Note: Descriptions are shown in the official language in which they were submitted.
CA 02286594 1999-10-13
1
PLANTS WITH CONTROLLED SIDE-SHOOT FORMATION
AND/OR ABSCISSION ZONE FORMATION
The present invention relates to nucleotide sequences encoding polypeptides
which are responsible for controlling side-shoot formation and/or petal
formation and/or
abscission zone formation as well as to the polypeptides and amino acid
sequences
encoded by said nucleotide sequences. Furthermore, the present invention
relates to
plants having controlled side-shoot formation and/or petal formation and/or
controlled
formation of abscission zones, wherein the expressible DNA sequence or
fragment or
1o derivative thereof responsible for side-shoot formation and/or petal
formation and/or
abscission zone formation is integrated in a stable manner into the genome of
the plant
cell or the plant tissue. Further, the invention relates to methods for the
production of
plants having controlled side-shoot formation and/or petal formation and/or
controlled
formation of abscission zones, wherein the expressible DNA sequence or
fragment or
derivative thereof responsible for side-shoot formation and/or petal formation
and/or
abscission zone formation is integrated in a stable manner into the genome of
plant cells
or plant tissues and the resulting plant cells or plant tissues are
regenerated to form
plants. Moreover, the invention relates to plants and seed stocks of plants,
which are
obtainable according to the method of the invention.
Technical Background
The performance characteristics of economic and ornamental plants are
considerably determined by their architecture. While the basic structure of a
plant
manifests in the embryonic development, the post-embryonic phase is
characterized by
the activity of apical meristems. Of fundamental importance is the ability of
the shoot
apical meristem (SAM) of higher plants to initiate shoot branches and to
control their
development. As a result, the habit of a plant and thus an essential
performance feature
is characterized by the number, arrangement and developmental intensity of its
side-
shoots. The branching of the shoot may occur terminally as well as laterally.
The
3o terminal branching in which the SAM is separated into two portions mainly
occurs in
lower cormophytes and has been described for only a few flowering plants
(Steeves and
Sussex, 1989, Patterns in Plant Development, 2°a Edition, Cambridge
University Press,
CA 02286594 1999-10-13
2
Cambridge). The lateral branching typical for flowering plants is based on the
formation
of new shoot apical meristems in the leaf axils, which are derived from SAM
cells, the
meristemic character of which remains preserved in contrast to surrounding
cells which
are involved in the development of leaf primordia. In the further course of
development,
a side bud is formed from said residual meristems, which besides some leaf
primordia
contains an apical meristem, the activity of which is subject to the control
by the main
shoot SAM.
The analysis of plant mutants revealed that branching of the shoot system is
controlled by genetic factors. Thus, in tomato (Lycopersicon esculentum) for
example,
1o there have been described a number of mutants, the side-shoot formation of
which is
inhibited in different stages (e.g. blind, blind 2, torosa, lateral
suppressor). A
morphological characterization showed that the production of axil buds is
disturbed in
the tomato mutants blind, blind-2 and torosa (Tucker, 1979, Ann. Bot. 43: 571-
577;
Mapelli and Lombardi, 1982, Plant & Cell Physiol. 23: 751-757). In contrast,
in plants
1 s which are homozygous for recessive lateral suppressor (Is) mutation, the
initiation of
most of the side buds does not occur (Brown, 1955, Rep. Tomato Genetics
Cooperative
5: 6-7). A histological analysis (Malayer and Guard, 1964, Amer. Jour. Bot.
51: 140-
143) shows that cells directly den~ived from SAM in the axils of the leaf
primordia, on
the meristemic activity of which the formation of side shoots is based, are
missing in the
20 lateral suppressor mutant. If a lack of side shoots in all leaf axils
results in a termination
of the shoot axis in the first inflorescence, the transition to floral
development shows
that the ability to establish axil meristems is not completely lost in the
mutant. In the
axil of the leaf primordium established directly before the inflorescence a
meristern
often is established in homozygous is mutants as well. The establishment of
this
25 meristem which is necessary for the sympodial structure of the shoot axis
is often
associated by the formation of a side bud in the axil of the next older leaf.
Following the
transition to the floral phase, the development of the is mutant is
characterized by a
smaller number of flowers per inflorescence (Williams, 1960, Heredity, 14: 285-
296),
the missing establishment of petal primordia (Szymkowiak and Sussex, 1993,
Plant J.,
3o 4: 1-7) and an aberrant number of stamens and carpets (Groot et al., 1994,
Sci. Hort.,
59: 157-162). Furthermore, a reduced fertility in the mutant is observed,
which also
CA 02286594 1999-10-13
3
results in the reduction of yield and which is the reason that the is mutant
did not reach
any significance for yield-oriented cultivation.
A further phenotypic change of the Is mutant relates to the formation of
abscission zones in the flower and fruit stems. While wild type plants have a
region of
5-10 layers of smaller cells, at the distal ends of which the non-pollinated
flower or the
ripe fruit comes off the plant (Roberts et al., 1984, Planta, 160: 159-163),
this abscission
zone is not formed in the Is mutant and during harvest the fruit comes off the
plant
without residues of the fruit stem and sepals.
The observed phenotypic changes are correlated with disorders in the
eqililibria
of particular plant hormones on a physiological level. In comparison with the
wild type,
lower cytokinin concentrations were measured in the shoot tips of Is mutants
(Maldiney
et al., 1986, Physiol. Plant, 68: 426-430; Sossountzov et al., 1988, Planta,
175: 291-
304), while the amounts of (3-indolylacetic acid (IAA)-like compounds as well
as
gibberellic and abscisic acids are markedly increased (Tucker, 1976, New
Phytol., 77:
561-568). Attempts to remedy the deficiencies of the is mutant by introducing
an
isopentenyl transferase gene from Agrobacterium tumefaciens resulted in an
increase of
endogenous cytokinine concentrations, but not in a normalization of the side-
shoot
development (Groot et al., 1995, Plant Growth Regulation, 16, 27-36).
Due to the great interest of breeders in single stem tomato varieties there
have
2o been early efforts to render the is mutant usable for commercial
cultivation. Since the
DNA sequence of the gene (Ls gene) responsible for side-shoot formation and/or
petal
formation and/or abscission zone formation has so far not been known, it was
repeatedly
attempted by genetic methods to separate the desired effects on the side-shoot
formation
from the non-desired effects on fertility and yield. However, up to now none
of these
efforts have been successful.
For the isolation of genes which are only characterized by a mutant phenotype
and their position on the genetic map, the strategies of insertional
mutagenesis and
positional cloning have been preferably used during the past years. The
insertional
mutagenesis uses mutant alleles formed by the insertion of a known sequence
for the
3o isolation of genes which in this manner are labeled on a molecular level.
In plants, the
T-DNA from Agrobacterium tumefaciens (Koncz et al., 1992, Plant Mol. Biol.,
20: 963-
976) as well as transposable elements (Gierl and Saedler, 1992, Plant Mol.
Biol., 19; 39-
CA 02286594 1999-10-13
4
49) were used for insertional mutagenesis (Jones et al., 1994, Science 266:
789-793).
Since the transposable elements Ac and Ds from maize preferentially transpose
to
coupled positions on the same chromosome (Knapp et al., 1994, Mol. Gen.
Genet., 243:
666-673) a transposon mutagenesis is particularly promising when a starting
line is
available in which the transposable element is present in close coupling with
the gene of
interest. Since such a tomato line is not available, a transposon mutagenesis
for the
isolation of the Ls gene is not very promising.
The strategy for positional cloning was developed for the analysis of the
molecular principles of hereditary diseases in mammals and inter alia used for
the
isolation of human genes for Duchenne's muscular dystrophy (Koenig et al.,
1987, Cell,
S0: 509-517), Cystic Fibrosis (Rommens et al., 1989, Science, 245: 1059-1065)
and
Huntington's Disease (Huntington's Disease Research Group, 1993, Cell 72: 971-
983).
Figure 1 schematically illustrates the course of a positional cloning. For
this strategy the
integration of the classical genetic locus into a map of molecular markers is
of
fundamental importance. The use of restriction fragment length polymorphisms
(RFLPs)
as genetic markers (Botstein et al., 1980, Am. J. Hum. Genet., 32: 314-331)
enables the
identification of closely coupled DNA fragments from the environment of the
gene to be
isolated. These fragments subsequently serve as hybridizing probes in Southern
analysis
by means of pulsed field gel electrophoresis (Chu et al., 1986, Science, 234,
1582-1585)
of separated high molecular weight DNA to transform the relative genetic
distance into
an absolute value for the physical distance which has to be bridged by the so-
called
"chromosome walk". Starting with flanking markers as starting points the
environment
of the desired gene is isolated in the form of overlapping DNA fragments.
