRNA [gb I16762] Arabidopsis farnesyl diphosphate synthase 1 [gb L46367] None None None None None None None Arabidopsis receptor-like protein kinase [gb M84660] None None Arabidopsis cDNA clone 204G13T7 [gb H77156] None None None None Arabidopsis cDNA clone H6B5T7 [gb W43818] None Brassica mRNA for chitinase [emb X61488] None Linum mRNA for fis1 protein [emb X86733] None None Arabidopsis 25S-18S ribosomal DNA spacer [emb X15550] None Tomato Pto kinase [gb U59316] None Yeast probable calcium-binding protein [sp. P36132] None None None Arabidopsis mRNA for J-domain protein [emb Z49238] Arabidopsis mRNA for J-domain protein [emb Z49238] Arabidopsis mRNA for J-domain protein [emb Z49238] None Arabidopsis mRNA for RNA helicase [emb X97970] Arabidopsis mRNA for RNA helicase [emb X97970] Arabidopsis 81 kb genomic sequence [emb X98130] Arabidopsis mRNA for unknown ORF02 [emb X97484] Arabidopsis cDNA clone G5C10T7 [gb N96129] None None Arabidopsis cyc3b mRNA [emb Z31402] None None Zea mays mRNA for porin [emb X73429] 0864xd.pcr AF005791 084xdea.pbr AF005792 0941e1.lb4 AF005793 0941e1.pbr AF005794 1562x2.lb4 AF005795 1772x3.lb4 AF005796 1851xa.lb4 1851xa.pcr 1851xb.lb4 1851xb.pcr 1884xa.lb4 1884xa.pcr 2031e2.lb4 2031e2.pbr AF005797 AF005798 AF005799 AF005800 AF005801 AF005802 AF005803 AF005804 2046x4.lb4 2051e3.lb4 2051e3.pbr AF005805 AF005806 AF005807 2276xa.lb4 2276xa.pcr 2322e10.lb4 2322e10.pbr AF005808 AF005809 AF005810 AF005811 AF005812 2322e6.lb4 2322e6.pbr AF005813 AF005814 2454ec.lb4 2454ec.pbr AF005815 AF005816 2454xa.lb4 AF005817 2454xa.pcr AF005818 2454xaeb.pbr AF005819 2462ea.lb4 2462ea.pbr AF005820 AF005821 2761xapcr.revAF005828 2761xc.lb4 AF005829 2761xc.pcr AF005830 2981xae1.pbr AF005839 2981xa.pcr AF005840 2981xa.lb4 AF005841 AF005831 3242e1.lb4 AF005832 3242e1.pbr 3322e1.lb4 AF005833 AF005834 AF005835 3322e1.pbr Okae6.lb4 Okae6.pbr AF005836 AF005837 AF005838 Data libraries: gb, Genbank, sp., Swissprot, emb, EMBL. Underlined: T-DNA tags in genes. half of the HindIII-digested and self-circularized DNAs were transformed into E. coli to rescue plasmids, and the other half of the samples were linearized by SmaI and subjected to long-range PCR. The plasmid and PCR DNA templates were sequenced with primers facing the left and right T-DNA ends (Figure 3, Lb2/4 and Km1/2, respectively). Alternatively, the PCR fragments were subcloned in pBluescript before sequencing (see Experimental procedures). The T-DNA rescue protocol was controlled by physical mapping of T-DNA insertions using Southern hybridization of plant DNAs (data not shown). Because about 40% of the T-DNA insertions were found in tandem repeats, the rescued plasmid clones and PCR fragments were always hybridized with T-DNA end-probes derived from the promoter region of gene 5 and the aph(3 )II gene of pPCV6NFHyg (Figure 3) to discard templates carrying selfjoined T-DNA borders without plant DNA. Due to preferential use of the right 25 bp border sequence, about 10% of © Blackwell Science Ltd, The Plant Journal, (1998), 13, 707–716 Gene identification with sequenced T-DNA tags 713 the T-DNA inserts also contained an intact left border still linked to vector sequences located outside the T-DNA. These T-DNA inserts were also discarded after hybridization with a probe from the pPCV6NFHyg vector backbone (i.e. RK2 sequences; Koncz et al., 1994) because they usually contained only short segments of plant DNA. In the remaining templates, the ends of plant DNA segments were automatically sequenced covering an average of 450– 650 bp per reaction. The availability of overlapping EcoRI and XbaI clones facilitated the assembly of longer sequence