U.S. patent application number 11/834332 was filed with the patent office on 2008-07-31 for potato transcription factors, methods of use thereof, and a method for enhancing tuber development.
This patent application is currently assigned to IOWA STATE UNIVERSITY RESEARCH FOUNDATION, INC.. Invention is credited to Hao CHEN, David J. HANNAPEL, Faye M. ROSIN.
Application Number | 20080184396 11/834332 |
Document ID | / |
Family ID | 38456882 |
Filed Date | 2008-07-31 |
United States Patent
Application |
20080184396 |
Kind Code |
A1 |
HANNAPEL; David J. ; et
al. |
July 31, 2008 |
POTATO TRANSCRIPTION FACTORS, METHODS OF USE THEREOF, AND A METHOD
FOR ENHANCING TUBER DEVELOPMENT
Abstract
The present invention relates to isolated nucleic acid molecules
which encode a BEL transcription factor from potato (Solanum
tuberosum L.) and the amino acid sequences encoded by such nucleic
acid molecules. Additional aspects of the present invention relate
to methods of using isolated nucleic acid molecules which encode
BEL transcription factors from potato to enhance growth and to
regulate flowering in plants. The present invention is also
directed to a method for enhancing tuber development in a plant.
This method includes transforming a tuberous plant with a DNA
construct including a nucleic acid molecule encoding a BEL
transcription factor or a KNOX transcription factor, and an
operably linked promoter and 3' regulatory region, whereby tuber
development is enhanced in the plant.
Inventors: |
HANNAPEL; David J.; (Ames,
IA) ; CHEN; Hao; (St. Louis, MO) ; ROSIN; Faye
M.; (Highland Park, NJ) |
Correspondence
Address: |
NIXON PEABODY LLP - PATENT GROUP
1100 CLINTON SQUARE
ROCHESTER
NY
14604
US
|
Assignee: |
IOWA STATE UNIVERSITY RESEARCH
FOUNDATION, INC.
Ames
IA
|
Family ID: |
38456882 |
Appl. No.: |
11/834332 |
Filed: |
August 6, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10624201 |
Jul 21, 2003 |
7265263 |
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11834332 |
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60397423 |
Jul 19, 2002 |
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Current U.S.
Class: |
800/290 |
Current CPC
Class: |
C12N 15/8261 20130101;
C07K 14/415 20130101; C12N 15/827 20130101; Y02A 40/146 20180101;
C12N 15/8297 20130101 |
Class at
Publication: |
800/290 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Goverment Interests
[0002] The subject matter of this application was made with support
from the United States Government under USDA/CSREES Grant Nos.
2002-31100-06019 and 2001-31100-06019. The government may have
certain rights.
Claims
1-25. (canceled)
26. A method for enhancing tuber development in a plant comprising:
transforming a tuberous plant with a first DNA construct
comprising: a first nucleic acid molecule encoding a BEL
transcription factor or a KNOX transcription factor, and a first
operably linked promoter and first 3' regulatory region, whereby
tuber development in the plant is enhanced.
27. The method according to claim 26, wherein the first nucleic
acid molecule encodes a BEL transcription factor.
28. The method according to claim 27, wherein the BEL transcription
factor is from Solanum tuberosum.
29. The method according to claim 28, wherein the first nucleic
acid molecule has a nucleotide sequence selected from the group
consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:13.
30. The method according to claim 28, wherein the first nucleic
acid molecule encodes a protein that is at least 85% similar to a
homeodomain region, a SKY box, a BELL domain, and a VSLTLGL-box in
either SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID
NO:10, SEQ ID NO:12, or SEQ ID NO:14 by basic BLAST using default
parameters analysis.
31. The method according to claim 28, wherein the first nucleic
acid molecule hybridizes to the nucleotide sequence of SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,
or SEQ ID NO:13 under stringent conditions characterized by a
hybridization buffer comprising 5.times.SSC at a temperature of
55.degree. C.
32. The method according to claim 28, wherein the first nucleic
acid molecule encodes a protein or polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:2,
SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,
and SEQ ID NO:14.
33. The method according to claim 28, wherein the first nucleic
acid molecule encodes a protein or polypeptide having a molecular
mass of about 56 kDa to about 76 kDa.
34. The method according to claim 26, wherein the first nucleic
acid molecule encodes a KNOX transcription factor.
35. The method according to claim 34, wherein the KNOX
transcription factor is from Solanum tuberosum.
36. The method according to claim 35, wherein the first nucleic
acid molecule has a nucleotide sequence of SEQ ID NO:16.
37. The method according to claim 35, wherein the first nucleic
acid molecule hybridizes to the nucleotide sequence of SEQ ID NO:16
under stringent conditions characterized by a hybridization buffer
comprising 5.times.SSC at a temperature of 55.degree. C.
38. The method according to claim 35, wherein the first nucleic
acid molecule encodes a protein or polypeptide having an amino acid
sequence of SEQ ID NO:17.
39. The method according to claim 26, wherein the first DNA
construct comprises a first nucleic acid molecule encoding a BEL
transcription factor and a second nucleic acid molecule encoding a
KNOX transcription factor.
40. The method according to claim 39, wherein the BEL transcription
factor and the KNOX transcription factor are from Solanum
tuberosum.
41. The method according to claim 26, wherein the first nucleic
acid molecule encodes a BEL transcription factor and the method
further comprises: transforming the tuberous plant with a second
DNA construct comprising: a second nucleic acid molecule encoding a
KNOX transcription factor, and a second operably linked promoter
and second 3' regulatory region.
42. The method according to claim 26, wherein the tuberous plant is
selected from the group consisting of potato, wild potato, dahlia,
caladium, Jerusalem artichoke, yam, sweet potato, cassaya, tuberous
begonia, and cyclamen.
43-56. (canceled)
Description
[0001] The present invention claims benefit of U.S. Provisional
Application Ser. No. 60/397,423, filed Jul. 19, 2002, which is
hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to isolated BEL transcription
factors from Solanum tuberosum, a method of enhancing tuber
development in plants, and methods of regulating flowering and
growth in plants.
BACKGROUND OF THE INVENTION
[0004] The primary developmental events of plants originate from
the shoot apical meristem (SAM) (Clark, "Organ Formation at the
Vegetative Shoot Meristem," Plant Cell 9:1067-1076 (1997);
Kerstetter et al., "Shoot Meristem Formation in Vegetative
Development," Plant Cell 9:1001-1010 (1997)). The shoot apical
meristem (SAM) is responsible for the formation of vegetative
organs such as leaves, and may undergo a phase change to form the
inflorescence or floral meristem. Many of these events are
controlled at the molecular level by transcription factors.
Transcription factors (TFs) are proteins that act as developmental
switches by binding to the DNA (or to other proteins that bind to
the DNA) of specific target genes to modulate their expression. An
important family of TFs involved in regulating the developmental
events in apical meristems is the knox (knotted-like homeobox gene
family (Reiser et al., "Knots in the Family Tree Evolutionary
Relationships and Functions of Knox Homeobox Genes," Plant Mol Biol
42:151-166 (2000)). Knox genes have been isolated from several
plant species (reviewed in Reiser et al., "Knots in the Family
Tree: Evolutionary Relationships and Functions of knox Homeobox
Genes," Plant Mol. Biol. 42:151-166 (2000)) and can be divided into
two classes based on expression patterns and sequence similarity
(Kerstetter et al., "Sequence Analysis and Expression Patters
Divide the Maize knotted1-like Homeobox Genes into Two Classes,"
Plant Cell 6:1888-1887 (1994)). Class I knox genes have high
similarity to the kn1 homeodomain and generally have a
meristem-specific mRNA expression pattern. Class II knox genes
usually have a more widespread expression pattern.
[0005] Knox genes belong to the group of TFs known as the TALE
superclass (Burglin, "Analysis of TALE Superclass Homeobox Genes
(MEIS, PBC, KNOX, Iroquois, TGIF) Reveals a Novel Domain Conserved
Between Plants and Animals," Nucleic Acids Res 25:4173-4180
(1997)). These TFs are distinguished by a very high level of
sequence conservation in the DNA-binding region, designated the
homeodomain, and consisting of three .alpha.-helices similar to the
bacterial helix-loop-helix motif (Kerstetter et al., "Sequence
Analysis and Expression Patterns Divide the Maize knotted1-like
Homeobox Genes into Two Classes," Plant Cell 6:1877-1887 (1994)).
The third helix, the recognition helix, is involved in DNA-binding
(Mann et al., "Extra Specificity From extradenticle: the
Partnership Between HOX and PBX/EXD Homeodomain Proteins," Trends
in Genet. 12:258-262 (1996)). TALE TFs contain a three amino acid
loop extension (TALE), proline-tyrosine-proline, between helices I
and II in the homeodomain, that has been implicated in protein
interactions (Passner et al., "Structure of DNA-Bound
Ultrabithorax-Extradenticle Homeodomain Complex," Nature
397:714-719 (1999)). There are numerous TFs from plants and animals
in the TALE superclass and the two main groups in plants are the
KNOX and BEL types (Burglin, "Analysis of TALE Superclass Homeobox
Genes (MEIS, PBC, KNOX, Iroquois, TGIF) Reveals a Novel Domain
Conserved Between Plants and Animals," Nucleic Acids Res
25:4173-4180 (1997)). Related genes in animal systems play an
important role in regulating gene expression.
[0006] Expression patterns and functional analysis of mutations
support the involvement of knox genes in specific developmental
processes of the shoot apical meristem. Kn1 from maize, the first
plant homeobox gene to be discovered (Vollbrecht et al., "The
Developmental Gene Knotted-1 is a Member of a Maize Homeobox Gene
Family," Nature 350:241-243 (1991)), is involved in maintenance of
the shoot apical meristem and is implicated in the switch from
indeterminate to determinate cell fates (Chan et al., "Homeoboxes
in Plant Development," Biochim Biophys Acta 1442:1-19 (1998);
Kerstetter et al., "Loss-of-Function Mutations in the Maize
Homeobox Gene, knotted1, are Defective in Shoot Meristem
Maintenance," Development 124:3045-3054 (1997); Clark et al., The
CLAVATA and SHOOT MERISTEMLESS Loci Competitively Regulate Meristem
Activity in Arabidopsis," Development 122:1567-1575 (1996)).
Transcripts of kn1 in maize (Jackson et al., "Expression of Maize
KNOTTED1 Related Homeobox Genes in the Shoot Apical Meristem
Predicts Patterns of Morphogenesis in the Vegetative Shoot,"
Development 120:405-413 (1994)), OSH1 in rice (Sentoku et al.,
"Regional Expression of the Rice KN1-type Homeobox Gene Family
During Embryo, Shoot, and Flower Development," Plant Cell
11:1651-1663 (1999)), and NTH15 in tobacco (Tamaoki et al.,
"Ectopic Expression of a Tobacco Homeobox Gene, NTH15, Dramatically
Alters Leaf Morphology and Hormone Levels in Transgenic Tobacco,"
Plant Cell Physiol 38:917-927 (1997)) were localized by in situ
hybridization to undifferentiated cells of the corpus and the
developing stem, but were not detected in the tunica or leaf
primordia. Overexpression of kn1 in Arabidopsis (Lincoln et al., "A
knotted1-like Homeobox Gene in Arabidopsis is Expressed in the
Vegetative Meristem and Dramatically Alters Leaf Morphology When
Overexpressed in Transgenic Plants," Plant Cell 6:1859-1876 (1994))
and in tobacco (Sinha et al., "Overexpression of the Maize Homeobox
Gene, KNOTTED-1, Causes a Switch From Determinate to Indeterminate
Cell Fates," Genes Dev 7:787-795 (1993)), resulted in plants with
altered leaf morphologies including lobed, wrinkled or curved
leaves with shortened petioles and decreased elongation of veins.
Plants were reduced in size and showed a loss of apical dominance.
In plants with a severe phenotype, ectopic meristems formed near
the veins of leaves indicating a reversion of cell fate back to the
indeterminate state (Sinha et al., "Overexpression of the Maize
Homeobox Gene, KNOTTED-1, Causes a Switch From Determinate to
Indeterminate Cell Fates," Genes Dev 7:787-795 (1993)).
Overexpression of OSH1 or NTH15 in tobacco resulted in altered
morphologies similar to the 35S-kn1 phenotype (Sato et al.,
"Abnormal Cell Divisions in Leaf Primordia Caused by the Expression
of the Rice Homeobox Gene OSH1 Lead to Altered Morphology of Leaves
in Transgenic Tobacco," Mol Gen Genet. 251:13-22 (1996); Tamaoki et
al., "Ectopic Expression of a Tobacco Homeobox Gene, NTH15,
Dramatically Alters Leaf Morphology and Hormone Levels in
Transgenic Tobacco," Plant Cell Physiol 38:917-927 (1997)).