Depending on
the distance of the flanking markers in the genetic map the DNA fragments are
YAC or
cosmid clones (Burke et al., 1987, Science, 236: 806-812). RFLP maps with high
marker density have been developed by Nam et al., 1989, Plant Cell, 1, 699-
705, and
Tanksley et al., 1992, Genetics, 132: 1141-1160. Grill and Somerville, 1991,
Mol. Gen.
Genet., 226: 484-490, and Martin et al., 1992, Mol. Gen. Genet, 233: 25-32,
describe
the preparation of YAC-libraries.
3o In the classical genetic map of tomato the Ls locus is mapped on the long
arm of
chromosome 7 (Taylor and Rossall, 1982, Planta, 154: 1-5). Schumacher et al.,
1995,
Mol. Gen. Genet, 246: 761-766, describe an integration of the Ls locus into
the RFLP
CA 02286594 1999-10-13
map, wherein the Ls locus was mapped within a 0.8 cM interval near the distal
end of
chromosome 7. Furthermore, Schumacher et al. describe that the Ls locus is
bounded by
the RFLP markers CD61 and CD65. The physical mapping by means of pulsed field
gel
electrophoresis showed that CD61 and CD65 are not more than 375 kb apart from
each
5 other.
With respect to agricultural cultivation the formation of side shoots is not
desired
in many economic plants due to various reasons:
1. Firstly, the young side shoots are "sink" organs (organs of consumption)
and thus reduce the yield of the main shoot. '
2. Highly branched shoot systems often represent a hardly surmountable
obstacle for mechanical treatment (e.g. harvest with machines).
For these reasons there have been early attempts to cultivate varieties
without
side shoots in a conventional manner. This has been successful in individual
economic
plants (e.g. sun flower). However, in many other dicotyledonous economic
plants (e.g.
tomato, cucumber, apple-tree, pear-tree) the single stem would be desirable,
but this has
so far not been realized in efficient culture varieties. Also in
monocotyledonous
economic plants, such as maize and sugar cane, suppression of side shoot
formation is
advantageous and highly desired for commercial use. At present, the single
stem e.g. of
tomato is achieved in green house cultivation common in Central and Northern
Europe
2o by manually removing the side shoots. Since the removal of the side shoots
cannot be
done with machines this is associated with enormous costs. Furthermore, at the
wound
site the plants are very susceptible of infections by pathogens, such as
pathogenic
bacteria, viruses and fungi. Thus, the removal of side shoots contributes to
the spreading
of diseases in green house.
In many ornamental plants, however, the additional formation of side shoots
and
thus an enhanced formation of flowers is desired. enhanced formation of side
shoots is
also highly beneficial in many economic plants, such as potato, coffee or tea
plant. Thus,
there is a need for cost-effective, efficient economic plants and ornamental
plants, in
which the formation of side shoots is increased or suppressed.
3o Inhibition of the formation of abscission zones is of interest in a number
of
plants. Thus, the premature abscission of fruits in citrus plants resulted in
losses of yield
which could be prevented if no abscission zones were formed. Similar results
may be
CA 02286594 1999-10-13
6
found in other fruit species, such as cherry, peach or black currant. Further,
an inhibition
of the formation of abscission zones, e.g. in tomato, is advantageous. If the
abscission
zones are not formed, the fruit comes off the plant during harvest without
residues of the
fruit stem and sepals. This feature is desired when tomatoes are harvested
with machines
and are subsequently processed to products such as tomato puree, since sepals
and fruit
stems deteriorate the quality of the tomato products.
In ornamental plants, an increased formation of abscission zones may be
useful,
since flowers would fall off by themselves after fading and there would be no
need to
remove them manually, such as with many balcony and garden plants. If this
does not
occur, the formation of new flowers is suppressed.
Short Description of the Invention
Isolation and cloning of the Ls gene would offer the possibility to change the
activity of said gene in a targeted manner and thus to suppress or increase
the formation
of side shoots in transgenic plants. Further, one may suppress or increase the
formation
of abscission zones and/or petals by changing the activity of the Ls gene in a
targeted
manner. Accordingly, the object underlying the present invention is to isolate
the Ls
gene or a DNA fragment containing said gene, determine its sequence and
provide a
method for the preparation of transgenic plants in which the activity of the
Ls gene was
2o varied in a targeted manner to suppress or increase the formation of side
shoots and/or
the formation of abscission zones and/or petals.
The object of the present invention is solved by providing the nucleotide
sequences according to SEQ ID NO: 1, 9 or 13 and the nucleotide sequences
hybridizing
to the nucleotide sequence according to SEQ ID NO: 1, 9 or 13, wherein said
nucleotide
sequences according to SEQ ID NO: 1, 9 or 13 and said nucleotide sequences
hybridizing to the nucleotide sequence according to SEQ ID NO: 1, 9 or 13
encode
polypeptides which are responsible for controlling side-shoot formation and/or
petal
formation andlor abscission zone formation. According to the present
invention, the
term "hybridization" is directed to conventional hybridization conditions,
preferably
"hybridization" is directed to such hybridization conditions in which the TM
value is in
the range from TM 45°C to TM 68°C. The term "hybridization" is
particularly preferably
CA 02286594 1999-10-13
7
directed to stringent hybridization conditions. The invention further relates
to
polypeptide and amino acid sequences encoded by said nucleotide sequences.
A further object of the invention is solved by a method for preparing plants
having controlled side-shoot formation and/or petal formation and/or
abscission zone
formation, wherein the expressible DNA sequence or fragment or derivative
thereof
responsible for controlling side-shoot formation and/or petal formation andlor
abscission zone formation is integrated in a stable manner into the genome of
plant cells
or plant tissues and the resulting plant cells or plant tissues are
regenerated to form
plants.
to In the present invention a method is preferred in which the integrated DNA
suppresses the side-shoot formation and/or petal formation and/or abscission
zone
formation. Particularly preferred is a method in which the integrated DNA is
expressed
in an antisense orientation with respect to the complementary endogenous
sequence
controlling side-shoot formation and/or petal formation and/or abscission zone
formation. Also particularly preferred is a method in which the integrated DNA
is
expressed in a sense orientation with respect to the complementary endogenous
sequence controlling side-shoot formation and/or petal formation and/or
abscission zone
formation. Furthermore, particularly preferred is a method in which side-shoot
formation and/or petal formation and/or abscission zone formation is
suppressed by a
2o ribozyme comprising the DNA sequences or fragment or derivative thereof
according to
the present invention. Particularly preferred is also a method in which the
DNA
sequences or fragment or derivative thereof according to the invention are
used to
switch off ("knock-out") the endogenous gene in plants by way of homologous
recombination.
In the present invention a method is further preferred wherein the DNA
integrated into the genome of the plants enhances side-shoot formation and/or
petal
formation and/or abscission zone formation. Particularly preferred is a method
in which
the DNA according to the invention is expressed in a sense orientation with
respect to
the endogenous sequence responsible for side-shoot formation and/or petal
formation
3o and/or abscission zone formation.
Particularly preferred is the method according to the invention for the
preparation of transgenic tomato, rape, potato or snapdragon plants.
Particularly
CA 02286594 1999-10-13
preferred is also a method according to the present invention for the
preparation of
transgenic plants, wherein the DNA integrated into the genome of the plants
comprises
the sequence according to SEQ ID NO: 1, 9 or 13 or fragment or derivative
thereof or
which is complementary to said sequence or fragment or derivative thereof, or
which
hybridizes with the sequence according to SEQ ID NO: 1, 9 or 13 or fragment or
derivative thereof and encodes a polypeptide having the biological activity of
side-shoot
formation and/or petal formation and/or abscission zone formation.
The invention further relates to transformed plant cells or transformed plant
tissue, wherein an expressible DNA sequence or fragment or derivative thereof
to responsible for controlling side-shoot formation and/or petal formation
and/or
abscission zone formation is integrated in a stable manner into the genome of
the plant
cell or plant tissue. Further, the invention relates to plants as well as to
seed stocks of
plants obtainable according to the method of the present invention.
The invention is further illustrated by the following figures, wherein:
Figure 1 schematically shows the course of a positional cloning.
Figure 2 illustrates in (a) a portion of the RFLP map published by Tanksley et
al., 1992, Genetics, 132: 1141-1160. In (b) the Ls region according to
Schumacher et al.,
1995, Mol. Gen. Genet., 246: 761-766, is integrated into this map.