segments, albeit at the cost of redundant sequencing. Thirty-four T-DNA-tagged chromosomal DNA fragments yielded total sequence information of 37 552 bp, which was subjected to BLASTX and BLASTN homology searches as described previously for the analysis of ESTs (Table 2; Hofte ¨ et al., 1993; Newman et al., 1994). Sequencing of 32 plant DNA templates with the T-DNA left border primers (Figure 3, lb2 and lb4) identified nine T-DNA-tagged genes. At a distance from the T-DNA tags, a significant sequence similarity to 16 Genbank accessions (genes, ESTs, proteins and BAC clones) was detected by sequencing of 29 templates with primers reading through endonuclease cleavage sites (Figure 3, primer pBR for EcoRI and primer PC3 for XbaI) which were used for self-circularization before plasmid rescue and LR-iPCR. Terminal sequences from 13 T-DNA-tagged plant DNA fragments shorter than 0.5 kb found no significant homology in the database. Initial prospectives of genome sequencing by T-DNA tags As expected from the comparison of estimated gene number and available unique ESTs in Arabidopsis, about onethird of sequenced T-DNA tags detected known Arabidopsis genes and ESTs (Table 2). Four of nine T-DNA inserts in genes were found in the promoter region of Arabidopsis genes encoding the ASK1 kinase (Swissprot P43291), cyclin 3b (EMBL accession number Z31402), ORF02 (EMBL accession number X97484) and EST mRNA (EMBL accession number N96129), respectively, whereas two tags were identified in the coding regions of a J-domain protein (EMBL accession number Z49238) and a farnesyl diphosphate synthase (FPS1, Genbank accession number L46367) gene. Furthermore, three T-DNA tags were localized in novel genes coding for orthologs of a copper amine oxidase, a peripheral Golgi-associated low density lipoprotein (LDLC, Genbank accession number U61947) and a pollen-specific maize mRNA (Genbank accession number I16762). From 16 sequences located at a distance from the T-DNA tags, only two showed nearly 100% identity with Arabidopsis ESTs, whereas a segment of identity with a common repeat element of 25S–18S ribosomal spacers was observed in another two sequences. Significant BLAST scores, but no identity, were detected between 12 sequenced Arabidopsis DNA segments and database © Blackwell Science Ltd, The Plant Journal, (1998), 13, 707–716 accessions from plants and other organisms (Table 2). In summary, 62% of sequenced T-DNA tags scored significant similarity in the database, and half of these proved to be novel in Arabidopsis. These data show that even a small set of sequenced TDNA tags may yield a significant number of new genes which remained undetected by the EST sequencing project, as well as gene mutations that could not be identified so far using the available genetic and molecular methods. Further studies of some of these mutations may thus unravel the function of cyclin 3b, ASK1 kinase, LDLC or Jdomain proteins in basic cellular processes. Although both plasmid rescue and LR-iPCR technologies proved to be equally efficient for the isolation of plant DNA junctions of any T-DNA tag (i.e. the frequency of obtaining no product was less than 1% using both methods), currently only the LR-iPCR method seems to be well adaptable to full automation. Our preliminary data indicate that, if appropriate controls were used to filter out PCR fragments resulting from amplification of tandem T-DNA insert junctions, up to 98% of PCR-amplified DNA fragments could yield suitable plant DNA sequence information, whereas the rest detect rare T-DNA insert rearrangements. Because technologies applied in current genome sequencing approaches are