[0007] Alterations in leaf and flower morphology in 35S-NTH15 or
OSH1 transgenic tobacco were accompanied by changes in hormone
levels. Whereas levels of all the hormones measured were changed
slightly, both gibberellin and cytokinin levels were dramatically
altered (Kusaba et al., "Alteration of Hormone Levels in Transgenic
Tobacco Plants Overexpressing the Rice Homeobox Gene OSH1," Plant
Physiol 116:471-476 (1998); Tamaoki et al., "Ectopic Expression of
a Tobacco Homeobox Gene, NTH15, Dramatically Alters Leaf Morphology
and Hormone Levels in Transgenic Tobacco," Plant Cell Physiol
38:917-927 (1997)). RNA blot analysis revealed that the
accumulation of GA 20-oxidase1 mRNA was reduced several fold in
transgenic plants (Kusaba et al., "Decreased GA.sub.1 Content
Caused by the Overexpression of OSH1 is Accompanied by Suppression
of GA 20-oxidase Gene Expression," Plant Physiol 117:1179-1184
(1998); Tanaka-Ueguchi et al., "Overexpression of a Tobacco
Homeobox Gene, NTH15, Decreases the Expression of a Gibberellin
Biosynthetic Gene Encoding GA 20-oxidase," Plant J 15:391-400
(1998)). A KNOX protein of tobacco binds to specific elements in
regulatory regions of the GA 20-oxidase1 gene of tobacco to repress
its activity (Sakamoto et al., KNOX Homeodomain Protein Directly
Suppresses the Expression of a Gibberellin Biosynthesis Gene in the
Tobacco Shoot Apical Meristem," Genes Dev 15:581-590 (2001)). GA
20-oxidase is a key enzyme in the GA biosynthetic pathway necessary
for the production of the physiologically inactive GA.sub.20
precursor of active GA.sub.1 (Hedden et al., "Gibberellin
Biosynthesis: Enzymes, Genes and Their Regulation," Annu Rev Plant
Physiol Plant Mol Biol 48:431-460 (1997)). GA.sub.1 and other
active GA isoforms are important regulators of stem elongation, the
orientation of cell division, the inhibition of tuberization,
flowering time, and fruit development (Jackson et al., "Control of
Tuberisation in Potato by Gibberellins and Phytochrome," B. Physiol
Plant 98:407-412 (1996); Hedden et al., "Gibberellin Biosynthesis:
Enzymes, Genes and Their Regulation," Annu Rev Plant Physiol Plant
Mol Biol 48:431-460 (1997); Rebers et al., "Regulation of
Gibberellin Biosynthesis Genes During Flower and Early Fruit
Development of Tomato," Plant J 17:241-250 (1999)).
[0008] Another plant homeobox gene family that is closely related
to the knox genes is the BEL (BELL) family (Chan et al.,
"Homeoboxes in Plant Development," Biochim Biophys Acta 1442:1-19
(1998); Burglin, "Analysis of TALE Superclass Homeobox Genes (MEIS,
PBC, KNOX, Iroquois, TGIF) Reveals a Novel Domain Conserved Between
Plants and Animals," Nucleic Acids Res 25:4173-4180 (1997)). BEL
TFs have been implicated in flower and fruit development (Reiser et
al., The BELL1 Gene Encodes a Homeodomain Protein Involved in
Pattern Formation in the Arabidopsis Ovule Primordium," Cell
83:735-742 (1995); Dong et al., "MDH1: an Apple Homeobox Gene
Belonging to the BEL1 Family," Plant Mol Biol 42:623-633 (2000)).
Genetic analysis of BEL1 in Arabidopsis showed that expression of
this TF regulated the development of ovule integuments and overlaps
the expression of AGAMOUS (Ray et al., "Arabidopsis Floral Homeotic
Gene BELL (BEL1) Controls Ovule Development Through Negative
Regulation of AGAMOUS Gene (AG)," Proc Natl Acad Sci USA
91:5761-5765 (1994); Reiser et al., The BELL1 Gene Encodes a
Homeodomain Protein Involved in Pattern Formation in the
Arabidopsis Ovule Primordium," Cell 83:735-742 (1995); Western et
al., "BELL1 and AGAMOUS Genes Promote Ovule Identity in Arabidopsis
thaliana," Plant J 18:329-336 (1999)). In COP1 mutants, the
photoinduced expression of ATH1, another BEL TF of Arabidopsis, was
elevated, indicating a possible role in the signal transduction
pathway downstream of COP1 (Quaedvlieg et al., "The Homeobox Gene
ATH1 of Arabidopsis is Depressed in the Photomorphogenic Mutants
cop1 and det1," Plant Cell 7:117-129 (1995)).
[0009] Plants must maintain a great deal of flexibility during
development to respond to environmental and developmental cues.
Responses to these signals, which include day length, light quality
or quantity, temperature, nutrient and hormone levels, are
coordinated within the meristem (Kerstetter et al., "Shoot Meristem
Formation in Vegatative Development," Plant Cell 9:1001-1010
(1997)). In potato, there is a specialized vegetative meristem
called the stolon meristem that develops as a horizontal stem and
under inductive conditions will form the potato tuber (Jackson,
"Multiple Signaling Pathways Control Tuber Induction in Potato,"
Plant Physiol. 119:1-8 (1999); Fernie et al., "Molecular and
Biochemical Triggers of Potato Tuber Development," Plant Physiol.
127:1459-1465 (2001)). Potato offers an excellent model system for
examining how vegetative meristems respond to external and internal
factors to control development at the molecular level. In model
tuberization systems, synchronous tuber formation occurs under
inductive conditions and shoot or stolon formation occurs under
noninductive conditions. The cellular and biochemical processes
that occur in these model systems have been examined extensively
(Vreugdenhil et al., "Initial Anatomical Changes Associated with
Tuber Formation on Single-Node Potato (Solanum tuberosum L.)
Cuttings: A Re-evaluation," Ann. Bot. 84:675-680 (1999); Xu et al.,
"The Role of Gibberellin, Abscisic Acid, and Sucrose in the
Regulation of Potato Tuber Formation In vitro," Plant Physiol.
117:575-584 (1998); Hannapel, "Characterization of Early Events of
Potato Tuber Development," Physiol. Plant 83:568-573 (1991);
Wheeler et al., "Comparison of Axillary Bud Growth and Patatin
Accumulation in Potato Leaf Cuttings as Assays for Tuber
Induction," Ann. Bot. 62:25-30 (1988)). In addition to being good
systems to examine integration of signals at the meristem,
understanding the molecular processes controlling tuberization in
potato is important. Potato is the fourth largest crop produced in
the world, ranking after maize, rice, and wheat, and is a major
nutritional source in many countries (Jackson, "Multiple Signaling
Pathways Control Tuber Induction in Potato," Plant Physiol. 119:1-8
(1999); Fernie et al., "Molecular and Biochemical Triggers of
Potato Tuber Development," Plant Physiol. 127:1459-1465 (2001));
therefore, research focusing on the process of tuber initiation and
development is very important.
[0010] Tuber formation in potatoes (Solanum tuberosum L.) is a
complex developmental process that requires the interaction of
environmental, biochemical, and genetic factors. Several important
biological processes like carbon partitioning, signal transduction,
and meristem determination are involved (Ewing et al., "Tuber
Formation in Potato: Induction, Initiation and Growth," Hort. Rev.
14:89-198 (1992)). Under conditions of a short-day photoperiod and
cool temperature, a transmissible signal is activated that
initiates cell division and expansion and a change in the
orientation of cell growth in the subapical region of the stolon
tip (Ewing et al., "Tuber Formation in Potato: Induction,
Initiation and Growth," Hort. Rev. 14:89-198 (1992); Xu et al.,
"Cell Division and Cell Enlargement During Potato Tuber Formation,"
J. Expt. Bot. 49:573-582 (1998)). In this signal transduction
pathway, perception of the appropriate environmental cues occurs in
leaves and is mediated by phytochrome and gibberellins (van den
Berg et al., "Morphology and (14C)gibberellin A-12 Metabolism in
Wild-Type and Dwarf Solanum tuberosum ssp. Andigena Grown Under
Long and Short Photoperiods," J. Plant Physiol. 146:467-473 (1995);
Jackson et al., "Phytochrome B Mediates the Photoperiodic Control
of Tuber Formation in Potato," Plant J. 9:159-166 (1996); Jackson
et al., "Control of Tuberisation in Potato by Gibberellins and
Phytochrome," B. Physiol Plant 98:407-412 (1996)). Tuber
development at the stolon tip is comprised of biochemical and
morphological processes. Both are controlled by differential gene
expression (Hannapel, "Characterization of Early Events of Potato
Tuber Development," Physiol. Plant 83:568-573 (1991); Bachem et
al., "Analysis of Gene Expression During Potato Tuber Development,"
Plant J. 9:745-753 (1996); Macleod et al., "Characterisation of
Genes Isolated from a Potato Swelling Stolon cDNA Library," Pot.
Res. 42:31-42 (1999)) with most of the work focusing on the
biochemical processes, including starch synthesis (Abel et al.,
"Cloning and Functional Analysis of a cDNA Encoding a Novel 139 kDa
Starch Synthase from Potato (Solanum tuberosum L.)," Plant J.
10:981-991 (1996); Preiss, "ADPglucose Pyrophosphorylase: Basic
Science and Applications in Biotechnology," Biotech. Annu. Rev.
2:259-279 (1996); Geigenberger et al., "Overexpression of
Pyrophosphatase Leads to Increased Sucrose Degradation and Starch
Synthesis, Increased Activities of Enzymes for Sucrose-Starch
Interconversions, and Increased Levels of Nucleotides in Growing
Potato Tubers," Planta 205:428-437 (1998)) and storage protein
accumulation (Mignery et al., "Isolation and Sequence Analysis of
cDNAs for the Major Potato Tuber Protein, Patatin," Nucl. Acid Res.
12:7989-8000 (1984); Hendriks et al., "Patatin and Four serine
Protease Inhibitor Genes are Differentially Expressed During Potato
Tuber Development," Plant Mol. Biol. 17:385-394 (1991); Suh et al.,
"Proteinase-Inhibitor Activity and Wound-Inducible Expression of
the 22-kDa Potato-Tuber Proteins," Planta 184:423-430 (1991)).
[0011] Much less is known about the morphological controls of
tuberization, although it is clear that phytohormones play a
prominent role (Koda et al., "Potato Tuber-Inducing Activities of
Jasmonic Acid and Related Compounds," Phytochemistry 30:1435-1438
(1991); Xu et al., "The Role of Gibberellin, Abscisic Acid, and
Sucrose in the Regulation of Potato Tuber Formation In vitro,"
Plant Physiol. 117:575-584 (1998), Sergeeva et al., "Tuber
Morphology and Starch Accumulation are Independent Phenomena:
Evidence from ipt-transgenic Potato Lines," Physiol. Plant
108:435-443 (2000)). Gibberellins (GA), in particular, play an
important role in regulating tuber development. High levels of GA
are correlated with the inhibition of tuberization, whereas low
levels are associated with the induction of tuber formation
(Jackson et al., "Control of Tuberisation in Potato by Gibberellins
and Phytochrome," B. Physiol Plant 98:407-412 (1996); Xu et al.,
"The Role of Gibberellin, Abscisic Acid, and Sucrose in the
Regulation of Potato Tuber Formation In vitro," Plant Physiol.
117:575-584 (1998)). Specific genes, such as lipoxygenases
(Kolomiets et al., "Lipoxygenase is Involved in the Control of
Potato Tuber Development," Plant Cell 13:613-626 (2001)) and MADS
box genes (Kang et al., "Nucleotide Sequences of Novel Potato
MADS-box cDNAs and their Expression in vegetative Organs," Gene
166:329-330 (1995)) that are involved in regulating tuber formation
have been identified.
[0012] Three independent research groups have recently confirmed
that BEL-like TFs interact via protein binding with their
respective knox-types in three separate species (Bellaoui et al.,
"The Arabidopsis BELL1 and KNOX TALE Homeodomain Proteins Interact
Through a Domain Conserved Between Plants and Animals," Plant Cell
13:2455-2470 (2001); Muller et al., "In vitro Interactions Between
Barley TALE Homeodomain Proteins Suggest a Role for Protein-Protein
Associations in the Regulation of Knox Gene Function," Plant J.
27:13-23 (2001); Smith et al., "Selective Interaction of Plant
Homeodomain Proteins Mediates High DNA-Binding Affinity," Proc.
Nat'l. Acad. Sci. USA 99:9579-9584 (2002)), but to date, there is
no published report on the function of this interaction. Moreover,
nothing is known about the role of either KNOX or the BEL TFs in
the regulation of development of tuberous plants, such as
potato.
[0013] The present invention is directed to overcoming these and
other deficiencies in the art.
SUMMARY OF THE INVENTION
[0014] The present invention relates to isolated nucleic acid
molecules which encode a BEL transcription factor from potato
(Solanum tuberosum L.) and the amino acid sequences encoded by such
nucleic acid molecules.
[0015] Another aspect of the present invention pertains to host
cells, DNA constructs, expression vectors, transgenic plants, and
transgenic plant seeds containing the isolated nucleic acid
molecules of the present invention.
[0016] The present invention is also directed to a method for
enhancing tuber development in a plant. This method includes
transforming a tuberous plant with a first DNA construct including
a first nucleic acid molecule encoding a BEL transcription factor
or a KNOX transcription factor, and a first operably linked
promoter and first 3' regulatory region, whereby tuber development
in the plant is enhanced.
[0017] A further aspect of the present invention relates to a
method for enhancing growth in a plant. This method includes
transforming a plant with a DNA construct including a nucleic acid
molecule encoding a BEL transcription factor from Solanum tuberosum
and an operably linked promoter and 3' regulatory region, whereby
growth in the plant is enhanced.