Figure 3 shows the mapping of cDNA and cosmid clones from the Ls region.
2o The cosmid clones A, B, C, D, E, F, G and L-as well as YAC clone CD61-5 are
symbolized by bars. The positions of the cDNA clones c10, c21, y25 and ET are
illustrated by open rectangles. The dashed lines represent recombination sites
in F2
plants 23, 24, 865 and 945.
Figure 4 shows the autoradiograph of a Southern blot analysis for the
detection
of Ls-related genes in different plant species. Genomic DNA from tomato
(Lycopersicon
esculentum), potato (Solanum tuberosum) and snapdragon (Antirrhinum majus) was
treated with the restriction enzyme EcoRI and hybridized with the cDNA clone
ET.
Figure 5 shows the nucleotide sequence and the amino acid sequence derived
therefrom (one letter code) of the Ls wild type gene from tomato (Lycopersicon
3o esculentum).
CA 02286594 1999-10-13
9
Figure 6 shows the nucleotide sequence and amino acid sequence derived
therefrom (one letter code) of the Ls homologous gene from potato (Solanum
tuberosum).
Figure 7 shows the nucleotide sequence and the amino acid sequence derived
therefrom (one letter code) of a 687 by DNA fragment of the Ls homologous gene
from
Arabidopsis thaliana.
Figure 8 shows an alignment of amino acid sequences of the Ls polypeptide from
Arabidopsis thaliana (LsAt), Lycopersicon esculentum (LsLe) and Sodanum
tuberosum
(LsSt). The one letter code was used for amino acids. Identical amino acids
are shaded
1o in black, similar amino acids are shaded in gray. The dash (-) represents
missing
sequence information, a dot (.) represents an additional amino acid in a
polypeptide. An
asterisk (*) represents a stop codon on nucleic acid level.
Detailed Description of the Invention
The method of cloning DNA fragments being several hundreds of kilobases in
length as artificial yeast chromosomes (Yeast Artificial Chromosome: YAC) in
Saccharomyces cerevisiae (Burke et al., 1987, Science, 236: 806-812) enables
the
transformation of the physical map into a number of overlapping YAC clones
spanning
the gene to be isolated. From a YAC library of tomato (Martin et al., 1992,
Mol. Gen.
Genet., 233: 25-32) clones containing the RFLP marker CD61 were isolated. By
mapping the YAC terminal fragments with respect to the RFLP markers flanking
the Ls
gene as well as to the recombination break points and to the Ls gene itself
the position
of the isolated DNA fragments in the Ls region was determined. Thus, YAC clone
CD61-5 was found to hybridize both with CD61 and with CD65 and therefore
contains
the entire genomic region including the Ls gene. Figure 3 schematically
illustrates the
position of the marker and of the YAC clone.
For identification of coding regions localized within the YAC clone this clone
was used as a radiolabeled probe to screen a cDNA library (Simon, 1990,
doctoral
thesis, University of Cologne, Cologne, Germany). The cDNA library used is
made
from RNA of both vegetative and floral shoot tips and thus represents
expressed genes
of the tissues in which the phenotype of the Ls mutation manifests itself. A
characterization of cDNA clones by cross hybridization revealed that the
purified clones
CA 02286594 1999-10-13
represented a total of 29 different transcripts. The subsequent fine mapping
of the cDNA
clones relative to the recombination break points in interval CD61-CD65
revealed that
only cDNA clone y25 cosegregated with the Ls gene and is a possible candidate
for said
gene. After the establishment of a cosmid contig also cosmid clones were used
as probes
5 to isolate further cDNA clones from the CD61-CD65 interval, which in
screening with
YAC clone CD61-5 as a probe were not detectable due to the high complexity of
the
probe. In these experiments three additional cDNA clones (c10, c21 and ET)
were
isolated which also cosegregated with the Ls gene and were possible other
candidates
for the Ls gene. Thus, a total of four cDNA clones were identified from the Ls
region,
10 which were candidates for the Ls gene. In Figure 3 said clones are
represented by open
rectangles.
In order to clone the Ls gene together with the promoter sequences necessary
for
the regulation of expression, the cDNA clone y25 was used as a starting point
for the
isolation of shorter genomic DNA fragments of the Ls region. For this purpose
a
genomic cosmid library from tomato was established in vector pCLD04541 (Bent
et al.,
1994, Science, 265: 1856-1860). Said vector contains the T-DNA border
sequences
necessary for plant transformation and thus allows for an introduction of
isolated DNA
fragments into plant cells without further cloning steps. From this library a
number of
overlapping cosmid clones was isolated in several typical cloning steps.
Mapping of said
2o cosmid clones relative to the recombination break points in the tested
interval showed
that the isolated genomic DNA fragments spanned a genomic region of about 60
kb. The
position of the cosmid clones is schematically illustrated in Figure 3.
To investigate the question whether a gene from the genomic DNA region
isolated as cosmid contig is able to compensate for the biological function
for formation
of side shoots, petals and abscission zones which is missing in the ds mutant
(complementation experiment), said Is mutant was transformed with the cosmid
clones
A, B, C, D, E, F, G and L. In all transgenes made by introduction of the
cosmids A, B,
C, D, E and F, no alteration of the phenotype could be observed. In contrast,
in eight
independent transgenic plants containing either cosmid G or L a partial or
complete
recovery of the wild type phenotype could be observed. The results of the
complementation experiments are illustrated in Table I.
CA 02286594 1999-10-13
11
Cosmid number of transformed number of complemented
plants plants
pCLD04541 8 0
A 5 ~ 0
B 15 0
C 5 0
D 7 0
E 2 0
F 8 0
G 5 3
L 11 5
Table I: Complementation experiments of is mutant via cosmid transformation
These transgenic plants form side shoots during vegetative development and
s again petals and abscission zones in the floral development. A Southern blot
analysis of
transgenic plants containing cosmid G or cosmid L revealed that in plants
showing no
complementation the T-DNA was only incompletely transferred. Thus, it has been
shown that introduced DNA fragments are able to complement the genetic
information
for formation of side shoots, petals and abscission zones, which is absent
from the
1 o mutant.
By using complementation experiments with subfragments of cosmid G the
DNA region in which the Ls gene is localized could be determined in more
detail. While
following transformation with DNA fragments containing the previously
identified gene
c21 no complementation of the is phenotype could be observed, the wild type
phenotype
15 could be recovered in eight independent transgenic plants by the
introduction of an
approx. 6 kb fragment bearing the ET gene. A DNA sequence analysis revealed
that the
ET gene of the ls~ mutant harbours a 1550 by deletion which removes the first
185
amino acids of the protein and 865 by of the sequence which is localized
upstream. A
second independent mutant allele Is' contains a 3 by insertion and several
point
2o mutations in a short DNA portion, one of which results in a termination of
the protein
after 24 amino acids. The complementation experiments and isolation and
mapping of
CA 02286594 1999-10-13
12
the cDNAs as well as the sequence analyses of the ET gene from the wild type
and two
independent is alleles revealed that the cDNA clone ET represents the entire
coding
sequence of the mRNA of the is gene.
To address the question whether similar or homologous genes are present also
in
other plant species the cDNA clone ET was employed as hybridization probe in
Southern experiments under reduced stringency. The term "plant", as used
herein,
comprises monocotyledonous and dicotyledonous economic and ornamental plants.
The
term "reduced stringency", as used herein, is directed to typical
hybridization conditions
with the modification that hybridization temperature was between 50°C
and 5S°C. In
to potato (Solanum tuberosum) and snapdragon (Antirrhinum majus) several DNA
fragments could be detected. From snapdragon several genomic clones were
isolated by
hybridization at 55°C. A DNA sequence analysis revealed that the
isolated snapdragon
clone has significant sequence homologies to the Ls gene. Thus, genes
homologous to
the tomato Ls gene may be isolated according to conventional methods by using
the
cDNA clone ET as a probe. Using gene specific primers the Ls homologous gene
was
isolated from genomic DNA of potato (Solarium tuberosum) via PCR. The Ls
homologous gene from potato shows a sequence identity of approx. 98% to the Ls
gene
of tomato on the DNA level as well as on the protein level. From genomic DNA
of
Arabidopsis (Arabidopsis thaliana) a 687 by DNA fragment of the Ls homologous
gene
2o was isolated via PCR using degenerate primers. On DNA level the Arabidopsis
thaliana
DNA fragment exhibits a sequence identity of about 63% to the tomato Ls gene.
On
protein level about 55% of the amino acids are identical.
