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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
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.
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 email@example.com). © 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
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
(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 (%)
Col-1 RLD 2
10.43 Diameter of microcalli
33.45 3.18 95.46
93.17 2.37 Upper section: Frequency of GUS-expressing cell clumps
following co-cultivation with Agrobacterium GV3101 (pMP90RK)
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
number Similarity to [accession number] T-DNA
number Similarity to [accession number] 0612ea.lb
AF005823 0612xa.pcr AF005783 0612xb.pcr
AF005827 0864xc.lb2 AF005788 0864xc.pcr AF005789 0864xd.lb4 AF005790 None
Amine oxidase (coppercontaining) [sp. P46881]
Copper amine oxidase
Arabidopsis ribosomal DNA
spacer (#3) [emb X52636]
Arabidopsis repeat region (clone
164 A) [emb X92080]
Arabidopsis CER3-like gene
Human Golgi protein LDLC
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
Arabidopsis farnesyl diphosphate
synthase 1 [gb L46367]
Arabidopsis receptor-like protein
kinase [gb M84660]
Arabidopsis cDNA clone
204G13T7 [gb H77156]
Arabidopsis cDNA clone H6B5T7
Brassica mRNA for chitinase
Linum mRNA for ﬁs1 protein
Arabidopsis 25S-18S ribosomal
DNA spacer [emb X15550]
Tomato Pto kinase [gb U59316] None
Yeast probable calcium-binding
protein [sp. P36132]
Arabidopsis mRNA for J-domain
protein [emb Z49238]
Arabidopsis mRNA for J-domain
protein [emb Z49238]
Arabidopsis mRNA for J-domain
protein [emb Z49238]
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
Arabidopsis cyc3b mRNA
Zea mays mRNA for porin
[emb X73429] 0864xd.pcr AF005791
0941e1.lb4 AF005793 0941e1.pbr AF005794 1562x2.lb4 AF005795 1772x3.lb4 AF005796 1851xa.lb4
AF005816 2454xa.lb4 AF005817
2761xc.lb4 AF005829 2761xc.pcr AF005830
2981xa.pcr AF005840 2981xa.lb4 AF005841 2981pc.km AF005831 3242e1.lb4 AF005832 3242e1.pbr
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.
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
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. msu.edu) 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
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
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). Acknowledgements
This work was supported as part of a joint project between the
Max-Planck Institut (Koln) and the Biological Research Center of
the Hungarian Academy of Sciences (Szeged) by the Deutsche
Forschungsgemeinschaft (DFG), as well as by the European Communities Biotech Programme BIO4-CT95–0183 ‘Arabidopsis insertional mutagenesis for the functional analysis of sequences
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