[0018] Yet another aspect of the present invention relates to a
method for regulating flowering in a plant. This method includes
transforming a plant with a DNA construct including a nucleic acid
molecule encoding a BEL transcription factor from Solanum tuberosum
and an operably linked promoter and 3' regulatory region, whereby
flowering in the plant is regulated.
[0019] The present invention relates to transcription factors which
can be used to enhance tuber formation, to enhance growth, or to
regulate flowering in a plant. In particular, accelerating tuber
growth in field plants shortens the time for field cultivation. It
can also be used to shorten the timing of a "late" potato variety
to produce an earlier harvest. Many desirable breeding lines of
potato produce tubers too late in the growing season or with too
low a yield. The method of the present invention circumvents these
problems, even under noninductive conditions. Enhanced tuberization
also has applications for producing food in space under a research
initiative directed by NASA (Food and Crop Systems Research, NASA's
Advanced Life Support Program). Potato tubers are also being
designed as biostorage organs for the production of pharmaceuticals
or bioproducts. Enhanced tuber growth would be advantageous in
these systems. Moreover, enhancement of growth in plants or
regulation of flowering in plants can be used to produce an earlier
harvest of plants/flowers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows Southern hybridization of POTH1. Genomic DNA
(10 .mu.g) was digested with the restriction enzymes, Hind III (H)
or Xba I (X) and hybridized to a .sup.32P-labeled POTH1 probe which
did not include the ELK or homeodomain. There is a restriction site
for Hind III within the coding sequence of POTH1. Size markers in
kb are shown on the right.
[0021] FIG. 2 shows POTH1 mRNA accumulation in various organs of
the potato plants. Poly(A)-enriched RNA (5 .mu.g in each lane) was
hybridized to a digoxygenin-rUTP-labeled POTH1 RNA antisense probe
with the ELK and homeodomain deleted. MT, mature tuber; S, stem; R,
root; IN, inflorescence; ML, mature leaf; SA, shoot apex; SS,
swollen stolon apex. Equal loading of intact poly(A)+ RNA in each
lane was confirmed by ethidium bromide staining. The hybridizing
bands are approximately 1.3 kb in length.
[0022] FIGS. 3A-F show the localization of POTH1 mRNA in potato
plants as revealed by in situ hybridization. The presence of POTH1
mRNA is indicated by an orange/brown stain under dark-field
microscopy. All micrographs are of equal magnification. Size
bar=300 .mu.m. FIG. 3A shows a longitudinal section through a
vegetative shoot apex, probed with antisense POTH1. AP=apical
meristem; L=leaf lamina; OL=older leaf lamina. Asterisks indicate
leaf primordia (beneath AP) and procambium (to left of AP). FIG. 3B
shows unswollen stolon apex, antisense POTH1. AP=apical meristem;
P=procambium; asterisk=lamina of young leaf; V=perimedullary
parenchyma associated with vascular tissue; X=xylem element. FIG.
3C shows unswollen stolon apex, sense POTH1. FIG. 3D shows swollen
stolon apex, antisense POTH1. AP=apical meristem; P=procambium;
V=perimedullary parenchyma and vascular tissue; L=lamina of young
leaf. FIG. 3E shows swollen stolon, subapical longitudinal section,
basal to section in 3D, antisense POTH1. IC=inner cortex;
V=perimedullary parenchyma and vascular tissue; PI=pith. FIG. 3F
shows swollen stolon, subapical section, sense POTH1.
[0023] FIGS. 4A-F show POTH1 mRNA accumulation in transgenic potato
plants and the evaluation of leaf and stem traits in POTH1
overexpression lines. FIG. 4A shows total RNA (5 .mu.g) from shoot
tips of wild-type (WT) and independent transgenic lines, potato
subsp. andigena 15, 18, 20, 29, and 11 that were hybridized to a
.sup.32P-labeled POTH1 probe with the ELK or homeodomain deleted.
In FIG. 4B, membranes were stripped and hybridized with
.sup.32P-labeled 1.2 kb wheat 18S rRNA to ascertain equal loading
and transfer. In FIGS. 4C-F, three plants each of wild-type and
overexpression lines, potato subsp. andigena 15, 18, 20, 29, and 11
were examined. Standard error is indicated for each mean. In FIG.
4C, plant height and in FIG. 4D, internode length were examined for
75-day old plants. In FIG. 4E, petiole length and in FIG. 4F, the
terminal leaflet length was measured for the sixth expanded leaf of
84-day old plants.
[0024] FIGS. 5A-Q show the phenotype of the leaves of POTH1
overexpression lines. FIG. 5A shows that the overall size and shape
of leaves from the andigena intermediate and severe overexpression
lines, line 20 and line 15, respectively, have been altered
compared to wild-type leaves (WT). In FIG. 5B, wild-type leaflets
(WT) have a prominent mid-vein (mv) and pinnate venation pattern.
The potato subsp. andigena intermediate overexpression mutant (line
20) has a mouse-ear shape, a shortened mid-vein, and palmate
venation pattern. FIG. 5C shows the shoot tip of WT potato subsp.
andigena line. FIG. 5D shows the severe mutant, potato subsp.
andigena line 15, which has a mouse-ear leaf phenotype and
shortened petioles causing leaves to cluster closely to the stem.
The bars in FIGS. 5C and D are 5 mm. In FIG. 5E, the rachis and
associated leaflets were detached from the petiole of a wild-type
(WT) and a representative sense line (19), to show a slight
increase in the proliferation of leaflets. FIG. 5F shows a
cross-section through a wild-type leaf showing the arrangement of
cell layers: e=epidermis; sp=spongy parenchyma; pp=palisade
parenchyma. Size bar=50 .mu.m. FIG. 5G shows a cross-section
through a potato subsp. andigena line 15 leaf after treatment with
GA.sub.3 showing an intermediate level of cell organization. Bar=50
.mu.m. FIG. 5H shows a cross-section through a potato subsp.
andigena line 15 leaf showing that the cell layers lack a palisade
parenchyma layer. Size bar=50 .mu.m. FIG. 5I shows a wild-type leaf
from potato subsp. andigena showing the morphology of a compound
leaf. In FIGS. 5J and K, the compound leaf structure is shown for
the overexpression mutant, potato subsp. andigena line 15. Shoot
tips were treated with either 10 .mu.M GA.sub.3 in 0.002% (v/v)
ethanol (FIG. 5J) or with 0.002% (v/v) ethanol alone (FIG. 5K).
Terminal leaflets from compound leaves of wild-type plants (FIG.
5L), GA.sub.3-treated line 15 (FIG. 5M), and untreated line 15
(FIG. 5N) are shown. The mid-vein is marked with an arrow in FIG.
5M. Note that the morphology and venation of the GA.sub.3-treated
leaf (FIGS. 5J and M) is more similar to the wild-type leaf (FIGS.
5I and L) than to the potato subsp. andigena line 15 untreated leaf
(FIGS. 5K and N). Bars in FIGS. 5I through 5K=1.0 mm. The second
expanded leaf was used for the leaf samples in FIGS. 5F through 5N.
FIG. 5O is a wild-type leaf from Solanum tuberosum cv. FL-1607
(`FL-1607`) showing the morphology of a compound leaf. In FIGS.
5P-Q, the compound leaf structure is shown for the overexpression
mutant, `FL-1607` line 5. Shoot tips were treated with either 10
.mu.M GA.sub.3 in 0.002% (v/v) ethanol (FIG. 5P) or with 0.002%
(v/v) ethanol alone (FIG. 5Q). The mid-vein is marked with an arrow
in FIG. 5P. Note that the morphology of the GA.sub.3-treated leaf
(FIG. 5P) is more similar to the wild-type leaf (FIG. 5O) than to
`FL-1607` line 5 control leaf (FIG. 5Q).
[0025] FIG. 6 shows the levels of intermediates in the GA
biosynthetic pathway. GAs were extracted from shoot tips down to
the sixth expanded leaf from wild-type and potato subsp. andigena
POTH1 overexpression lines 29, 20, and 11. GAs were separated by
HPLC and levels were measured by gas chromatography-mass
spectrometry (GC-MS). GA.sub.53, GA.sub.19, and GA.sub.20 are
precursors to GA.sub.1, the physiologically active GA, whereas
GA.sub.8 is the inactive metabolite. GA.sub.53 and GA.sub.19 levels
increased, whereas GA.sub.20, GA.sub.1, and GA.sub.8 levels
decreased in POTH1 overexpression lines. Measurements are the
average of three replications. Standard error is indicated for each
mean. Concentrations of GA.sub.53, GA.sub.19, GA.sub.20, GA.sub.1
and GA.sub.8 were determined by calculating the area of the peaks
at the correct Kovats retention indices (KRI) at 448/450
(KRI=2,497), 434/436 (2,596), 418/420 (2,482), 506/508 (2,669), and
594/596 (2,818), respectively.
[0026] FIGS. 7A-B show the accumulation of mRNA for GA 20-oxidase1
in transgenic plants that overexpress the potato knox gene, POTH1.
In FIG. 7A, 5 .mu.g of total RNA from the shoot tips of wild-type
lines (designated 2, 9, and 10) and the overexpression lines,
potato subsp. andigena 11, 15, and 18 were hybridized with a 1.2-kb
fragment of the potato GA 20-oxidase1 cDNA, StGA20ox1 (Carrera et
al., "Feedback Control and Diurnal Regulation of Gibberellin
20-oxidase Transcript Levels in Potato," Plant Physiol. 119:765-773
(1999), which is hereby incorporated by reference in its entirety).
In FIG. 7B, the membrane was stripped and re-probed with 18S wheat
rRNA to ascertain equal loading and efficient transfer.
[0027] FIG. 8 shows GA 20-oxidase1 mRNA accumulation in shoot tips
of POTH1 overexpressers (plants #11, 15, and 18) with a severe
phenotype (dwarf with small, curled leaves). Total RNA (10 .mu.g in
each lane) was hybridized to .sup.32P-labeled GA 20-oxidase1
(Carrera et al., "Feedback Control and Diurnal Regulation of
Gibberellin 20-oxidase Transcript Level in Potato," Plant Physiol.
119:765-773 (1999), which is hereby incorporated by reference)
probe. Standard procedures for RNA blot hybridization were used.
The plants shown are 8 weeks old. These same plants had reduced
levels of GA.sub.20 and GA.sub.1 and increased levels of GA.sub.53
and GA.sub.19.
[0028] FIGS. 9A-C show the specific interaction of POTH1 with seven
BEL1-like proteins of potato. FIG. 9A shows selection on a nutrient
carbon medium minus histidine, leucine, trytophan, and adenine. The
pAD plasmid provides leucine selection, the pBD plasmid (pBridge)
provides tryptophan selection, and histidine and adenine selection
are activated from the host strain (AH109) chromosomal DNA. The
asterisk (*) designation indicates yeast growth with both plasmids
transformed together, whereas the pAD plasmids (designated 5, 11,
13, 14, 22, 29, 30) are transformed alone (no growth). SIR4, a
transcriptional activator of yeast, is used as a positive control
and pBHD is POTH1 in pBridge alone. FIG. 9B shows that POTH1
interacts with all seven BELs as determined by a quantitative yeast
two-hybrid assay. LacZ induction in the yeast strain AH109 was
assayed in transformed yeast cultures using a quantitative yeast
.beta.-galactosidase assay method (Pierce Chemical Company). For
each pair, the dark bars on the left represent the pAD or pBHD
plasmid alone transformed into yeast. The white bars on the right
in each pair represent both plasmids (pAD and pBHD) transformed
together. The standard error of the mean is represented by error
bars. FIG. 9C shows immunoprecipitates of the in vitro binding of
POTH1 to BEL proteins of potato. .sup.35S-labeled GAD: POTH1 fusion
protein and the three BEL1 proteins (p11Z-5, -13, and -30) were
synthesized in separate in vitro transcription/translation
reactions (lanes 2, 3, 6, and 9, respectively). Each of the three
BEL1 proteins were incubated with the GAD:POTH1 protein and
immunoprecipitated with anti-GAD antibodies (lanes 5, 8, and 11).
None of the three BEL proteins bound to the GAD protein alone
(lanes 4, 7, and 10). Labeled proteins were visualized by
autoradiography after separation by SDS-PAGE. Molecular size
markers are shown on the right.
[0029] FIGS. 10A-B show a deletion analysis of the binding regions
of POTH1 and a potato BEL1-like protein using the yeast two-hybrid
system. In FIG. 10A, deletion constructs of POTH1 in pBridge were
tested for expression in the yeast strain AH109 and cotransformed
with the full-length BEL cDNA, StBEL-05, in pGAL4 to test for
interaction. In FIG. 10B, deletion constructs of StBEL-05 in pGAL4
were cotransformed with the full-length cDNA of POTH1 in pBridge.
Interaction was verified with both nutritional selection and
.beta.-galactosidase activity. The white box indicates the
homeodomain. The gray box indicates the putative protein/protein
interaction region (for POTH1, this is the conserved KNOX domain,
for StBEL5, the BELL domain). The black boxes are conserved
sequences identified in the BEL proteins (see FIG. 13A) and the
diagonal hatched boxes in POTH1 represent the ELK domain. The
numbers in parentheses represent the amino acids of the full-length
sequence included in each construct.