The present invention is further directed to DNA sequences which are derived
from a plant genome and code for a protein necessary for controlling side-
shoot
formation and/or petal formation and/or formation of abscission zones. Upon
introduction and expression in plant cells the information contained in the
nucleotide
sequence results in the formation of a ribonucleic acid. By means of said
ribonucleic
acid a protein activity may be introduced into the cells or an endogenous
protein activity
may be suppressed. Particularly preferred is a DNA sequence according to SEQ
ID NO:
3o 1 from Lycopersicon esculentum shown in Figure 5, a DNA sequence according
to SEQ
ID NO: 9 from Solarium tuberosum shown in Figure 6 and a DNA sequence
according
to SEQ ID NO: 13 from Arabidopsis thaliana shown in Figure 7.
CA 02286594 1999-10-13
r
13
Moreover, the present invention relates to the use of the DNA sequences or
fragments or derivatives according to the present invention which are derived
from said
DNA sequences by insertion, deletion or substitution in the transformation of
plant cells.
The DNA sequences according to the present invention may be employed using
different methods to suppress the formation of side-shoots and thus of
branches of the
shoot system and/or petals and/or abscission zones:
1. To suppress the formation of side-shoots and/or petals and/or abscission
zones the DNA sequence according to the present invention may be cloned in an
antisense or a sense orientation into conventional vectors (e.g. plasmids) and
thus
1o combined with control elements for expression in plant cells, such as
promoters and
terminators. By using the prepared vectors, plant cells may be transformed
with the aim
to prevent the synthesis of the endogenous protein. For this purpose, shorter
parts of the
DNA sequence according to the invention, i.e. fragments, or DNA sequences
having a
sequence similarity of from 50% to 100%, i.e. derivatives, may also be used.
Thus, the
I5 Ls homologous gene isolated from Arabidopsis may be employed for example to
suppress the formation of side-shoots and thus of branches of the shoot system
and/or
petals and/or abscission zones in the related species Brassica napus (rape).
The targeted
suppression of a genetic activity in plant cells by the introduction of
antisense or sense
constructs is a common method which has been successfully employed in many
cases
20 (Gray et al., 1992, Plant. Mol. Biol., 19: 69-87).
2. Furthermore, the formation of side shoots and/or petals and/or abscission
zones may be inhibited by expressing a ribozyme constructed for this purpose
using the
DNA sequences according to the present invention. Preparation and use of
ribozymes
are disclosed in de Feyter et al., 1996, Mol. Gen. Genet., 250: 329-338 for
tobacco
25 mosaic virus resistant tomato and tobacco plants.
3. Furthermore, the DNA sequence according to the present invention may
be used to inactivate the endogenous gene. By using the DNA sequences of the
present
invention oligonucleotides may be synthesized to test plants in the context of
mutagenesis experiments by means of PCR technique for the presence of
insertions (e.g.
3o transposable elements or T-DNA from Agrobacterium tumefaciens) in the Ls
gene.
Generally, the genetic activity will be blocked by such insertions (Koes et
al., 1995,
Proc. Natl. Acad. Sci. USA, 92: 8149-8153).
CA 02286594 1999-10-13
14
4. The DNA sequence according to the invention may be also employed to
switch off ("knock-out") the endogenous Ls gene by means of homologous
recombination. This method was successfully employed in mice and is also
described
for use in plants by Miao and Lam, 1995, Plant. J., 7, 359-365.
In contrast to tomato and other economic plants, in ornamental plants (e.g.
geraniums, fuchsias and chrysanthemums) phenotypes are often preferred which
exhibit
a bushy growth due to a strong development of the side shoots. In order to
generate said
growth forms today, the plants are either decapitated, which promotes the
initiation of
side axes, or are treated with particular chemicals. However, said practice is
also
1o associated with considerable costs. In these cases, the preparation of
transgenic plants
having bushy growth forms according to the present invention represents a more
cost-
effective alternative.
In ornamental plants an enhanced formation of abscission zones my be used such
that after fading the flowers fall off by themselves and must not be manually
removed as
with many balcony and garden plants. If this does not occur, the formation of
new
flowers often is suppressed.
For the preparation of transgenic plants with strong side-shoot formation
and/or
abscission zone formation the DNA sequence or fragment or derivative thereof
according to the invention which is derived from said sequence by insertion,
deletion or
2o substitution, is introduced into plasmids in a sense orientation and
combined with
control elements for expression in plant cells. Using said plasmids plant
cells may be
transformed such that a translatable messenger ribonucleic acid (mRNA) is
expressed
which enables the synthesis of a protein stimulating the formation and
development of
side shoots and/or petals and/or abscission zones.
The DNA sequence or fragments or derivatives thereof according to the present
invention which are derived from said sequence by insertion, deletion or
substitution
may be used to isolate homologous or similar DNA sequences from the genome of
tomato or other plants, which DNA sequences influence the formation of side
shoots
andlor petals and/or abscission zones as well. For this purpose the DNA
sequence or
3o fragments, e.g. oligonucleotides, or derivatives according to the present
invention may
be employed as probe molecules to screen cDNA libraries or genomic DNA
libraries of
the plants to be screened according to conventional methods. Alternatively,
degenerated
CA 02286594 1999-10-13
or non-degenerated oligonucleotides (primers) may be derived from the sequence
according to the present invention, which may be used to screen said cDNA
libraries or
genomic DNA libraries on a PCR basis. Similar to the DNA sequences according
to the
present invention, the thus isolated related DNA sequences may be employed for
5 inhibition or stimulation of side-shoot formation and/or petal formation
and/or
abscission zone formation in plants.
For expression of the DNA sequences according to the present invention in
sense
or antisense orientation in plant cells on the one hand transcription
promoters and on the
other hand transcription terminators are necessary. A great number of
promoters and
1o terminators have been described in the literature (e.g. Kbster-Topfer et
al., 1989, Mol.
Gen. Genet., 219: 390-6; Rocha-Sosa et al., 1989, EMBO J., 8: 23-29). The
transcriptional initiation and termination regions may be derived either from
the host
plant or from a heterologous organism. The DNA sequences of the transcription
initiation and transcription termination regions may be prepared synthetically
or
15 obtained naturally or may contain a mixture of synthetic and natural DNA
components.
Methods for genetic modification have been described for dicotyledonous and
monocotyledonous plants (Gasser and Fraley, 1989, Science 244: 1293-1299;
Potrykus,
1991, Ann. Rev. Plant. Mol. Biol. Plant. Physiol., 42: 205-226). In addition
to the
transformation by means of Agrobacterium tumefaciens (Hoekema, 1983, Nature,
303:
179-180; Filatti et al., 1987, Biotech, 5:726-730), DNA may be introduced by
transformation of protoplasts, microinjection, electroporation or ballistic
methods into
plant cells. For selection of transformed plant cells the DNA to be introduced
is coupled
with a selection marker which imparts resistance against antibiotics (e.g.
kanamycin,
hygromycin, bleomycin) to the cells. From the transformed plant cells whole
plants may
then be regenerated in a typical selection medium. Regeneration of plant cells
is
described for example in EP-B-0 242 236, which is incorporated herein by
special
reference. The plants thus obtained are tested for the presence and intactness
of the
introduced DNA by means of conventional molecular biological methods. Once the
introduced DNA is integrated into the genome, it is generally stable and is
transmitted to
3o the offspring. By using conventional methods seed stocks may be obtained
from the
resulting plants.
CA 02286594 1999-10-13
16
The following examples are meant to illustrate the present invention and are
not
construed to be limiting. If not mentioned otherwise, molecular biological
standard
procedures were used, as described by Sambrook et al., 1989, Molecular
Cloning: A
Laboratory Manual, 2°d Edition, Cold Spring Harbor Laboratory Press,
Cold Spring
Harbor, New York. Southern hybridizations were carried out in 6 x SSPE (0.9 M
NaCI,
50 mM NaH2P04 x H20, 5 mM EDTA, 0.1% BSA, 0.1% Ficoll, 0.1% PVP, 0.5% SDS,
100 ~,g/ml of calf thymus DNA) with a Hybond N+ membrane (Amersham). Plaque
hybridizations were performed in 6 x SSPE (1.08 M NaCI, 60 mM NaH2P04 x H20, 6
mM EDTA, 0.1 % BSA, 0.1 % Ficoll, 0.1 % PVP, 0.1 % SDS, 200 ~g/ml of calf
thymus
1 o DNA) with a Hybond N+ membrane (Amersham).
Example 1
Isolation of YAC clones from the Ls region of tomato
From a tomato YAC library (Martin et al., 1992, Mol. Gen. Genet., 233: 25-32)
clones were isolated containing CD61 marker (Schumacher et al., 1995, Mol.