fully compatible with the PCR-based TDNA end-sequencing approach (Huang and Miao, 1997; Miklos and Rubin, 1996), sequenced T-DNA tags may provide not only useful tools to extend the ‘genes galore’ accumulated by ESTs (Cooke et al., 1996; Hofte et al., 1993; ¨ Newman et al., 1994; Sasaki et al., 1994), but also to elucidate the function of plant genes by reverse genetics. Experimental procedures Culture media One litre of basal medium (BM, pH 5.8) contained 4.33 g of MS inorganic salts (Murashige and Skoog, 1962; Sigma M-5524), 3.0% sucrose and 2 B5 vitamins (Gamborg et al., 1968), including 2 mg nicotinic acid, 2 mg pyridoxin HCl, 20 mg thiamine–HCl, and 200 mg myo-inositol. Gelrite 0.2% (Phytagel, Sigma) was used as gelling agent in BM, CM and RM. Seeds were germinated in 0.5 MS containing half the concentration of MS inorganic salts, 0.5% sucrose and 0.8% agar (Koncz et al., 1994). Root cultures were maintained in callus medium (CM) containing 0.5 mg 2,4dichlorophenoxyacetic acid (2,4-D), 2.0 mg indole-3-acetic acid (IAA), and 0.5 mg 6-(γ,γ- dimethylallylamino)-purine riboside (IPAR) in 1 litre of BM. Shoots were induced in regeneration medium (RM), consisting of BM with 2.0 mg l–1 IPAR and 0.05 mg l–1 α-naphtaleneacetic acid (NAA), and transferred to test tubes containing 0.5 MS solidified with gelrite to obtain seeds. Agrobacterium media were as described by Koncz et al., 1994. Establishment and transformation of Arabidopsis cell suspensions About 5000 seeds (0.1 g) of each of Arabidopsis ecotypes Col-1, Col-5 (also known as C24), WS2 and RLD1 (Ohio Arabidopsis 714 Jaideep Mathur et al. Biological Resource Center, seeds@genes ys.cps. were surface sterilized in Eppendorf tubes with 1 ml of 5% calcium hypochlorite containing 0.02% Triton X-100 (or 10% (v/v) sodium hypochlorite, 0.1% Triton X-100) for 15 min, pelleted by centrifugation, washed five times with 1 ml of sterile water, dried, and germinated on 0.5 MS agar plates exposed to a 16 h light/8 h dark period at 25°C. Fifteen to twenty one-week-old seedlings were placed in an Erlenmeyer flask containing 35 ml of liquid BM and cultured on a rotary shaker (120 rev min–1) for 15–20 days under similar conditions. Roots of seedlings (™ 3 g) harvested from a flask were dissected, cut in pieces (™ 2 mm), transferred into 50 ml of liquid CM, and cultured in the dark for 15–21 days. Alternatively, approximately 1000 seeds were directly germinated in the dark in 50 ml of CM to initiate cell suspensions from seedling-derived calli in a similar fashion. Cell clumps released from root or seedling explants were separated by filtration through a sieve of 850 µm (or 500 µm). Root explants were returned to CM to generate more cells in suspension, and the filtered cells were pelleted, suspended in 30–50 ml of fresh CM, and allowed to proliferate for 7 days of regular sub-culture. During the first two sub-cultures, cells from a single flask were dispensed into two flasks of 250 ml, pelleted, and after removing the excess of medium were resuspended in 50 ml of fresh CM. During subsequent subcultures, 50 ml CM was added to each flask, and the resulting 100 ml of culture was equally dispensed into three flasks. Using this strategy, a fine cell suspension was obtained from each Arabidopsis ecotype after 5 weeks. The size of cell clumps was controlled by filtering the cultures through a sieve of 250 µm at every second sub-culture. Logarithmic growth of cultures was controlled by monitoring the increase of fresh weight as described (Street, 1973). An aliquot of 1.5 ml of Agrobacterium culture (OD550: 1.0) grown as described (Koncz et al., 1994) was pelleted in an