[0030] FIG. 11A shows a Northern blot analysis of the accumulation
of mRNA for four BEL1-like cDNAs (StBEL-05, -13, -14, and -30) in
potato organs. Ten .mu.g of total RNA from flowers, shoot tips
(SAM), leaves, stems, roots, unswollen stolons (U stolon), swollen
stolons (S stolon), and tubers were loaded per lane. Swollen
stolons represent an early stage of tuber formation. A probe for
the 18S ribosomal RNA was used to verify equal loading of RNA
samples (bottom panel).
[0031] FIG. 11B shows a Northern blot analysis of the accumulation
of the mRNA of StBEL-05 in leaves and stolons of WT plants grown
under long days (LD, 16 hours of light, 8 hours of dark) and short
days (SD, 8 hours of light, 16 hours of dark). Ten .mu.g of total
RNA from stolons were loaded per lane. Leaves and stolons were
harvested from the photoperiod-responsive potato species, Solanum
tuberosum ssp. andigena, 4 and 8 days after the plants were
transferred to short-day conditions. Samples were harvested one
hour after the end of the dark period. A gene-specific probe for
each BEL cDNA was used. Ethidium bromide-stained ribosomal RNA is
visualized as a loading control.
[0032] FIG. 11C shows a Northern blot analysis of the accumulation
of the mRNA of potato BEL-like cDNAs (StBEL-05, StBEL-13, StBEL-14,
and StBEL-30) in tuberizing stolons. Ten .mu.g of total RNA from
stolons were loaded per lane. Stolons were harvested from the
photoperiod-responsive potato species, Solanum demissum, 1, 2, 4,
or 7 days after the plants were transferred to short-day
conditions. A gene-specific probe for each BEL cDNA was used. A
probe for the 18S ribosomal RNA was used to verify equal loading of
RNA samples (bottom panels).
[0033] FIG. 12 shows the phylogenetic tree of the BEL1-like
proteins of potato (Solanum tuberosum L.). The amino acid sequence
of seven potato BEL-like proteins was analyzed and compared to BEL
proteins of plants. These data were organized into a phylogenetic
tree with the ME-Boot program of the MEGA package (version 1.0) and
the neighbor-joining program (Saitou and Nei, 1987). The numbers
listed at the branching points are boot-strapping values which
indicate the level of significance (%) for the separation of two
branches. The length of the branch line indicates the extent of
difference according to the scale at the lower left-hand side.
Databank accession numbers are listed on the dendrogram and the
common name of the species is listed in the right-hand column.
[0034] FIG. 13A shows a schematic of the amino acid sequence of the
BEL1-like proteins of potato. Boxed regions represent conserved
sequences identified by aligning all seven BELs. Helices I, II, and
III of the homeodomain are designated. The proline-tyrosine-proline
(PYP) loop extension is located between helices I and II. For
clarity in labeling, the sequence is not drawn to scale.
[0035] FIG. 13B shows predicted helices of the putative
protein-binding region (BELL domain) of the BEL1 protein StBEL-05.
The bold letters represent amino acids conserved in other plant
BEL1 proteins based on a BLAST analysis of StBEL-05. The underlined
portion of the sequence represents a predicted .alpha.-helix. A
consensus for the prediction of the sequence structure was derived
by using three software programs for amino acid sequence analysis:
sspal, ssp, and nnssp (http://www.softberry.com/protein.html). Four
deletion constructs from FIG. 14B are designated with arrows.
Construct pAD5-1 contains aa 230 through 653 of pAD-05 (interaction
with POTH1), and pAD5-2 contains aa 257 through 653 of pAD-05 (no
interaction). Construct pAD5-11 consists of aa 1 through 286 of
pAD-05 (no interaction), and pAD5-9 consists of aa 1 through 315
(interaction with POTH1).
[0036] FIG. 13C is a Southern blot analysis of BEL-like genes of
potato. Genomic DNA (10 .mu.g per lane) was digested with EcoRI,
HindIII, and PstI. Each blot was hybridized with a .sup.32P-labeled
gene-specific probe from each of the four StBEL cDNAs. DNA size
markers in kilobases are indicated on the right.
[0037] FIGS. 14A-C show in vitro tuberization of transgenic plants
that overexpress sense transcripts of StBEL-05. Northern blot
analysis for the accumulation of mRNA for StBEL-05 was performed by
using 10 .mu.g of total RNA/lane from vegetative meristems of in
vitro plantlets and gene-specific probes for StBEL-05 (see FIG.
14A). Equal loading of RNA samples was verified by visualizing
ethidium bromide-stained rRNA bands with UV light. The rate of
tuberization (days to tuberize) was determined by the first
appearance of tubers from among twenty-four replicates (see FIG.
14B). The number of tubers was scored after 2 weeks of LD
conditions (0 d), and after 7 (7 d) and 14 days (14 d) of SD
conditions (see FIG. 14B). Tubers were harvested and weighed after
21 days (see FIG. 14C) from the StBEL-05 overexpression (24 plants
each) and wild-type lines (35 plants). Cultured transgenic plants
of Solanum tuberosum ssp. andigena were grown on a Murashige and
Skoog medium with 6% sucrose under a long-day photoperiod (16 hours
of light, 8 hours of dark) in a growth chamber for two weeks. For
tuber induction, plants were transferred to a Murashige and Skoog
medium supplemented with 6% sucrose and evaluated daily for tuber
formation under a short-day photoperiod (8 hours of light, 16 hours
of dark) in the growth chamber until tubers formed. All numbered
lines were verified as transgenic by using PCR with
transgene-specific primers. Control plants were both nontransgenic
(WT) and transgenic (StBEL-05 line 6).
[0038] FIG. 15 shows overexpression mutant lines for the potato
KNOX gene, POTH1 (lines 15 and 18), and for the BEL1-like protein,
StBEL-05 (lines 12, 14, and 19). These StBEL-05 sense lines had a
leaf phenotype similar to wild-type plants (WT). These are 8-week
plants grown under long-day conditions (16 hours of light, 8 hours
of dark) in the greenhouse supplemented with high pressure sodium
HID lamps. The StBEL-05 plants ranged in height from 34 to 39 cm,
whereas, the POTH1 lines were 7 to 10 cm in height.
[0039] FIGS. 16A-B are a Northern blot analysis of the accumulation
of the mRNA of the GA 20-oxidase1 gene of potato (Carerra et al.,
"Feedback Control and Diurnal Regulation of Gibberellin 20-oxidase
Transcript Levels in Potato," Plant Physiol. 119:765-773 (1999),
which is hereby incorporated by reference in its entirety) in
wild-type plants and sense lines 11, 12, and 20 of StBEL-05 (FIG.
16A). Total RNA was extracted from the 2.0 mm distal tip of stolons
from plants grown under LD conditions (16 hours of light, 8 hours
of dark). Wild-type RNA (WT) was extracted from two separate pools.
Ten .mu.g of total RNA were loaded per lane. A gene-specific probe
for GA 20-oxidase1 was used for hybridization. All three StBEL-05
lines exhibited enhanced tuber formation. Ethidium bromide-stained
rRNA is visualized as a loading control (FIG. 16B).
[0040] FIG. 17A shows tubers harvested from independent lines of
StBEL-05 transgenic plants (Solanum tuberosum spp. andigena) grown
in soil under a short-day photoperiod. Plants were grown under long
days (LD) (16 hours of light, 8 hours of dark) in 10 cm pots until
they reached the 16-leaf stage and then transferred to short days.
After 14 days under short days, tubers from three plants per
independent line were harvested and photodocumented. Tuber numbers
and yields increased by at least threefold in these StBEL-05 lines
relative to control plants. Starting from the upper left-hand
corner and proceeding clockwise are tubers harvested from control
plants (WT) and from each of the StBEL-05 overexpression lines 14,
19, and 12. Other than the increase in the rate of tuber formation,
the phenotype of these sense lines was similar to wild-type.
Reference bar is equivalent to 1.0 cm.
[0041] FIG. 17B shows tubers from the same StBEL-05 lines from FIG.
17A harvested after 21 days of culture in vitro under inductive
conditions of a short-day photoperiod (8 hours of light, 16 hours
of dark) and 6% sucrose in the media. Tubers from 35 control plants
and from 25 plants of the StBEL-05 lines are displayed in the same
order as shown in FIG. 17A. Tuber yield per plant of line 14 was
sixteenfold greater than wild-type. The tubers showed an intense
purple color, which is the result of anthocyanin accumulation
characteristic of this subspecies. Reference bar is equivalent to
1.0 cm.
[0042] FIG. 17C shows tuber production for stolons from
overexpression lines of POTH1. Excised stolon tips from plants
grown under LD conditions were grown in vitro in the dark in media
supplemented with 8% sucrose. Tubers were harvested after 35 days
of culture. Starting from the upper left-hand corner and proceeding
clockwise are tubers harvested from control plants (WT) and from
each of the POTH1 overexpression lines 11, 18, and 20. Twelve
stolon tips per independent line were evaluated for tuber
production. Reference bar is equivalent to 1.0 cm.
[0043] FIG. 17D shows the rate of tuberization for stolons from
overexpression lines 11, 18, 20, 29, and 15 of POTH1 and from
wild-type plants (WT). Excised stolon tips (approximately 1.5 cm in
length) from plants grown under long-day conditions were grown in
vitro in the dark in media supplemented with 8% (w/v) sucrose and
monitored for 20 days.
[0044] FIGS. 18A-B show gel mobility shift assays (FIG. 18A) for
the binding of two transcription factors of potato, POTH1 (HD) and
StBEL-05, to regions of the GA20 oxidase1 promoter and the first
intron (FIG. 18B). Each DNA probe is tested for binding in four
sets: DNA alone, with StBEL-05 only, with POTH1 (HD) only, and with
both StBEL-05 and POTH1. The two proteins appear to bind in tandem
to the P1 region. Two-hundred ng of purified protein and
.sup.32P-labeled DNA fragments were used in each binding reaction.
The protein/DNA mix was run on a nondenaturing polyacrylamide gel.
These results are representative of several replications. The GA20
ox1 promoter was provided by Salome Prat, Barcelona.
[0045] FIG. 19 shows the effect of binding two transcription
factors to the GA20 oxidase1 promoter on the rate of transcription.
The potato GA20 oxidase1 promoter (1170 bp) plus an enhancer was
fused to a GUS marker (GAPGUS, gray bars). The two transcription
factors, POTH1 and StBEL-05, were cloned and expressed in separate
protein expression vectors. All constructs were transformed into
tobacco protoplasts through electroporation. Whereas, repression of
transcription was affected by each TF alone, expression of the
proteins in tandem resulted in the greatest repression of
transcription. Activity of the 35SGUS construct (black bars) was
used as a baseline control. The "no protein" protoplasts are
designated as 100% transcriptional activity. All activities are
calculated in relation to a luciferase internal control.
[0046] FIG. 20 shows GA20 oxidase1 mRNA accumulation in stolon tips
of plants grown under long-day conditions. Ten .mu.g of total RNA
was probed with a .sup.32P-fragment specific for the potato GA20
oxidase1 cDNA. These StBEL-05 lines all exhibited enhanced tuber
formation.
[0047] FIGS. 21A-B show a competition gel-retardation assay of P1
with cold P1 or P3 in the presence of StBEL-05 (FIG. 21A) or POTH1
(FIG. 21B). Lane 1 is labeled P1 alone, lane 2 is the labeled P1
with either StBEL-05 (FIG. 21A) or POTH1 (FIG. 21B). Increased
amounts (10.times., 25.times., 50.times., 100.times.) of unlabeled
P1 or P3 were added to lanes 3 to 6 and 7 to 10, respectively. The
DNA-protein complexes are indicated with arrowheads.
[0048] FIG. 22 shows a dissociation rate analysis of StBEL-05-P1,
POTH1-P1, and StBEL-05-POTH1-P1 complexes. Labeled P1 was incubated
on ice for 30 minutes with recombinant proteins, as indicated on
the top. Then a 500-fold molar excess of unlabeled P1 was added and
aliquots analyzed by gel mobility shift assay after the indicated
time. The arrows show the DNA-protein complexes.
[0049] FIGS. 23A-B show the protein structures of POTH1 (FIG. 23A)
and StBEL-05 (FIG. 23B). Conserved regions are labeled. These
include the protein-binding regions for POTH1, KNOX I and KNOX II,
and for StBEL-05, the Sky box and the BELL domains. The DNA-binding
domains (HD) consisting of three helices and the characteristic
proline-tyrosine-proline TALE are also designated. POTH1 is 345 aa
in length, whereas StBEL-05 is 688 aa. The schematics of protein
structure presented here are not drawn to scale to enhance visual
clarity.
[0050] FIGS. 24A-C show schematics of constructs (FIG. 24A) and the
repression effect of StBEL-05 and POTH1 on the ga20ox1 promoter
(FIG. 24B) and on the 35S CaMV promoter (FIG. 24C). The construct
with the LUC gene under the control of the cauliflower mosaic virus
(CaMV) 35S promoter was used as an internal control. Each
transfection was performed three times. Relative GUS-LUC activity
was calculated with reporter alone set as 100%. Data are means
.+-.SE.