Gen.
Genet., 246: 761-766). For this, DNA mixtures which were derived from a
microtiter
plate with 96 YAC clones were first tested by using the conventional PCR
method.
Thus, from 144 of such DNA mixtures nine could be identified which yielded a
PCR
product with the CD61-F and CD61-R primers (Schumacher et al., 1995, Mol. Gen.
2o Genet., 246: 761-766). The isolation of single clones was carried out by
means of
colony hybridization or PCR, wherein the DNA of clones of a row or column of a
microtiter plate was used as a mixture. Thus, from 96 clones of a plate single
clones
were identified using 20 PCR reactions. In total, five YAC clones were
identified, the
insert size of which was determined to be 280 - 320 kb by pulsed field gel
electrophoresis (Chu et al., 1986, Science, 234: 1582-1585). It was shown in
PCR and
Southern experiments that YAC CD61-5, in addition to CD61, also carried the
second
flanking marker CD65 and thus spanned the Ls locus.
Example 2
3o Isolation of cDNA clones of the Ls region from tomato
For preparation of a hybridization probe DNA from the YAC clone CD61-5 was
isolated following separation by means of pulsed field gel electrophoresis.
However,
CA 02286594 1999-10-13
17
separation on said pulsed field gel only allowed for a relatively rough
preparation, such
that the probe used, in addition to the YAC clone CD61-5, also contained
portions of the
DNA from yeast chromosome III (360 kb) and VI (280 kb). Following radio-
labeling
said DNA was used as a probe to screen S x 105 pfu (plaque forming units) in a
conventional plaque hybridization. Hybridization with the YAC probe provided a
plurality of signals of different intensity. For rescreening 50 plaques of
different signal
intensities were selected and 44 purified clones could then be grouped by
means of cross
hybridization. 23 of 44 clones which resulted from rescreening were present
only once.
In total, 29 different transcripts were identified in this screening.
Following
1o establishment of a cosmid contig the cDNA library was again screened with
the cosmid
clones to isolate additional cDNA clones which were not detectable in
screening with
YAC61-5 as a probe due to the high complexity of the probe. In these
experiments,
three additional cDNA clones were isolated. In total 32 different transcripts
were
detected.
is
Example 3
RFLP mapping of isolated cDNA clones from tomato
Of 30 identified transcripts 22 showed typical hybridization patterns for
single or
low-copy sequences which enabled RFLP mapping. In a first RFLP analysis the
isolated
2o cDNA clones were hybridized against filters which carried DNA from L.
esculentum, L.
penellii as well as from the back crossing line IL83 digested with the
restriction
endonuclease enzymes EcoRI, EcoRV and XbaI (Eshed et al., 1992, Theor. Appl.
Genet., 83: 1027-1034). This line, in which the distal terminus of chromosome
7 is
derived from L. pennellii while the rest of the genome is composed of L.
esculentum
25 chromosomes, enables a first rough mapping in the presence of a
polymorphism
between L. esculentum and L. pennellii. If a polymorphous DNA fragment was
derived
from the Ls region, the line IL83 exhibited the L. pennellii allele, whereas
the L.
esculentum allele was present for fragments from the remaining genome. In this
manner
four cDNA clones were identified which were not derived from chromosome 7.
Fine
3o mapping of the 18 remaining cDNA clones derived from chromosome 7 was
carried out
via RFLP analysis of the plants W23 and W24 which contained recombination
events in
the interval CD61-Ls and Ls-CD65, respectively. Since in this analysis
candidates for
CA 02286594 1999-10-13
18
the Ls gene in plant W23 exhibited the L. esculentum as well as the L.
pennellii specific
fragment, while in plant W24 only the L. esculentum specific fragment was
present, the
cDNA clones were hybridized against filters carrying genomic DNA digested with
EcoRI, EcoRV or XbaI of both parental species as well as of both recombinants
W23
and W24. In this manner a total of four cDNA clones was identified which
cosegregated
with the Ls gene and thus, were possible candidates for the Ls gene.
Example 4
Preparation and screening of a genomic cosmid library of tomato
to DNA of the T-DNA/cosmid vector pCLD04541 (Bent et al., 1994, Science, 265:
1856-1860) was isolated according to the protocol of Sambrook et al., 1989,
Molecular
Cloning: A Laboratory Manual, 2°d Edition, Cold Spring Harbor
Laboratory Press, Cold
Spring Harbor, New York, purified via two CsCI gradients and dialyzed against
TE for
3 days. The DNA was completely digested with BamHI and subsequently
1 s dephosphorylated with alkaline phosphatase to prevent self ligation of the
vector. 200 ng
of genomic tomato DNA partially digested with MboI and 2 mg of vector DNA were
ligated with T4 DNA ligase in 10 ml at 16°C over night. 3 ml of said
ligation assay were
employed for packaging and transfected into E. coli SURE (Stratagene). This
assay
resulted in 6 x 106 independent recombinant bacteria. Each of 100 plates were
plated
20 with 2500 cfu (colony forming units) and rinsed off with 10 ml each of LB
medium. In
each case a glycerol culture was made from this material and a DNA preparation
was
carried out. These 100 DNA pools were screened by means of PCR analysis.
Positive
pools were then subjected to colony filter hybridization to identify positive
single
clones.
Example 5
Cloning and sequencing of the Ls gene from tomato
The insert of the cDNA clone ET which was isolated as a probe in screening of
the cDNA library with cosmid G was cut out with EcoRI and cloned into vector
pGEM-
llZf(+). The missing 5' terminus of the gene was isolated by means of the RACE
technique (Frohman et al., 1988, Proc. Natl. Acad. Sci. USA 85: 8998-9002).
Here,
starting from an oligonucleotide specifically binding to known regions of the
gene, a
CA 02286594 1999-10-13
19
DNA complementary to RNA (cDNA) was prepared. Subsequently deoxycytosin
nucleotides were attached to the cDNA using terminal transferase. With a
second gene
specific primer and a primer binding to the polydeoxycytosin tail the 5' end
of the cDNA
was amplified via PCR and cloned into the plasmid vector pGEM-T. Subsequently
the
longest of the RACE clones were sequenced. Simultaneously with the analysis of
cDNA
clone ET subfragments of the respective genomic region of cosmid G were
isolated and
recloned into the plasmid vectors pGEM-4Z and pSPORTI. Overlapping
subfragments
were then sequenced. The genomic sequence did not show any difference from the
sequence of the cDNA clone, which means that the Ls gene does not contain any
intron.
to Moreover, the respective genome regions of both mutants Is~ and ls2 were
amplified
from the genomic DNAs via PCR using suitable primers and cloned into the pGEM-
T
vector. Sequence analysis of said products exhibited a deletion of 1.5 kb in
the lsl allele
compared to the wild type sequence. Besides the loss of nucleotides 1-685 of
the open
reading frame the ls~ mutant also lacks 865 base pairs of the region located
5' of the
open reading frame, which is thought to have a regulatory function (promoter)
for
expression. Therefore, it may be assumed that the lsl mutant is no longer able
to form a
functional protein from the Ls gene. In the ls2 allele an insertion of 3 base
pairs as well
as 3 base exchanges were found in the 5' region of the open reading frame. One
of these
base exchanges leads to a stop codon resulting in a termination of the amino
acid chain
2o after 24 amino acids. Again a protein without any function is to be
assumed. The vectors
pGEM-l lzf(+), pGEM-4z, pGEM-T were purchased from the company Promega Corp.,
Madison, U.S.A., vector pSPORTI was purchased from the company Life
Technologies,
Eggenstein, and used according to the manufacturer's instructions.
Example 6
Transformation of plants with Ls cDNA constructs of tomato
Ls cDNA was isolated with gene specific primers CD61-13 (5'-
TTAGGGTTTTCACTCCACGC-3 ; SEQ ID ~ NO: 3) and CD61-28 (5'-
TCCCCTTTTTTTCCTTTCTCTC-3'; SEQ ID NO: 4) by means of the conventional
3o PCR method and cloned into plasmid vector pGEM-4z (GSETB). For preparation
of the
transformation constructs the Ls cDNA was cut off from plasmid GSET8 with
SaII/SstI
(for sense construct) and XbaI/SstI (for antisense construct) and ligated into
the plant
CA 02286594 1999-10-13
transformation vector pBIR digested with SaII/SstI (sense construct) and
XbaI/SstI
(antisense construct), respectively (Meissner, 1990, doctoral thesis,
University of
Cologne, Cologne). In the resulting clones the cDNA is present either in sense
or in
antisense orientation between promoter and polyadenylation site of the 35S
gene of
5 cauliflower mosaic virus. The resulting sense and antisense plasmids were
introduced
into the Agrobacterium tumefaciens strain GV3101 (Koncz and Shell et al.,
1986, Mol.