Eppendorf tube by centrifugation, resuspended in 1.5 ml CM, and added to 35 ml of cell suspension at the time of sub-culturing. The co-cultivation was carried out for 48 h, then claforan and tricarcillin-clavulanic acid (150 mg l–1 of each) were added to kill Agrobacterium. The cells were harvested 5 days later, and either subcultured for another 7 days, or directly plated on RM. Cells from Agrobacterium infected cultures were collected in a 50 ml Flacon tube by centrifugation at 1000 g for 3 min, washed twice with 3.0% sucrose, and counted on a 2 cm2 grid. Cells suspended in liquid RM at a density of 103 cells ml–1 were plated on RM with gelrite containing antibiotics for selection of transformed cells (hygromycin 15 mg l–1 or kanamycin 100 mg ml–1) and to control Agrobacterium. Alternatively, cells were suspended in 0.2% gelrite dissolved in 3.0% sucrose solution (pH 5.8), and layered on the top of RM plates. The regenerating calli were cultured under continuous light at 22°C until green colonies appeared, which were counted after 35 days by placing a 2 cm2 grid on the Petri dishes. Colonies in at least three randomly chosen squares from ten plates per experiment were counted. Regenerated shoots were individually transferred to test tubes (length: 20 cm; diameter: 3 cm) containing 10–15 ml of 0.5 MS with gelrite, closed with loose cotton plugs, and cultured for 6–7 weeks to set seeds at 25°C using a 16 h light/8 h dark period. Agrobacterium vectors and histochemical GUS-staining Plasmid pPCV6NFGUS was constructed by cloning of a HindIII fragment of p35SGUSINT from pBIN19 (Vancanneyt et al., 1990) into the XbaI site of pPCV6NFHyg (Koncz et al., 1989) after fillingin the ends of DNA fragments with T4 DNA polymerase to regenerate the XbaI site (Sambrook et al., 1989). In addition to known features of pPCV6NFHyg (Figure 3; Koncz et al., 1989), pPCV6NFGUS carried a uidA (β-glucuronidase, GUS) reporter gene with a portable intron from the potato ST-LS1 gene under the control of the 35S RNA promoter of cauliflower mosaic virus (CaMV 35S). This uidA reporter gene therefore yielded no detectable GUS activity in Agrobacterium (Vancanneyt et al., 1990). Transfer of pPCV6NFGUS and pPCV6NFHyg from E. coli to Agrobacterium GV3101 (pMP90RK), and the use of Agrobacterium strains for plant transformation were as described by Koncz et al., 1994. To monitor the transformation rates with pPCV6NFGUS, aliquots of cell cultures (0.5 ml) sampled at various times after Agrobacterium co-cultivation were stained with X-gluc (1 mg ml– 1 X-gluc in 50 mM sodium phosphate buffer (pH 7.0) containing 0.5 mM potassium ferricyanide and 0.5 mM potassium ferrocyanide) for 6 h, then plated on grids to count the number of GUS-expressing microcalli in at least three squares of 1 cm2 using ten samples from each experiment. As control, changes in GUS activity were also assayed after co-cultivation by fluorimetric enzyme assays (Jefferson, 1987). Isolation of T-DNA tagged chromosomal DNA fragments by plasmid rescue Plants grown aseptically in 0.5 MS containing hygromycin (15 mg ml–1) were harvested prior to flowering to purify DNA on a large scale using CsCl-banding or on a mini-scale with or without CTAB precipitation (Dellaporta et al., 1983; Taylor and Powell, 1983). Other methods yielding high molecular weight genomic DNA free of protein and RNA (e.g. Souer et al., 1995; Krysan et al., 1996) were also found to be applicable. An aliquot of 5 µg of total plant DNA was digested with 100 U of restriction endonucleases EcoRI, XbaI or HindIII for at least 6 h at 37°C in a volume of 200 µl of enzyme buffer (Sambrook et al., 1989). After testing 