[0051] FIG. 25 A-C show schematics of constructs (FIG. 25A) and the
effect of dominant negative constructs of either StBEL-05 or POTH1
on the repression activity of StBEL-05 (FIG. 25B) or POTH1 (FIG.
25C), respectively. The construct with the LUC gene under the CaMV
35S promoter was used as a control. Each transfection was performed
three times. Relative GUS-LUC activity was calculated with reporter
alone set as 100%. Data are means .+-.SE.
[0052] FIGS. 26A-C show a schematic of the mutated base in a 9-bp
motif (FIG. 26A) and that mutation in the StBEL-05-POTH1
heterodimer binding site deprived the ga20ox1 promoter of its
response to StBEL-05 and POTH1 repression (FIGS. 26B-C). The
construct with the LUC gene under the CaMV 35S promoter was used as
control. Each transfection was performed three times. Relative
GUS-LUC activity was calculated with reporter alone set as 100%.
Data are means .+-.SE.
[0053] FIG. 27 shows a model of BEL/KNOX binding to target DNA.
Light grey=StBEL-05 homeodomain; dark grey=POTH1 homeodomain. The
three helices are indicated as I, II, or III. The schematics of
protein structure presented here are not drawn to scale to enhance
visual clarity. The third helix of the homeodomains of both POTH1
and StBEL-05 fit in the major groove of the DNA double helix.
DETAILED DESCRIPTION OF THE INVENTION
[0054] The present invention relates to nucleic acid molecules
encoding BEL transcription factors from potato (Solanum tuberosum
L.). BEL transcription factor is a general term used herein to mean
a member of the BEL-1-like family of transcription factors, which
includes a BELL domain (Bellaoui et al., "The Arabidopsis BELL1 and
KNOX TALE Homeodomain Proteins Interact Through a Domain Conserved
Between Plants and Animals," Plant Cell 13(11):2455-70 (2001),
which is hereby incorporated by reference in its entirety) and
which regulates growth, in particular, floral development.
[0055] In a first embodiment, the BEL transcription factor from
Solanum tuberosum is identified herein as StBEL-05 and is encoded
by a nucleic acid molecule having a nucleotide sequence of SEQ ID
NO:1 as follows:
TABLE-US-00001 1 catgcagaga taaaaatata gatcagtctg acaagaaggc
aacttctcaa agcttagaga 61 gctaccaccc gaagatagac agttagttac
atgtactgtt atagataaaa ggagaaatcc 121 gaagaagaaa gaattttttt
tgcagatatg tactatcaag gaacctcgga taatactaat 181 atacaagctg
atcatcaaca acgtcataat catgggaata gtaataataa taatattcag 241
acactttatt tgatgaaccc taacaattat atgcaaggct acactacttc tgacacacag
301 cagcagcagc agttactttt cctgaattct tcaccagcag caagcaacgc
gctttgccat 361 gcgaatatac aacacgcgcc gctgcaacag cagcactttg
tcggtgtgcc tcttccggca 421 gtaagtttgc acgatcagat caatcatcat
ggacttttac agcgcatgtg gaacaaccaa 481 gatcaatctc agcaggtgat
agtaccatcg tcgacggggg tttctgccac gtcatgtggc 541 gggatcacca
cggacttggc gtctcaattg gcgtttcaga ggccgattcc gacaccacaa 601
caccgacagc agcaacaaca gcaaggcggt ctatctctaa gcctttctcc tcagctacaa
661 cagcaaatta gtttcaataa caatatttca tcctcatcac caaggacaaa
taatgttact 721 attaggggaa cattagatgg aagttctagc aacatggttt
taggctctaa gtatctgaaa 781 gctgcacaag agcttcttga tgaagttgtt
aatattgttg gaaaaagcat caaaggagat 841 gatcaaaaga aggataattc
aatgaataaa gaatcaatgc ctttggctag tgatgtcaac 901 actaatagtt
ctggtggtgg tgaaagtagc agcaggcaga aaaatgaagt tgctgttgag 961
cttacaactg ctcaaagaca agaacttcaa atgaaaaaag ccaagcttct tgccatgctt
1021 gaagaggtgg agcaaaggta cagacagtac catcaccaaa tgcaaataat
tgtattatca 1081 tttgagcaag tagcaggaat tggatcagcc aaatcataca
ctcaattagc tttgcatgca 1141 atttcgaagc aattcagatg cctaaaggat
gcaattgctg agcaagtaaa ggcgacgagc 1201 aagagtttag gtgaagagga
aggcttggga gggaaaatcg aaggctcaag actcaaattt 1261 gtggaccatc
atctaaggca acaacgcgcg ctgcaacaga taggaatgat gcaaccaaat 1321
gcttggagac cccaaagagg tttacctgaa agagctgtct ctgtccttcg tgcttggctt
1381 ttcgagcatt ttcttcatcc ttacccaaag gattcagaca aaatcatgct
tgctaagcaa 1441 acggggctaa caaggagcca ggtgtctaac tggttcataa
atgctcgagt tcgattatgg 1501 aagccaatgg tagaagaaat gtacttggaa
gaagtgaaga atcaagaaca aaacagtact 1561 aatacttcag gagataacaa
aaacaaagag accaatataa gtgctccaaa tgaagagaaa 1621 catccaatta
ttactagcag cttattacaa gatggtatta ctactactca agcagaaatt 1681
tctacctcaa ctatttcaac ttcccctact gcaggtgctt cacttcatca tgctcacaat
1741 ttctccttcc ttggttcatt caacatggat aatactacta ctactgttga
tcatattgaa 1801 aacaacgcga aaaagcaaag aaatgacatg cacaagtttt
ctccaagtag tattctttca 1861 tctgttgaca tggaagccaa agctagagaa
tcatcaaata aagggtttac taatccttta 1921 atggcagcat acgcgatggg
agattttgga aggtttgatc ctcatgatca acaaatgacc 1981 gcgaattttc
atggaaataa tggtgtctct cttactttag gacttcctcc ttctgaaaac 2041
ctagccatgc cagtgagcca acaaaattac ctttctaatg acttgggaag taggtctgaa
2101 atggggagtc attacaatag aatgggatat gaaaacattg attttcagag
tgggaataag 2161 cgatttccga ctcaactatt accagatttt gttacaggta
atctaggaac atgaatacca 2221 gaaagtctcg tattgatagc tgaaaagata
aaaggaagtt agggatactc ttatattgtg 2281 tgaggccttc tggcccaagt
cggaggaccc aatttgatac aacctatcat aggagaaaag 2341 aagtggagac
taaattaaag taacaaaatt ttaaagcaca ctttctagta tatatacttc 2401
ttttttttat agtatagaaa agaagagatt ttgtgcttta gtgtatagat agagtctact
2461 tagtasaggt tatacttcta gttccttgag aagattgata caactagtag
tatttttttt 2521 cttttgggtt ggcttggagt actattttaa gttattggaa
actagctata gtaaatgttg 2581 taaagttgtg atattgttcc tctcaatttg
catataattt gaaatatttt gtacctacta 2641 gctagtctct aaattatgtt
tccattgctt gtaattgcaa ttttatttga attttgtgct 2701 atcattatta
gattagcaaa aaaaaaaaaa aaaaa
[0056] The nucleic acid sequence corresponding to SEQ ID NO:1
encodes a BEL transcription factor isolated from Solanum tuberosum
identified herein as StBEL-05, which has a deduced amino acid
sequence corresponding to SEQ ID NO:2 as follows:
TABLE-US-00002 Met Tyr Tyr Gln Gly Thr Ser Asp Asn Thr Asn Ile Gln
Ala Asp His 1 5 10 15 Gln Gln Arg His Asn His Gly Asn Ser Asn Asn
Asn Asn Ile Gln Thr 20 25 30 Leu Tyr Leu Met Asn Pro Asn Asn Tyr
Met Gln Gly Tyr Thr Thr Ser 35 40 45 Asp Thr Gln Gln Gln Gln Gln
Leu Leu Phe Leu Asn Ser Ser Pro Ala 50 55 60 Ala Ser Asn Ala Leu
Cys His Ala Asn Ile Gln His Ala Pro Leu Gln 65 70 75 80 Gln Gln His
Phe Val Gly Val Pro Leu Pro Ala Val Her Leu His Asp 85 90 95 Gln
Ile Asn His His Gly Leu Leu Gln Arg Met Trp Asn Asn Gln Asp 100 105
110 Gln Ser Gln Gln Val Ile Val Pro Ser Ser Thr Gly Val Ser Ala Thr
115 120 125 Ser Cys Gly Gly Ile Thr Thr Asp Leu Ala Ser Gln Leu Ala
Phe Gln 130 135 140 Arg Pro Ile Pro Thr Pro Gln His Arg Gln Gln Gln
Gln Gln Gln Gly 145 150 155 160 Gly Leu Ser Leu Ser Leu Ser Pro Gln
Leu Gln Gln Gln Ile Ser Phe 165 170 175 Asn Asn Asn Ile Ser Ser Ser
Ser Pro Arg Thr Asn Asn Val Thr Ile 180 185 190 Arg Gly Thr Leu Asp
Gly Ser Ser Ser Asn Met Val Leu Gly Ser Lys 195 200 205 Tyr Leu Lys
Ala Ala Gln Glu Leu Leu Asp Glu Val Val Asn Ile Val 210 215 220 Gly
Lys Ser Ile Lys Gly Asp Asp Gln Lys Lys Asp Asn Ser Met Asn 225 230
235 240 Lys Glu Ser Met Pro Leu Ala Ser Asp Val Asn Thr Asn Ser Ser
Gly 245 250 255 Gly Gly Glu Ser Ser Ser Arg Gln Lys Asn Glu Val Ala
Val Glu Leu 260 265 270 Thr Thr Ala Gln Arg Gln Glu Leu Gln Met Lys
Lys Ala Lys Leu Leu 275 280 285 Ala Met Leu Glu Glu Val Glu Gln Arg
Tyr Arg Gln Tyr His His Gln 290 295 300 Met Gln Ile Ile Val Leu Ser
Phe Glu Gln Val Ala Gly Ile Gly Ser 305 310 315 320 Ala Lys Ser Tyr
Thr Gln Leu Ala Leu His Ala Ile Ser Lys Gln Phe 325 330 335 Arg Cys
Leu Lys Asp Ala Ile Ala Glu Gln Val Lys Ala Thr Ser Lys 340 345 350
Ser Leu Gly Glu Glu Glu Gly Leu Gly Gly Lys Ile Glu Gly Ser Arg 355
360 365 Leu Lys Phe Val Asp His His Leu Arg Gln Gln Arg Ala Leu Gln
Gln 370 375 380 Ile Gly Met Met Gln Pro Asn Ala Trp Arg Pro Gln Arg
Gly Leu Pro 385 390 395 400 Glu Arg Ala Val Ser Val Leu Arg Ala Trp
Leu Phe Glu His Phe Leu 405 410 415 His Pro Tyr Pro Lys Asp Ser Asp
Lys Ile Met Leu Ala Lys Gln Thr 420 425 430 Gly Leu Thr Arg Ser Gln
Val Ser Asn Trp Phe Ile Asn Ala Arg Val 435 440 445 Arg Leu Trp Lys
Pro Met Val Glu Glu Met Tyr Leu Glu Glu Val Lys 450 455 460 Asn Gln
Glu Gln Asn Ser Thr Asn Thr Ser Gly Asp Asn Lys Asn Lys 465 470 475
480 Glu Thr Asn Ile Ser Ala Pro Asn Glu Glu Lys His Pro Ile Ile Thr
485 490 495 Ser Ser Leu Leu Gln Asp Gly Ile Thr Thr Thr Gln Ala Glu
Ile Ser 500 505 510 Thr Ser Thr Ile Ser Thr Ser Pro Thr Ala Gly Ala
Ser Leu His His 515 520 525 Ala His Asn Phe Ser Phe Leu Gly Ser Phe
Asn Met Asp Asn Thr Thr 530 535 540 Thr Thr Val Asp His Ile Glu Asn
Asn Ala Lys Lys Gln Arg Asn Asp 545 550 555 560 Met His Lys Phe Ser
Pro Ser Ser Ile Leu Ser Ser Val Asp Met Glu 565 570 575 Ala Lys Ala
Arg Glu Ser Ser Asn Lys Gly Phe Thr Asn Pro Leu Met 580 585 590 Ala
Ala Tyr Ala Met Gly Asp Phe Gly Arg Phe Asp Pro His Asp Gln 595 600
605 Gln Met Thr Ala Asn Phe His Gly Asn Asn Gly Val Ser Leu Thr Leu
610 615 620 Gly Leu Pro Pro Ser Glu Asn Leu Ala Met Pro Val Ser Gln
Gln Asn 625 635 640 Tyr Leu Ser Asn Asp Leu Gly Ser Arg Ser Glu Met
Gly Ser His Tyr 645 650 655 Asn Arg Met Gly Tyr Glu Asn Ile Asp Phe
Gln Ser Gly Asn Lys Arg 660 665 670 Phe Pro Thr Gln Leu Leu Pro Asp
Phe Val Thr Gly Asn Leu Gly Thr 675 680 685
The BEL transcription factor has a molecular mass of approximately
75.7 kDa. StBEL-05, isolated from Solanum tuberosum, has a single
open reading frame ("ORF") of 2067 bp, extending between
nucleotides 148-2214.