Gen. Genet., 204: 383-396) by direct transformation. Subsequently the T-DNAs
of the
two different constructs were transformed into leaf pieces of tomato and
tobacco
according to Fillatti et al., 1987, Biotech, 5: 726-730. Different transgenic
'plants
1o containing the Ls antisense construct show a reduction of side-shoot
formation
Example 7
Isolation of a Ls related gene from snapdragon (Antirrhinum majus)
With cDNA clone ET as a probe a genomic phage library from Antirrhinum
15 majus was screened. Hybridization was carried out at 55°C, i.e.
under reduced
stringency. In this experiment 14 clones were isolated, clone HH13 of which
showing
the strongest hybridization signals was further characterized. The sequence
analysis
carried out following recloning the phage insert into the plasmid vector pGEM-
l lzf(+)
showed that the isolated Antirrhinum majus gene has high sequence homology to
the Ls
2o gene from tomato. Within both sequences regions could be identified, in
which the
derived amino acid sequence is totally conserved.
Example 8
Isolation of an Ls related gene from potato (Solanum tuberosum)
In a Southern blot experiment under reduced stringency at 55°C using
cDNA of
the Ls gene as a hybridization probe, a DNA fragment could be detected in
genomic
DNA from Solanum tuberosum (Fig. 4). Using gene specific primers CD61-24 (5'-
TTTCCCACTCAAGCCAACTC-3 ; SEQ ID NO: 5), CD61-6 (5'-
GGTGGCAATGTAGCTTCCAG-3 ; SEQ ID NO: 6), PO1 (5'-
3o TCGAGGCGTTGGATTATTATAC-3'; SEQ ~ ID NO: 7) and POS (5'-
GGCCCCCATATCTTTTTCC-3'; SEQ ID NO: 8) from Ls gene overlapping genomic
DNA fragments were isolated from conventionally isolated DNA from Solanum
CA 02286594 1999-10-13
21
tuberosum by using the PCR method. The PCR reactions were carried out as
follows:
Denaturation at 95°C for 30 seconds, annealing at 60°C for 1
minute, elongation at 72°C
for 2 minutes. This cycle was repeated 30 times. The resulting PCR products
were
cloned into the plasmid vector pGEM-T. A sequence analysis revealed that the
isolated
DNA fragments from Solanum tuberosum bear the sequence information for an open
reading frame having a coding capacity of 431 amino acids (Fig. 6). The DNA
sequence
is shown in SEQ ID NO: 9 and the amino acid sequence encoded by the DNA
sequence
is illustrated in SEQ ID NO: 10. On DNA level as well as on protein level the
Ls
homologous gene of potato exhibits a sequence identity of about 98% to the Ls
gene of
1 o tomato.
Example 9
Isolation of an Ls related gene from Arabidopsis thaliana
For the isolation of the Ls homologous gene from Arabidopsis thaliana the
degenerated primers CD61-38 (5'-CARTGGCCNCCNYTNATGCA-3'; SEQ ID NO:
11)* and CD61-41 (5'-TGRTTYTGCCANCCNARRAA-3'; SEQ ID NO: 12)* were
made and used for PCR reactions with genomic DNA from Arabidopsis thaliana
isolated in a usual manner. The PCR reactions were carried out as follows:
Denaturation
at 95°C for 30 seconds, annealing at 50°C for 1 minute,
elongation at 72°C for 1 minute.
2o This cycle was repeated 35 times. In this manner a DNA fragment of about
700 by could
be amplified which was subsequently cloned into the plasmid vector pGEM-T. A
sequence analysis showed that the isolated DNA fragment from Arabidopsis
thaliana
(SEQ ID NO: 13) was 687 by in length and has a high sequence similarity to the
Ls gene
from Lycopersicon esculentum. On the DNA level the Arabidopsis thaliana DNA
fragment shows a sequence identity of about 63% to the Ls gene of tomato. On
the
protein level about 55% of the amino acids are identical The amino acid
sequence
encoded by the isolated DNA fragment (SEQ ID NO: 13) is illustrated in SEQ ID
NO:
14. By using the isolated DNA fragment the Ls homologous gene from Arabidopsis
thaliana may be isolated using conventional molecular biological standard
methods.
* In the description of the degenerated primers the WIPO standard St. 23 was
used:
CA 02286594 1999-10-13
22
R=A+G
N=A+G+C+T
Y=C+T
CA 02286594 1999-10-13
23
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Nikolaus (Klaus) Theres
(B) STREET: Schiffgesweg 30
(C) CITY: Pulheim -
(D) STATE: NRW
(E) COUNTRY: Germany
(F) POSTAL CODE: 50259
(G) TELEPHONE: + 49 2234 89386
(ii) TITLE OF INVENTION: PLANTS WITH CONTROLLED SIDE-SHOOT FORMATION
AND/OR ABSCISSION ZONE FORMATION
(iii) NUMBER OF SEQUENCES: 14
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPA)
(2) INFORMATION FOR SEQ ID N0: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1729 Base pairs
(B) TYPE: Nucleotide
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETIC: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Lycopersicon esculentum
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 1:
CCTCTGTCCTTCCCCCCAGGTCCCCTTTTTTTCCTTTCTCTCTCTCCTTTATTTCTCTTT 60
TCATAAGCATATTCTTTCTCTCTCTAGGGTTTTCACTTTCACCTGAAATAGTGTTGTTAA 120
ATTGAATGATATGTTAGGATCCTTTGGTTCTTCATCATCTCAATCTCACCCTCATCATGA 180
TGAAGAATCTTCTGATCATCATCAACAGCGTAGATTCACCGCTACTGCTACAACTATCAC 240
CACCACCACCATCACTACCTCACCAGCTATTCAAATCCGCCAGCTACTCATTAGCTGTGC 300
GGAGTTGATTTCGCAGTCCGATTTCTCGGCCGCGAAAAGACTCCTTACTATATTATCAAC 360
TAACTCATCTCCTTTTGGTGATTCAACTGAACGGTTAGTCCATCAATTTACTCGCGCACT 920
TTCCCTTCGTCTCAACCGCTATATATCGTCAACCACCAATCATTTCATGACACCTGTTGA 480
AACAACTCCAACTGATTCTTCTTCTTCGTCATCATTAGCTCTAATTCAATCATCATATCT 540
ATCTCTAAACCAAGTTACCCCTTTCATAAGGTTTACTCAATTAACCGCTAATCAAGCGAT 600
CA 02286594 1999-10-13
24
TTTAGAAGCGATTAACGGTAATCATCAAGCAATCCACATCGTTGATTTCGACATTAATCA 660
CGGGGTTCAATGGCCACCGTTAATGCAAGCACTAGCTGATCGTTACCCTGCTCCCACTCT 720
TCGAATCACCGGTACTGGAAATGACCTTGATACCCTTCGTAGAACAGGTGATCGTTTAGC 780
TAAATTTGCTCACTCATTAGGGTTGAGATTTCAATTCCATCCTCTTTATATAGCCAATAA 840
TAACCACGATCACGATGAAGATCCTTCTATTATTTCCTCCATTGTACTACTCCCTGATGA 900
AACCCTAGCTATCAACTGTGTTTTCTACCTCCACCGCCTTTTAAAAGACCGCGAAAAGTT 960
AAGGATTTTTTTGCATAGGGTTAAGTCAATGAACCCTAAAATTGTTACAATCGCGGAGAA 1020
GGAAGCAAATCATAACCATCCTCTTTTTTTACAAAGATTCATCGAGGCGTTGGATTATTA 1080
TACAGCTGTGTTTGATTCACTGGAAGCTACATTGCCACCGGGTAGTCGAGAGAGGATGAC 1140