10 µl aliquots by agarose gel electrophoresis, the samples were phenol/ chloroform extracted and precipitated with i-propanol (Sambrook et al., 1989). To isolate self-circularized plant fragments by T-DNA-mediated plasmid rescue, 1 µg of digested DNA samples were ligated in a volume of 200 µl ligase buffer (Sambrook et al., 1989) containing 10% polyethylene glycol (PEG 4000) at 15°C for 8 h. The samples were phenol/chloroform extracted, precipitated by i-propanol, washed twice by 70% ethanol, dried, and dissolved in water before electroporation into E. coli DH1 cells (Zabarovsky and Weinberg, 1990) using a BioRad Gene Pulser (settings 2.5 kV, pulse controller 200 Ω, (capacitance extender 250 µFD for a 0.2 ml BioRad cuvette). Plasmids carrying a segment of a tandem T-DNA repeat or intact left T-DNA border were identified by colony hybridization (Sambrook et al., 1989) using 32P-labelled probes of XbaI–BamHI and XhoI–KpnI fragments from pPCV6NFHyg which contained sequences from the right border-linked aph(3 )II gene and vector backbone outside of the T-DNA, respectively (Koncz et al., 1994). All rescued plasmids were subjected to physical mapping, and sequenced using a PCR kit (ABI Prism- Dye Terminator Cycle Sequencing) and an automatic sequencer (ABI 377). The positions of sequencing primers lb2 (5 -GACCCTTACCGCTTTAGTTCCGTAGCTAGCACTTC-3 ) and lb4 (5 -AGAGGTATAACTGGTAGTATGAG-3 ) facing the left T-DNA border, as well as that of primer pBR (5 -CCTATAAAAATAGGCGTATCACGAGGCCC-3 ) at the EcoRI site and primer PC3 (5 -CCTTGCGCCCTGAGTGCTTGCGGCAGC3 ) at the XbaI site are shown in Figure 3. HindIII fragments of plant DNA rescued as plasmids by self-circularization were sequenced with the lb2/lb4 and Km1/Km2 primers (see below). © Blackwell Science Ltd, The Plant Journal, (1998), 13, 707–716 Gene identification with sequenced T-DNA tags 715 Amplification of T-DNA tagged plant DNA fragments by LR-iPCR Plant DNA sequences flanking the right arm of pPCV6NFHyg TDNA were isolated by long-range inverse PCR (LR-iPCR). After digestion with EcoRI or XbaI and self-circularization by ligation, 0.5 µg of plant DNA was digested with either SmaI or SphI to linearize the DNA templates by cleaving the right T-DNA arm (see Figure 3). These DNA samples were subjected to PCR amplification using primer Km1 (5 -CAAAGCGAACCACCAGCTTACCCGTCCATCGGC-3 ) facing the T-DNA right border, and either primer EH1 (5 -TCCTGCGGGTAAATAGCTGCGCCGATGG-3 ) at the EcoRI site, or primer XH1 (5 -TAGCTCAGATCCTTACCGCCGGTTTCGG-3 ) at the XbaI site. PCR reactions were performed in 50 µl using either Elongase PCR (BRL) or LA-PCR (Takara Shuzo Co.) kits as recommended by the suppliers. DNA samples were denatured at 95°C for 2 min, and amplified using 35 cycles (94°C for 30 sec, 65°C for 30 sec, 68°C for 8 min) followed by elongation at 68°C for 10 min. The PCR products were resolved on agarose gels and isolated (Sambrook et al., 1989). When no amplified DNA fragment was detected, a second PCR amplification was performed using 1 µl from a 500-fold diluted first PCR reaction mixture in combination with the nested primers Km2 (5 -CAGTCATAGCCGAATAGCCTCTCCACCC-3 ), and EH2 (5 -CGTTATGTTTATCGGCACTTTGCATCGG3 ), or XH2 (5 -CCGTTCAATTTACTGATTGTCCAAGCTC-3 ). The isolated PCR fragments were either used directly as templates for sequencing, or digested with BamHI and EcoRI or XbaI (see Figure 3) for subcloning in pBluescript (Stratagene) before sequencing. Sequence analyses were performed using the GCG and BLAST computer program packages as described for Genbank database searches with ESTs (Newman et al., 1994). 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