[0057] In a second embodiment, the BEL transcription factor from
Solanum tuberosum is identified herein as StBEL-11 and is encoded
by a nucleic acid molecule having a nucleotide sequence of SEQ ID
NO:3 as follows:
TABLE-US-00003 1 atgactttca ggtctagtct tccactagac ctccgtgaaa
tttcaacaac aaatcatcaa 61 gttggaatac tatcatcatc accattacca
tcaccaggaa caaataccaa taatatcaat 121 catactcgag gattaggggc
atcatcatct ttttcgattt ctaatgggat gatattgggt 181 tctaagtacc
taaaagttgc acaagatctt cttgatgaag ttgttaatgt tggaaaaaac 241
atcaaattat cagatggctt agagagtggt gcaaaggaga aacacaaatt ggacaatgaa
301 ttaatatctt tggctagtga tgatgttgaa agcagcagcc aaaaaaatag
tggtgttgaa 361 cttacaacag ctcaaagaca agaacttcaa atgaagaaag
ccaagcttgt tagcatgctt 421 gatgaggtgg atcaaaggta tagacaatac
catcaccaaa tgcaaatgat tgcaacatca 481 tttgagcaaa caacaggaat
tggatcatca aaatcataca cacaacttgc tttgcacaca 541 atttcaaagc
aatttagatg tttaaaagat gcaatttctg ggcaaataaa ggacactagc 601
aaaactttag gggaagaaga aaacattgga ggcaaaattg aaggatcaaa gttgaaattt
661 gtggatcatc atttacgcca acaacgtgca ctacaacaat tagggatgat
gcaaaccaat 721 gcatggaagc ctcaaagagg tttgccagaa agagcggttt
cagttctccg cgcttggctt 781 ttcgagcatt ttcttcatcc gtatcccaaa
gattcagata aaatcatcct tgctaagcaa 841 acagggctaa caaggagcca
ggtatcaaat tggtttataa atgctagagt tagactatgg 901 aagccaatgg
tagaagaaat gtacatggaa gaagtgaaga aaaacaatca agaacaaaat 961
attgagccta ataacaatga aattgttggc tcaaaatcaa gtgttccaca agagaaatta
1021 ccaattagta gcaatattat tcataatgct tctccaaatg atatttctac
ttccaccatt 1081 tcaacatctc cgacgggtgg cggcggttcg attccgactc
agacggttgc aggtttctcc 1141 ttcattaggt cattaaacat ggagaacatt
gatgatcaaa ggaacaacaa aaaggcaaga 1201 aatgagatgc aaaattgttc
aactagtact attctctcaa tggaaagaga aatcataaat 1261 aaagttgtgc
aagatgagac aatcaaaagt gaaaagttca acaacacaca aacaagagaa 1321
tgttactctc taatgactcc aaattacaca atggatgatc aatttggaac aaggttcaat
1381 aatcaaaatc atgaacaatt ggcaacaaca acaacttttc atcaaggaaa
tggtcatgtt 1441 tctcttactt tagggcttcc accaaattct gaaaaccaac
acaattacat tggattggaa 1501 aatcattaca atcaacctac acatcatcca
aatattagct atgaaaacat tgattttcag 1561 agtggaaagc gatacgccac
tcaactatta caagattttg tttcttgatg atatatataa 1621 tttgcaggta
aatcagcttg aaattacatc atgacaggtc ttgaataaaa gaaggggagt 1681
tgagatttag tgatcatata aatatgtata ggtagaaatt ttagttagta tatataggtt
1741 atacttctag tttcttaatg aagatacaag ttttgttgtt atttttgtat
tgaggtaact 1801 agctagcttg gattatttaa agttggtgca tgcaactaaa
gaagaagaaa aaataatcta 1861 tatatgcaaa ctacagtata ttgtaaattt
tgtgcttc
[0058] The nucleic acid sequence corresponding to SEQ ID NO:3
encodes a BEL transcription factor isolated from Solanum tuberosum
identified herein as StBEL-11, which has a deduced amino acid
sequence corresponding to SEQ ID NO:4 as follows:
TABLE-US-00004 Met Thr Phe Arg Ser Ser Leu Pro Leu Asp Leu Arg Glu
Ile Ser Thr 1 5 10 15 Thr Asn His Gln Val Gly Ile Leu Ser Ser Ser
Pro Leu Pro Ser Pro 20 25 30 Gly Thr Asn Thr Asn Asn Ile Asn His
Thr Arg Gly Leu Gly Ala Ser 35 40 45 Ser Ser Phe Ser Ile Ser Asn
Gly Met Ile Leu Gly Ser Lys Tyr Leu 10 55 60 Lys Val Ala Gln Asp
Leu Leu Asp Glu Val Val Asn Val Gly Lys Asn 65 70 75 80 Ile Lys Leu
Ser Asp Gly Leu Glu Ser Gly Ala Lys Glu Lys His Lys 85 90 95 Leu
Asp Asn Glu Leu Ile Ser Leu Ala Ser Asp Asp Val Glu Ser Ser 100 105
110 Ser Gln Lys Asn Ser Gly Val Glu Leu Thr Thr Ala Gln Arg Gln Glu
115 120 125 Leu Gln Met Lys Lys Ala Lys Leu Val Ser Met Leu Asp Glu
Val Asp 130 135 140 Gln Arg Tyr Arg Gln Tyr His His Gln Met Gln Met
Ile Ala Thr Ser 145 150 155 160 Phe Glu Gln Thr Thr Gly Ile Gly Ser
Ser Lys Ser Tyr Thr Gln Leu 165 170 175 Ala Leu His Thr Ile Ser Lys
Gln Phe Arg Cys Leu Lys Asp Ala Ile 180 185 190 Ser Gly Gln Ile Lys
Asp Thr Ser Lys Thr Leu Gly Glu Glu Glu Asn 195 200 205 Ile Gly Gly
Lys Ile Glu Gly Ser Lys Leu Lys Phe Val Asp His His 210 215 220 Leu
Arg Gln Gln Arg Ala Leu Gln Gln Leu Gly Met Met Gln Thr Asn 225 230
235 240 Ala Trp Lys Pro Gln Arg Gly Leu Pro Glu Arg Ala Val Ser Val
Leu 245 250 255 Arg Ala Trp Leu Phe Glu His Phe Leu His Pro Tyr Pro
Lys Asp Ser 260 265 270 Asp Lys Ile Ile Leu Ala Lys Gln Thr Gly Leu
Thr Arg Ser Gln Val 275 280 285 Ser Asn Trp Phe Ile Asn Ala Arg Val
Arg Leu Trp Lys Pro Met Val 290 295 300 Glu Glu Met Tyr Met Glu Glu
Val Lys Lys Asn Asn Gln Glu Gln Asn 305 310 315 320 Ile Glu Pro Asn
Asn Asn Glu Ile Val Gly Ser Lys Ser Ser Val Pro 325 330 335 Gln Glu
Lys Leu Pro Ile Ser Ser Asn Ile Ile His Asn Ala Ser Pro 340 345 350
Asn Asp Ile Ser Thr Ser Thr Ile Ser Thr Ser Pro Thr Gly Gly Gly 355
360 365 Gly Ser Ile Pro Thr Gln Thr Val Ala Gly Phe Ser Phe Ile Arg
Ser 370 375 380 Leu Asn Met Glu Asn Ile Asp Asp Gln Arg Asn Asn Lys
Lys Ala Arg 385 390 395 400 Asn Glu Met Gln Asn Cys Ser Thr Ser Thr
Ile Leu Ser Met Glu Arg 405 410 415 Gln Ile Ile Asn Lys Val Val Gln
Asp Gln Thr Ile Lys Ser Glu Lys 420 425 430 Phe Asn Asn Thr Gln Thr
Arg Glu Cys Tyr Ser Leu Met Thr Pro Asn 435 440 445 Tyr Thr Met Asp
Asp Gln Phe Gly Thr Arg Phe Asn Asn Gln Asn His 450 455 460 Gln Gln
Leu Ala Thr Thr Thr Thr Phe His Gln Gly Asn Gly His Val 465 470 475
480 Ser Leu Thr Leu Gly Len Pro Pro Asn Ser Gln Asn Gln His Asn Tyr
485 490 495 Ile Gly Leu Glu Asn His Tyr Asn Gln Pro Thr His His Pro
Asn Ile 500 505 510 Ser Tyr Glu Asn Ile Asp Phe Gln Ser Gly Lys Arg
Tyr Ala Thr Gln 515 520 525 Leu Leu Gln Asp Phe Val Ser 530 535
The BEL transcription factor has a molecular mass of approximately
59 kDa. StBEL-11, isolated from Solanum tuberosum, has a single
open reading frame ("ORF") of 1608 bp, extending between
nucleotides 1-1608.
[0059] In a third embodiment, the BEL transcription factor from
Solanum tuberosum is identified herein as StBEL-13 and is encoded
by a nucleic acid molecule having a nucleotide sequence of SEQ ID
NO:5 as follows:
TABLE-US-00005 1 ggggagcgag tggttccgac aaggtatggt aatgggtgga
ggtgcaagta 51 gtcaacaatt gggatatgca aaaaatcata ctcctaatgt
ggcggagtcc 101 atgcaacttt ttctaatgaa tccacaacca aggtcacctt
ctccatctcc 151 tcctaattca acttcttcta cgcttcacat gttgttacca
aacccatcat 201 ctacttcaac acttcaaggg tttcctaatc cggccgaagg
atctttcggt 251 caattcatta catgggggaa tggaggaqca agtgctgcca
cagccaccca 301 tcatctcaat gcccagaatg aaatcggagg agtaaacgtt
gtagaaagtc 351 aaggcctatc tctatccttg tcttcttcgt tacagcacaa
ggcggaggaa 401 ttacaaatga gcggagaagc tggaggaatg atgttcttca
atcaaggagg 451 gtctagtact tccgggcagt atcgatacaa gaatttgaat
atgggtggat 501 caggagtaag cccaaacatt catcaagtcc atgttgggta
tgggtcatca 551 ttaggagtgg tcaatgtgtt gaggaattcc aaatacgcga
aagctgccca 601 agaactactg gaagaattct gcagtgttgg aagaggtaaa
ttgaagaaga 651 ctaacaacaa agcagcagcc aataacccta atacgaaccc
tagtggcgct 701 aacaatgaag cttcttcaaa agatgttcct actttgtccg
ctgctgatag 751 aattgagcat cagagaagga aggtcaaact tttatctatg
gttgatgagg 801 tagataggag gtacaatcat tactgtgaac aaatgcagat
ggttgtaaat 851 tcgtttgatt tagtgatggg tttcggcaca gcagttccct
acacagcact 901 tgcacagaag gcaatgteaa gacatttcag gtgtttaaag
gatgcaatag 951 gagcacaatt gaagcagagt tgtgagttat taggagagaa
agatgcagga 1001 aattcgggat tgactaaagg agaaactccg aggcttaaga
tgcttgaaca 1051 aagtttgagg caacaaaggg cgtttcacca aatgggaatg
atggaacaag 1101 aagcttggag accacaaaga ggcttacctg aacgttctgt
caacatttta 1151 agagcttggc tttttgagca ttttctacac ccgtatccaa
gtgatgctga 1201 taaacatctg ttggcaagac agactggtct ctccagaaat
caggtatcaa 1251 attggttcat taatgctagg gttcggttgt ggaaacccat
ggtagaagat 1301 atgtatcaac aagaagccaa agatgaagat ggagatggag
atgagaagag 1351 ccaaagccaa aacagtggca ataacataat tgcacaaaca
ccaacgccta 1401 atagcctgac taacacttca tctactaata tgacgacgac
aacagcccct 1451 acaactacga cagctctagc tgctgcagag acaggaacag
ctgccactcc 1501 cataactgtt acctcaagca aaagatccca aatcaatgcc
acggatagtg 1551 acccttcact tgtagcaatc aattccttct ctgaaaacca
agctactttt 1601 ccgaccaaca ttcatgatcc cgacgattgc cgtcgcggca
acttatccgg 1651 tgacgacggg accaccacac atgatcatat ggggtccacc
atgataaggt 1701 ttgggaccac tgctggtgac gtgtcactca ccttagqgtt
acgacatgca 1751 ggaaatttac cagagaatac tcatttcttt ggttaattaa
tacgtatttt 1801 ccccatagta attaattaaa actgaatttg cttgagctca
tcataattta 1851 tgcattgctt tttgttataa gaaattccat aaattagctt
tgtgttaaaa 1901 aaaaaaaaaa aaaaaaaaaa
[0060] The nucleic acid sequence corresponding to SEQ ID NO:5
encodes a BEL transcription factor isolated from Solanum tuberosum
identified herein as StBEL-13, which has a deduced amino acid
sequence corresponding to SEQ ID NO:6 as follows:
TABLE-US-00006 Met Val Met Gly Gly Gly Ala Ser Ser Gln Gln Leu Gly
Tyr Ala Lys 1 5 10 15 Asn His Thr Pro Asn Val Ala Glu Ser Met Gln
Leu Phe Leu Met Asn 20 25 30 Pro Gln Pro Arg Ser Pro Ser Pro Ser
Pro Pro Asn Ser Thr Ser Ser 35 40 45 Thr Leu His Met Leu Leu Pro
Asn Pro Ser Ser Thr Ser Thr Leu Gln 50 55 60 Gly Phe Pro Asn Pro
Ala Glu Gly Ser Phe Gly Gln Phe Ile Thr Trp 65 70 75 80 Gly Asn Gly