~
AGTTGAACAAGTGTGGTTTGGGAGAGAGATTGTTGATATCGTTGCGATGGAAGGAGATAA 1200
AAGGAAAGAAAGACATGAAAGGTTTAGATCATGGGAAGTTATGTTGAGGAGTTGTGGATT 1260
TAGTAATGTTGCTTTAAGCCCTTTTGCATTATCACAAGCTAAGCTTCTTTTGAGACTTCA 1320
TTATCCTTCTGAAGGCTATCAACTCGGAGTTTCGAGTAATTCTTTCTTCTTAGGTTGGCA 1380
AAATCAACCCCTTTTCTCCATCTCGTCTTGGCGTTGAGAAAAACTATCAAATAGCCAACT 1440
TCAGAGGGTAATTAAGACTACTGATAGTTTAGGAGGGATCTGAAGAAAACGCGTGGAGTG 1500
AAAACCCTAAATAACCAGATTTTCTAATGAAGTTGTAGTAGTAGAAATTTGCATGGTGAA 1560
GAACAATATTGAAGAGGTATTGAAATTTCATGTTTTTTTTGTTTTACTTATTGATATGAA 1620
TGTTTTAAAATTTTTAACATAGAGGACTAGGTTGATGATATATAGTATTTAAGTTAACTA 1680
GTCTTTGTATAACGCAAGATCTTGATCAACTTATTTTTATTTTTAATTA 1729
(2) INFORMATION FOR SEQ ID N0: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 428 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
vi) ORIGINAL SOURCE:
(A) ORGANISM: Lycopersicon esculentum
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:-2:
Met Leu Gly Ser Phe Gly Ser Ser Ser Ser Gln Ser His Pro His His
1 5 10 15
Asp Glu Glu Ser Ser Asp His His Gln Gln Arg Arg Phe Thr Ala Thr
20 25 30
Ala Thr Thr Ile Thr Thr Thr Thr Ile Thr Thr Ser Pro Ala Ile Gln
35 40 45
Ile Arg Gln Leu Leu Ile Ser Cys Ala Glu Leu Ile Ser Gln Ser Asp
50 55 60
CA 02286594 1999-10-13
25
Phe Ser Ala Ala Lys Arg Leu Leu Thr Ile Leu Ser Thr Asn Ser Ser
65 70 75 80
Pro Phe Gly Asp Ser Thr Glu Arg Leu Val His Gln Phe Thr Arg Ala
85 90 95
Leu Ser Leu Arg Leu Asn Arg Tyr Ile Ser Ser Thr Thr Asn His Phe
100 105 110
Met Thr Pro Val Glu Thr Thr Pro Thr Asp Ser Ser Ser Ser Ser Ser
115 120 125
Leu Ala Leu Ile Gln Ser Ser Tyr Leu Ser Leu Asn Gln Val Thr Pro
130 135 140
Phe Ile Arg Phe Thr Gln Leu Thr Ala Asn Gln Ala Ile Leu Glu Ala
145 150 155 160
Ile Asn Gly Asn His Gln Ala Ile His Ile Val Asp Phe Asp Ile Asn
165 170 175
His Gly Val Gln Trp Pro Pro Leu Met Gln Ala Leu Ala Asp Arg Tyr
180 185 190
Pro Ala Pro Thr Leu Arg Ile Thr Gly Thr Gly Asn Asp Leu Asp Thr
195 200 205
Leu Arg Arg Thr Gly Asp Arg Leu Ala Lys Phe Ala His Ser Leu Gly
210 215 220
Leu Arg Phe Gln Phe His Pro Leu Tyr Ile Ala Asn Asn Asn His Asp
225 230 235 240
His Asp Glu Asp Pro Ser Ile Ile Ser Ser Ile Val Leu Leu Pro Asp
245 250 255
Glu Thr Leu Ala Ile Asn Cys Val Phe Tyr Leu His Arg Leu Leu Lys
260 265 270
Asp Arg Glu Lys Leu Arg Ile Phe Leu His Arg Val Lys Ser Met Asn
275 280 285
Pro Lys Ile Val Thr Ile Ala Glu Lys Glu Ala Asn His Asn His Pro
290 295 300
Leu Phe Leu Gln Arg Phe Ile Glu Ala Leu Asp Tyr Tyr Thr Ala Val
305 310 315 320
Phe Asp Ser Leu Glu Ala Thr Leu Pro Pro Gly Ser Arg Glu Arg Met
325 330 335
Thr Val Glu Gln Val Trp Phe Gly Arg Glu Ile Val Asp Ile Val Ala
340 345 350
Met Glu Gly Asp Lys Arg Lys Glu Arg His Glu Arg Phe Arg Ser Trp
355 360 365
Glu Val Met Leu Arg Ser Cys Gly Phe Ser Asn Val Ala Leu Ser Pro
370 375 380
Phe Ala Leu Ser Gln Ala Lys Leu Leu Leu Arg Leu His Tyr Pro Ser
385 390 395 400
CA 02286594 1999-10-13
26
Glu Gly Tyr Gln Leu Gly Val Ser Ser Asn Ser Phe Phe Leu Gly Trp
405 410 415
Gln Asn Gln Pro Leu Phe Ser Ile Ser Ser Trp Arg
420 425
(2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 Base pairs
(B) TYPE: Nucleotide
(C) STRANDEDNESS:single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic DNA .
(iii) HYPOTHETIC: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
TTAGGGTTTT CACTCCACGC 20
(2) INFORMATION FOR SEQ ID N0: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 Base pairs
(B) TYPE: Nucleotide
(C) STRANDEDNESS:single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic DNA
(iii) HYPOTHETIC: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4:
TCCCCTTTTT TTCCTTTCTC TC 22
(2) INFORMATION FOR SEQ ID N0: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 Base pairs
(B) TYPE: Nucleotide
(C) STRANDEDNESS:single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic DNA
(iii) HYPOTHETIC: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:
TTTCCCACTC AAGCCAACTC 20
(2) INFORMATION FOR SEQ ID N0: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 Base pairs
(B) TYPE: Nucleotide
(C) STRANDEDNESS:single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: synthetic DNA -
(iii) HYPOTHETIC: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 6:
GGTGGCAATG TAGCTTCCAG 20
(2) INFORMATION FOR SEQ ID N0: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 Base pairs
(B) TYPE: Nucleotide
(C) STRANDEDNESS:single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic DNA
(iii) HYPOTHETIC: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:~7:
TCGAGGCGTT GGATTATTAT AC 22
(2) INFORMATION FOR SEQ ID N0: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 Base pairs
(B) TYPE: Nucleotide
(C) STRANDEDNESS:single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic DNA
(iii) HYPOTHETIC: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 8:
GGCCCCCATA TCTTTTTCC 19
(2) INFORMATION FOR SEQ ID N0: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1296 Base pairs
(B) TYPE: Nucleotide
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETIC: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Solanum tuberosum
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 9:
ATGTTAGGAT CCTTTGGTTC TTCATCATCT CAATCTCACC CTCATCATGA TGAAGAATCT 60
TCTGATCATC ATCAACGGCG TAGATTCACC GCTACTACTA CAACTATCAC CACCACCACC 120
CA 02286594 1999-10-13
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ACAACGACCTCACCAGCTATTCAAATCCGCCAGCTACTCATTAGCTGTGCGGAGTTGATT 180
TCGCGGTCCGATTTCTCGGCCGCGAAAAGACTCCTTACCATATTATCAACTAACTCTTCT 240
CCTTTTGGTGATTCAACTGAACGGTTAGTCCATCAGTTTACTCGCGCACTTTCCCTTCGT 300
CTCAACCGCTATATATCGTCAACCACCAATCATTTCATGACACCTGTTGAAACAACTCCA 360
ACTGATTCTTCATCTTCGTTGCCATCGTCATCATTAGCTCTAATTCAATCATCATATCAT 420
TCTCTAAATCAAGTTACCCCTTTTATAAGGTTTACTCAATTAACCGCTAATCAAGCGATT 480
TTAGAAGCGATTAACGGTAATCATCAAGCAATCCACATCGTTGATTTCGACATTAATCAC 540
GGGGTTCAATGGCCACCGTTAATGCAAGCACTAGCTGATCGTTACCCTGCTCCTACTCTT 600
CGAATCACCGGTACTGGAAATGACCTTGATACCCTTCGTAGAACAGGTGATCGTTTAGCT .