Gly Ala Ser Ala Ala Thr Ala Thr His His Leu Asn Ala 85 90 95 Gln
Asn Gln Ile Gly Gly Val Asn Val Val Glu Ser Gln Gly Leu Ser 100 105
110 Leu Ser Leu Ser Ser Ser Leu Gln His Lys Ala Glu Glu Leu Gln Met
115 120 125 Ser Gly Gln Ala Gly Gly Met Met Phe Phe Asn Gln Gly Gly
Ser Ser 130 135 140 Thr Ser Gly Gln Tyr Arg Tyr Lys Asn Leu Asn Met
Gly Gly Ser Gly 145 150 155 160 Val Ser Pro Asn Ile His Gln Val His
Val Gly Tyr Gly Ser Ser Leu 165 170 175 Gly Val Val Asn Val Leu Arg
Asn Ser Lys Tyr Ala Lys Ala Ala Gln 180 185 190 Glu Leu Leu Gln Gln
Phe Cys Ser Val Gly Arg Gly Lys Leu Lys Lys 195 200 205 Thr Asn Asn
Lys Ala Ala Ala Asn Asn Pro Asn Thr Asn Pro Ser Gly 210 215 220 Ala
Asn Asn Gln Ala Ser Ser Lys Asp Val Pro Thr Leu Ser Ala Ala 225 230
235 240 Asp Arg Ile Glu His Gln Arg Arg Lys Val Lys Leu Leu Ser Met
Val 245 250 255 Asp Glu Val Asp Arg Arg Tyr Asn His Tyr Cys Gln Gln
Met Gln Met 260 265 270 Val Val Asn Ser Phe Asp Leu Val Met Gly Phe
Gly Thr Ala Val Pro 275 280 285 Tyr Thr Ala Leu Ala Gln Lys Ala Met
Ser Arg His Phe Arg Cys Leu 290 295 300 Lys Asp Ala Ile Gly Ala Gln
Leu Lys Gln Ser Cys Gln Leu Leu Gly 305 310 315 320 Gln Lys Asp Ala
Gly Asn Ser Gly Leu Thr Lys Gly Glu Thr Pro Arg 325 330 335 Leu Lys
Met Leu Gln Gln Ser Leu Arg Gln Gln Arg Ala Phe His Gln 340 345 350
Met Gly Met Met Gln Gln Glu Ala Trp Arg Pro Gln Arg Gly Leu Pro 355
360 365 Gln Arg Ser Val Asn Ile Leu Arg Ala Trp Leu Phe Glu His Phe
Leu 370 375 380 His Pro Tyr Pro Ser Asp Ala Asp Lys His Leu Leu Ala
Arg Gln Thr 385 390 395 400 Gly Leu Ser Arg Asn Gln Val Per Asn Trp
Phe Ile Asn Ala Arg Val 405 410 415 Arg Leu Trp Lys Pro Met Val Glu
Asp Met Tyr Gln Gln Glu Ala Lys 420 425 430 Asp Glu Asp Gly Asp Gly
Asp Glu Lys Per Gln Ser Gln Asn Ser Gly 435 440 445 Asn Asn Ile Ile
Ala Gln Thr Pro Thr Pro Asn Ser Leu Thr Asn Thr 450 455 460 Ser Ser
Thr Asn Met Thr Thr Thr Thr Ala Pro Thr Thr Thr Thr Ala 465 470 475
480 Leu Ala Ala Ala Glu Thr Gly Thr Ala Ala Thr Pro Ile Thr Val Thr
485 490 495 Ser Ser Lys Arg Ser Gln Ile Asn Ala Thr Asp Ser Asp Pro
Ser Leu 500 505 510 Val Ala Ile Asn Ser Phe Ser Glu Asn Gln Ala Thr
Phe Pro Thr Asn 515 520 525 Ile His Asp Pro Asp Asp Cys Arg Arg Gly
Asn Leu Ser Gly Asp Asp 530 535 540 Gly Thr Thr Thr His Asp His Met
Gly Ser Thr Met Ile Arg Phe Gly 545 550 555 560 Thr Thr Ala Gly Asp
Val Per Leu Thr Leu Gly Leu Arg His Ala Gly 565 570 575 Asn Leu Pro
Glu Asn Thr His Phe Phe Gly 580 585
The BEL transcription factor has a molecular mass of approximately
64.5 kDa. StBEL-13, isolated from Solanum tuberosum, has a single
open reading frame ("ORF") of 1759 bp, extending between
nucleotides 26-1784.
[0061] In a fourth embodiment, the BEL transcription factor from
Solanum tuberosum is identified herein as StBEL-14 and is encoded
by a nucleic acid molecule having a nucleotide sequence of SEQ ID
NO:7 as follows:
TABLE-US-00007 1 aaccnaaaaa agagatcqaa ttcggcacga gtgatcatgg
tccttcgtct 51 tctaagaaca ttattagtga acaattttac caacatggta
gtcatgaaaa 101 tatgttgaca acaacaacta ctcatcatga tgatcatcaa
ggctcgtggc 151 atcacgataa taacagaaca ttacttgttg atgatccatc
tatgagatgt 201 gttttccctt gtgaaggaaa tgaaaggcca agteatggac
tttcattatc 251 tctttgttcc tcaaatccat caagtattgg tttacaatct
tttgaactta 301 gacatcaaga tttgcaacaa ggattaatac atgatggatt
tttgggtaaa 351 tctacaaata tacaacaagg gtattttcat catcatcatc
aagttaggga 401 ctcgaaatat ttaggtccgg ctcaagagtt gctcagtgag
ttctgtagtc 451 tcggaataaa qaagaataat gatcattctt cttcaaaagt
acttctaaag 501 caacatgaga gtactqctag tacttcaaaa aagcaacttt
tacagtctct 551 tgaccttttg gaacttcaaa aaagaaagac aaaattgctt
caaatgcttg 601 aagaggtgga tagaaggtac aagcattatt gtgatcaaat
gaaggctgtt 651 gtatcatcat ttgaagcagt ggctggaaat ggagcagcaa
cagtttactc 701 agccttagca tcaagggcta tgtcaaggca ttttagatgt
ttaagagatg 751 gaattgtggc acaaattaag gccacaaaaa tggctatggg
agaaaaagac 601 agtactagta ctcttattcc tggttcaaca agaggtgaaa
caccaagact 851 cagacttctt gatcaaactt taaggcaaca aaaggctttc
caacagatga 901 atatgatgga gacteateca tggagaccgc aacgtggtct
cccagaaaga 951 tcagtctccg ttctccgcgc ttggctcttt gaacactttc
ttcacccgta 1001 cccaagtgat gttgataaac acattttagc tcgccaaact
ggtctttcaa 1051 gaagccaggt gtctaattgg ttcattaatg caagggtaag
gctatggaag 1101 ccaatggtgg aagaaatgta cttagaagaa acaaaagaag
aagaaaatgt 1151 tggatctcca gatggatcaa aagccctaat tgatgacatg
acaattcatc 1201 aatcacacat tgatcatcat caagctgatc aaaagccaaa
tcttgtaaga 1251 attgactctg aatgcatatc ttccatcata aatcatcaac
ctcatgagaa 1301 aaatgatcaa aactatggag taattagagg tggagatcaa
tcgtttggcg 1351 cgattgagct agatttttca acaaatattg cttatggtac
tagtggtggt 1401 gaccatcatc atcatggagg gggtgtttct ttaacattgg
gattacaaca 1451 acatggtgga agtggtggat catcaatggg gttaactaca
ttttcatcac 1501 aaccatctca taatcaaagt tcactttttt atccaagaga
tgatgatcaa 1551 gttcaatatt catcactttt ggatagtgaa aatcagaatt
tgccatatag 1601 aaaccttgat gggggcacaa cttcttcatg atttggctgg
ttaaaaaatg 1651 acagagatte ttcattttgg accttattat atactctaat
tttaatatat 1701 attggtgatg aatgatgata aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1751 aaaaaaaaaa acctcgancc cggtcgactn tanancecta
tagngagtcg 1801 tnttnctgca nanatctntg aatcgtaaat nctgaaaaac
cccgcaagtt 1851 cacttcaact gngcatcgng cnccatctca atttctttca
tttatncatc 1901 gttttgcctt nttttatgta actatnctcc tntaagtttc
aatcttggcc 1951 atgtaacctn tgatctntaa aattttttaa atgactanaa
ttaatgccca 2001 tntttttttt ggacctaaat tnttcatgaa aatntnttnc
nagggcttnt 2051 tcaaaanctt tggacttntt cnccanaggt ttggtcaagt
ntccaatcaa 2101 ggt
[0062] The nucleic acid sequence corresponding to SEQ ID NO:7
encodes a BEL transcription factor isolated from Solanum tuberosum
identified herein as StBEL-14, which has a deduced amino acid
sequence corresponding to SEQ ID NO:8 as follows:
TABLE-US-00008 Met Val Asn His Gln Leu Gln Asn Phe Glu Thr Asn Pro
Glu Met Tyr 1 5 10 15 Asn Leu Ser Ser Thr Thr Ser Ser Met Asp Gln
Met Ile Gly Phe Pro 20 25 30 Pro Asn Asn Asn Asn Pro His His Val
Leu Trp Lys Gly Asn Phe Pro 35 40 45 Asn Lys Ile Asn Gly Val Asp
Asp Asp Asp His Gly Pro Ser Ser Ser 50 55 60 Lys Asn Ile Ile Ser
Glu Gln Phe Tyr Gln His Gly Ser His Glu Asn 65 70 75 80 Met Leu Thr
Thr Thr Thr Thr His His Asp Asp His Gln Gly Ser Trp 85 90 95 His
His Asp Asn Asn Arg Thr Leu Leu Val Asp Asp Pro Ser Met Arg 100 105
110 Cys Val Phe Pro Cys Glu Gly Asn Glu Arg Pro Ser His Gly Leu Ser
115 120 125 Leu Ser Leu Cys Ser Ser Asn Pro Ser Ser Ile Gly Leu Gln
Ser Phe 130 135 140 Glu Leu Arg His Gln Asp Leu Gln Gln Gly Leu Ile
His Asp Gly Phe 145 150 155 160 Leu Gly Lys Ser Thr Asn Ile Gln Gln
Gly Tyr Phe His His His His 165 170 175 Gln Val Arg Asp Ser Lys Tyr
Leu Gly Pro Ala Gln Glu Leu Leu Ser 180 185 190 Glu Phe Cys Ser Leu
Gly Ile Lys Lys Asn Asn Asp His Ser Ser Ser 195 200 205 Lys Val Leu
Leu Lys Gln His Glu Ser Thr Ala Ser Thr Ser Lys Lys 210 215 220 Gln
Leu Leu Gln Ser Leu Asp Leu Leu Glu Leu Gln Lys Arg Lys Thr 225 230
235 240 Lys Leu Leu Gln Met Leu Glu Glu Val Asp Arg Arg Tyr Lys His
Tyr 245 250 255 Cys Asp Gln Met Lys Ala Val Val Ser Ser Phe Glu Ala
Val Ala Gly 260 265 270 Asn Gly Ala Ala Thr Val Tyr Ser Ala Leu Ala
Ser Arg Ala Met Ser 275 280 285 Arg His Phe Arg Cys Leu Arg Asp Gly
Ile Val Ala Gln Ile Lys Ala 290 295 300 Thr Lys Met Ala Met Gly Glu
Lys Asp Ser Thr Ser Thr Leu Ile Pro 305 310 315 320 Gly Ser Thr Arg
Gly Glu Thr Pro Arg Leu Arg Leu Leu Asp Gln Thr 325 330 335 Leu Arg
Gln Gln Lys Ala Phe Gln Gln Met Asn Met Met Glu Thr His 340 345 350
Pro Trp Arg Pro Gln Arg Gly Leu Pro Glu Arg Ser Val Ser Val Leu 355
360 365 Arg Ala Trp Leu Phe Glu His Phe Leu His Pro Tyr Pro Ser Asp
Val 370 375 380 Asp Lys His Ile Leu Ala Arg Gln Thr Gly Leu Ser Arg
Ser Gln Val 385 390 395 400 Ser Asn Trp Phe Ile Asn Ala Arg Val Arg
Leu Trp Lys Pro Met Val 405 410 415 Gln Glu Met Tyr Leu Gln Glu Thr
Lys Gln Glu Gln Asn Val Gly Ser 420 425 430 Pro Asp Gly Ser Lys Ala
Leu Ile Asp Asp Met Thr Ile His Gln Ser 435 440 445 His Ile Asp His
His Gln Ala Asp Gln Lys Pro Asn Leu Val Arg Ile 450 455 460 Asp Ser
Gln Cys Ile Ser Ser Ile Ile Asn His Gln Pro His Gln Lys 465 470 475
480 Asn Asp Gln Asn Tyr Gly Val Ile Arg Gly Gly Asp Gln Ser Phe Gly
485 490 495 Ala Ile Gln Leu Asp Phe Ser Thr Asn Ile Ala Tyr Gly Thr
Ser Gly 500 505 510 Gly Asp His His His His Gly Gly Gly Val Ser Leu
Thr Leu Gly Leu 515 520 525 Gln Gln His Gly Gly Ser Gly Gly Ser Ser
Met Gly Leu Thr Thr Phe 530 535 540 Ser Ser Gln Pro Ser His Asn Gln
Ser Ser Leu Phe Tyr Pro Arg Asp 545 550 555 560 Asp Asp Gln Val Gln
Tyr Ser Ser Leu Leu Asp Ser Gln Asn Gln Asn 565 570 575 Leu Pro Tyr
Arg Asn Leu Asp Gly Gly Thr Thr Ser Ser 580 585
The BEL transcription factor has a molecular mass of approximately
64.8 kDa. StBEL-14, isolated from Solanum tuberosum, has a single
open reading frame ("ORF") of 1768 bp, extending between
nucleotides 85-1852.