660
AAATTTGCTCACTCATTAGGGTTGAGATTTCAATTCCATCCTCTTTATATCGCCAATAAT 720
AACCGCGATCACGGTGAAGATCCTTCTATTATTTCCTCCATTGTACTTCTCCCTGATGAA 780
ACCCTAGCTATCAACTGTGTTTTCTATCTCCACCGCCTTTTAAAAGACCGCGAAAAATTA 840
AGGATTTTTTTGCATAGGGTTAAGTCAATGAACCCTAAAATTGTTACAATCGCGGAGAAG 900
_
GAAGCAAATCATAACCATCCTCTTTTTTTACAAAGATTTATCGAGGCGTTGGATTATTAT 960
ACAGCTGTGTTTGATTCATTGGAAGCTACATTGCCACCGGGTAGTCGTGAGAGGATGACA 1020
GTTGAACAAGTGTGGTTTGGGAGAGAAATTGTTGATATCGTGGCGATGGAAGGAGATAAA 1080
AGGAAAGAAAGACATGAAAGGTTTAGATCATGGGAAGTTATGTTGAGGAGTTGTGGATTT 1140
AGTAATGTTGCTTTAAGCCCTTTTGCATTATCACAAGCTAAGCTTCTTTTGAGACTACAT 1200
TATCCTTCTGAAGGCTATCAACTCGGAGTTTCGAGTAATTCTTTCTTCTTAGGTTGGCAA 1260
AATCAACCTCTTTTCTCCATCTCGTCTTGGCGTTGA 1296
(2) INFORMATION FOR SEQ ID N0: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 431 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Solanum tuberosum
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 10:
Met Leu Gly Ser Phe Gly Ser Ser Ser Ser Gln Ser His Pro His His
1 5 10 15
Asp Glu Glu Ser Ser Asp His His Gln Arg Arg Arg Phe Thr Ala Thr
20 25 30
Thr Thr Thr Ile Thr Thr Thr Thr Thr Thr Thr Ser Pro Ala Ile Gln
35 40 45
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Ile Arg Gln Leu Leu Ile Ser Cys Ala Glu Leu Ile Ser Arg Ser Asp
50 55 60
Phe Ser Ala Ala Lys Arg Leu Leu Thr Zle Leu Ser Thr Asn Ser Ser
65 70 75 80
Pro Phe Gly Asp Ser Thr Glu Arg Leu Val His Gln Phe Thr Arg Ala
85 90 95
Leu Ser Leu Arg Leu Asn Arg Tyr Ile Ser Ser Thr Thr Asn His Phe
100 105 110
Met Thr Pro Val Glu Thr Thr Pro Thr Asp Ser Ser Ser Ser Leu Pro
115 120 125
Ser Ser Ser Leu Ala Leu Ile Gln Ser Ser Tyr His Ser Leu Asn Gln
130 135 140 .
Val Thr Pro Phe Ile Arg Phe Thr Gln Leu Thr Ala Asn Gln Ala Ile
145 150 155 160
Leu Glu Ala Ile Asn Gly Asn His Gln Ala Ile His Ile Val Asp Phe
165 170 175
Asp Ile Asn His Gly Val Gln Trp Pro Pro Leu Met Gln Ala Leu Ala
180 185 190
Asp Arg Tyr Pro Ala Pro Thr Leu Arg Ile Thr Gly Thr Gly Asn Asp
195 200 205
Leu Asp Thr Leu Arg Arg Thr Gly Asp Arg Leu Ala Lys Phe Ala His
210 215 220
Ser Leu Gly Leu Arg Phe Gln Phe His Pro Leu Tyr Ile Ala Asn Asn
225 230 235 240
Asn Arg Asp His Gly Glu Asp Pro Ser Ile Ile Ser Ser Ile Val Leu
245 250 255
Leu Pro Asp Glu Thr Leu Ala Ile Asn Cys Val Phe Tyr Leu His Arg
260 265 270
Leu Leu Lys Asp Arg Glu Lys Leu Arg Ile Phe Leu His Arg Val Lys
275 280 285
Ser Met Asn Pro Lys Ile Val Thr Ile Ala Glu Lys Glu Ala Asn His
290 295 300
Asn His Pro Leu Phe Leu Gln Arg Phe Ile Glu Ala Leu Asp Tyr Tyr
305 310 315 320
Thr Ala Val Phe Asp Ser Leu Glu Ala Thr Leu Pro Pro Gly Ser Arg
325 330 335
Glu Arg Met Thr Val Glu Gln Val Trp Phe Gly Arg Glu Ile Val Asp
340 345 350
Ile Val Ala Met Glu Gly Asp Lys Arg Lys Glu Arg His Glu Arg Phe
355 360 365
Arg Ser Trp Glu Val Met Leu Arg Ser Cys Gly Phe Ser Asn Val Ala
370 375 380
Leu Ser Pro Phe Ala Leu Ser Gln Ala Lys Leu Leu Leu Arg Leu His
385 390 _ 395 400
CA 02286594 1999-10-13
Tyr Pro Ser Glu Gly Tyr Gln Leu Gly Val Ser Ser Asn Ser Phe Phe
405 410 415
Leu Gly Trp Gln Asn Gln Pro Leu Phe Ser Ile Ser Ser Trp Arg
420 425 430
(2) INFORMATION FOR SEQ ID N0: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 Base pairs
(B) TYPE: Nucleotide
(C) STRANDEDNESS:single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic DNA
(iii) HYPOTHETIC: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 11:
CARTGGCCNC CNYTNATGCA 20
(2) INFORMATION FOR SEQ ID N0: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 Base pairs
(B) TYPE: Nucleotide
(C) STRANDEDNESS:single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: synthetic DNA
(iii) HYPOTHETIC: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 12:
TGRTTYTGCC ANCCNARRAA 20
(2) INFORMATION FOR SEQ ID N0: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 687 Base pairs
(B) TYPE: Nucleotide
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETIC: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
GAGAGGTCAT CAAACCCTAG CAGTCCACCT CCATCTCTCC GCATAACCGG ATGCGGTCGA 60
GATGTAACCG GATTAAACCG AACTGGAGAC CGGTTAACCC GGTTCGCTGA CTCTTTAGGT 120
CTCCAATTCC AGTTTCACAC GCTAGTGATC GTAGAAGAAG ATCTCGCCGG ACTTTTGCTA 180
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CAGATCCGATTGTTAGCTCTCTCAGCCGTACAAGGAGAGACCATTGCCGTCAATTGTGTT 240
CACTTCCTCCACAAAATATTTAACGACGATGGAGATATGATCGGTCACTTCTTGTCAGCG 300
ATCAAGAGCTTAAACTCTAGAATCGTTACAATGGCAGAGAGAGAAGCTAATCATGGAGAT 360
CACTCGTTCTTGAATAGATTCTCTGAGGCAGTGGATCATTACATGGCGATCTTTGATTCG 420
TTGGAAGCGACGTTGCCGCCAAATAGCCGAGAGAGACTAACCCTAGAGCAACGGTGGTTC 480
GGTAAGGAGATTTTGGATGTTGTGGCGGCGGAAGAGACGGAGAGAAAGCAAAGACATCGG 540
AGGTTTGAGATTTGGGAAGAGATGATGAAGAGGTTTGGTTTCGTTAACGTTCCTATTGGA 600
AGCTTTGCTTTGTCTCAAGCTAAGCTTCTTCTTAGACTTCATTATCCTTCAGAAGGTTAT 660
,
AATCTTCAGTTCCTTAACAATTCTTTG 687
(2) INFORMATION FOR SEQ ID N0: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 229 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 19:
Glu Arg Ser Ser Asn Pro Ser Ser Pro Pro Pro Ser Leu Arg Ile Thr
1 5 10 15
Gly Cys Gly Arg Asp Val Thr Gly Leu Asn Arg Thr Gly Asp Arg Leu
20 25 30
Thr Arg Phe Ala Asp Ser Leu Gly Leu Gln Phe Gln Phe His Thr Leu
35 40 45
Val Ile Val Glu Glu Asp Leu Ala Gly Leu Leu Leu Gln Ile Arg Leu
50 55 60
Leu Ala Leu Ser Ala Val Gln Gly Glu Thr Ile Ala Val Asn Cys Val
65 70 75 80
His Phe Leu His Lys Ile Phe Asn Asp Asp Gly Asp Met Ile Gly His
85 90 95
Phe Leu Ser Ala Ile Lys Ser Leu Asn Ser Arg Ile Val Thr Met Ala
100 105 110
Glu Arg Glu Ala Asn His Gly Asp His Ser Phe Leu Asn Arg Phe Ser
115 120 125
Glu Ala Val Asp His Tyr Met Ala Ile Phe Asp Ser Leu Glu Ala Thr
130 135 140
Leu Pro Pro Asn Ser Arg Glu Arg Leu Thr Leu Glu Gln Arg Trp Phe
145 150 155 160
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Gly Lys Glu Ile Leu Asp Val Val Ala Ala Glu Glu Thr Glu Arg Lys
165 170 175
Gln Arg His Arg Arg Phe Glu Ile Trp Glu Glu Met Met Lys Arg Phe
180 185 190
Gly Phe Val Asn Val Pro Ile Gly Ser Phe Ala Leu Ser Gln Ala Lys
195 200 205
Leu Leu Leu Arg Leu His Tyr Pro Ser Glu Gly Tyr Asn Leu Gln Phe
210 215 220
Leu Asn Asn Ser Leu
225