[0063] In a fifth embodiment, the BEL transcription factor from
Solanum tuberosum is identified herein as StBEL-22 and is encoded
by a nucleic acid molecule having a nucleotide sequence of SEQ ID
NO:9 as follows:
TABLE-US-00009 1 acgagcgttt atgagacagc cgggttgttg tctgaaatgt
tcaattttca gacaacatcc 61 acggctgcaa ctgaattgtt gcagaatcaa
ttgtcaaata actatagaca cccgaatcaa 121 cagccacatc atcaacctcc
gaccagggag tggtttggta acagacaaga gatcgtagtt 181 ggtggaagtt
tgcaggtaac atttggggat acaaaagatg atgtgaatgc gaaggtatta 241
ttgagtaacc gtgatagtgt aactgattat tatcagcgtc aacacaatca agtaccaagt
301 ataaataccg cggagtccat gcaacttttt cttatgaatc cacaaccaag
ttcaccatca 361 caatctactc cttcaactct tcatcaaggg ttttctagcc
cggtcggagg gcattttagt 421 caattcatgt gtggaggagc aagtacttct
tcaaatccaa ttggaggagt aaatgtgatt 481 gatcaagggc aaggtctttc
attgtccttg tcatctactt tacaacattt ggaagcatcc 541 aaagtggaag
atttgaggat gaatagtgga ggagaaatgt tgtttttcaa tcaagaaagt 601
caaaatcatc ataatattgg ttttgggtca tcactaggac tagtcaatgt gttgaggaat
661 tcaaagtatg tcaaagcaac acaagagttg ttggaagagt tttgttgtgt
tgggaagggt 721 caattgttca agaaaatcaa caaagtttct aggaataaca
acacaagtac atcacccatt 781 attaacccta gtggaagtaa taacaataat
tcatcttctt caaaggctat tatccctcct 841 aatttgtcaa ctgcagagag
acttgatcat caaagaagga aggtcaaact tttatccatg 901 cttgatgagg
tagagaaaag atacaaccac tattgtgaac aaatgcagat ggtagtaaac 961
tcattcgatc tagtgatggg ttttggagct gcagttcctt acacagcact agcacagaaa
1021 gccatgtcta ggcatttcaa gtgtttaaaa gatggcgtgg cggcgcaatt
gaagaagaca 1081 tgtgaggcac taggtgaaaa agatgcaagc agtagttcag
gactgactaa aggagaaaca 1141 ccaaggctta aggtgcttga acaaagcttg
aggcaacaaa gagcttttca acaaatggga 1201 atgatggaac aagaagcttg
gaggccacaa agaggattgc ctgaacgatc tgtcaatatt 1261 ttaagagctt
ggcttttcga acattttcta catccgtatc caagtgatgc agataagcat 1321
cttttggcac gacagactgg tctctccaga aaccaggtag caaactggtt cataaatgcg
1381 agggtgagat tgtggaaacc catggtagaa gaaatgtatc aaagagaggt
taatgaagat 1441 gatgttgatg acatgcaaga aaaccaaaac agtacaaata
cacaaatacc aacgcctaat 1501 attattatta caaccaattc taacattaca
gaaacaaaat cagctgccac tgccacaatt 1561 gcttcagaca aaaaacccca
aatcaatgtc tctgaaattg acccttcaat tgtcgcaatg 1621 aatacacatt
attcttcctc tatgccaact caattaacca atttccccac tattcaagat 1681
gagtccgacc acatcttata tcgccgcagt ggagcggaat atgggaccac aaatatggct
1741 agtaattctg aaattggatc caacatgata acatttggga ccactacggc
tagtgatgtt 1801 tcacttacct taggactgcg ccatgcgggt aatttacctg
agaatactca tttttccggt 1861 taattaagat agtgtattca aacactgcta
cataaattat gattttatat atatatatat 1921 tgtcatccga ttagtttat
[0064] The nucleic acid sequence corresponding to SEQ ID NO:9
encodes a BEL transcription factor isolated from Solanum tuberosum
identified herein as StBEL-22, which has a deduced amino acid
sequence corresponding to SEQ ID NO:10 as follows:
TABLE-US-00010 Thr Ser Val Tyr Glu Thr Ala Gly Leu Leu Ser Gln Met
Phe Asn Phe 1 5 10 15 Gln Thr Thr Ser Thr Ala Ala Thr Glu Leu Leu
Gln Asn Gln Leu Ser 20 25 30 Asn Asn Tyr Arg His Pro Asn Gln Gln
Pro His His Gln Pro Pro Thr 35 40 45 Arg Gln Trp Phe Gly Asn Arg
Gln Glu Ile Val Val Gly Gly Ser Leu 50 55 60 Gln Val Thr Phe Gly
Asp Thr Lys Asp Asp Val Asn Ala Lys Val Leu 65 70 75 80 Leu Ser Asn
Arg Asp Ser Val Thr Asp Tyr Tyr Gln Arg Gln His Asn 85 90 95 Gln
Val Pro Ser Ile Asn Thr Ala Glu Ser Met Gln Leu Phe Leu Met 100 105
110 Asn Pro Gln Pro Ser Ser Pro Ser Gln Ser Thr Pro Ser Thr Leu His
115 120 125 Gln Gly Phe Ser Ser Pro Val Gly Gly His Phe Ser Gln Phe
Met Cys 130 135 140 Gly Gly Ala Ser Thr Ser Ser Asn Pro Ile Gly Gly
Val Asn Val Ile 145 150 155 160 Asp Gln Gly Gln Gly Leu Ser Leu Ser
Leu Ser Ser Thr Leu Gln His 165 170 175 Leu Gln Ala Ser Lys Val Gln
Asp Leu Arg Met Asn Ser Gly Gly Gln 180 185 190 Met Leu Phe Phe Asn
Gln Glu Ser Gln Asn His His Asn Ile Gly Phe 195 200 205 Gly Ser Ser
Leu Gly Leu Val Asn Val Leu Arg Asn Per Lys Tyr Val 210 215 220 Lys
Ala Thr Gln Gln Leu Leu Glu Gln Phe Cys Cys Val Gly Lys Gly 225 230
235 240 Gln Leu Phe Lys Lys Ile Asn Lys Val Ser Arg Asn Asn Asn Thr
Ser 245 250 255 Thr Ser Pro Ile Ile Asn Pro Ser Gly Ser Asn Asn Asn
Asn Ser Ser 260 265 270 Ser Ser Lys Ala Ile Ile Pro Pro Asn Leu Per
Thr Ala Gln Arg Leu 275 280 285 Asp His Gln Arg Arg Lys Val Lys Leu
Leu Ser Met Leu Asp Gln Val 290 295 300 Gln Lys Arg Tyr Asn His Tyr
Cys Gln Gln Met Gln Met Val Val Asn 305 310 315 320 Ser Phe Asp Leu
Val Met Gly Phe Gly Ala Ala Val Pro Tyr Thr Ala 325 330 335 Leu Ala
Gln Lys Ala Met Ser Arg His Phe Lys Cys Leu Lys Asp Gly 340 345 350
Val Ala Ala Gln Leu Lys Lys Thr Cys Gln Ala Leu Gly Gln Lys Asp 355
360 365 Ala Ser Ser Ser Ser Gly Leu Thr Lys Gly Gln Thr Pro Arg Leu
Lys 370 375 380 Val Leu Gln Gln Ser Leu Arg Gln Gln Arg Ala Phe Gln
Gln Met Gly 385 390 395 400 Met Met Glu Gln Glu Ala Trp Arg Pro Gln
Arg Gly Leu Pro Glu Arg 405 410 415 Ser Val Asn Ile Leu Arg Ala Trp
Leu Phe Gln His Phe Leu His Pro 420 425 430 Tyr Pro Ser Asp Ala Asp
Lys His Leu Leu Ala Arg Gln Thr Gly Leu 435 440 445 Ser Arg Asn Gln
Val Ala Asn Trp Phe Ile Asn Ala Arg Val Arg Leu 450 455 460 Trp Lys
Pro Met Val Glu Glu Met Tyr Gln Arg Gln Val Asn Glu Asp 465 470 475
480 Asp Val Asp Asp Met Gln Glu Asn Gln Asn Ser Thr Asn Thr Gln Ile
485 490 495 Pro Thr Pro Asn Ile Ile Ile Thr Thr Asn Ser Asn Ile Thr
Glu Thr 500 505 510 Lys Ser Ala Ala Thr Ala Thr Ile Ala Ser Asp Lys
Lys Pro Gln Ile 515 520 525 Asn Val Ser Glu Ile Asp Pro Ser Ile Val
Ala Met Asn Thr His Tyr 530 535 540 Ser Ser Ser Met Pro Thr Gln Leu
Thr Asn Phe Pro Thr Ile Gln Asp 545 550 555 560 Glu Ser Asp His Ile
Leu Tyr Arg Arg Ser Gly Ala Glu Tyr Gly Thr 565 570 575 Thr Asn Met
Ala Ser Asn Ser Glu Ile Gly Ser Asn Met Ile Thr Phe 580 585 590 Gly
Thr Thr Thr Ala Ser Asp Val Ser Leu Thr Leu Gly Leu Arg His 595 600
605 Ala Gly Asn Leu Pro Glu Asn Thr His Phe Ser Gly 610 615 620
The BEL transcription factor has a molecular mass of approximately
67.3 kDa. StBEL-22, isolated from Solanum tuberosum, has a single
open reading frame ("ORF") of 1863 bp, extending between
nucleotides 1-1863.
[0065] In a sixth embodiment, the BEL transcription factor from
Solanum tuberosum is identified herein as StBEL-29 and is encoded
by a nucleic acid molecule having a nucleotide sequence of SEQ ID
NO:11 as follows:
TABLE-US-00011 1 caagggcttt cacttagcct gtcctcgtcc cagcagccgg
ggtttgggaa cttcacggcg 61 gcgcgtgagc ttgtttcttc gccttcgggt
tcggcttcag cttcagggat acaacaacaa 121 caacagcaac aacagagtat
tagtagtgtg cctttgagtt ctaagtacat gaaggctgca 181 caagagctac
ttgatgaagt tgtaaatgtt ggaaaatcaa tgaaaagtac taatagtact 241
gatgttgttg ttaataatga tgtcaagaaa tcgaagaata tgggcgatat ggacggacag
301 ttagacggag ttggagcaga caaagacgga gctccaacaa ctgagctaag
tacaggggag 361 agacaagaaa ttcaaatgaa gaaagcaaaa cttgttaaca
tgcttgacga ggtggagcag 421 aggtatagac attatcatca ccaaatgcag
tcagtgatac attggttaga gcaagctgct 481 ggcattggat cagcaaaaac
atatacagca ttggctttgc agacgatttc gaagcaattt 541 aggtgtctta
aggacgcgat aattggtcaa atacgatcag caagccagac gttaggcgaa 601
gaagatagtt tgggagggaa gattgaaggt tcaaggctta aatttgttga taatcagcta
661 agacagcaaa gggctttgca acaattggga atgatccagc ataatgcttg
gagacctcag 721 agaggattgc ccgaacgagc tgtttctgtt cttcgcgctt
ggctttttga acatttcctc 781 catccttatc ccaaggattc agacaaaatg
atgctagcaa aacaaacagg actaactagg 841 agtcaggtgt cgaattggtt
catcaatgct cgagttcgtc tttggaagcc aatggtggaa 901 gagatgtact
tggaagagat aaaagaacac gaacagaatg ggttgggtca agaaaagacg 961
agcaaattag gtgaacagaa cgaagattca acaacatcaa gatccattgc tacacaagac
1021 aaaagccctg gttcagatag ccaaaacaag agttttgtct caaaacagga
caatcatttg 1081 cctcaacaca accctgcttc accaatgccc gatgtccaac
gccacttcca tacccctatc 1141 ggtatgacca tccgtaatca gtctgctggt
ttcaacctca ttggatcacc agagatcgaa 1201 agcatcaaca ttactcaagg
gagtccaaag aaaccgagga acaacgagat gttgcattca 1261 ccaaacagca
ttccatccat caacatggat gtaaagccta acgaggaaca aatgtcgatg 1321
aagtttggtg atgataggca ggacagagat ggattctcac taatgggagg accgatgaac
1381 ttcatgggag gattcggagc ctatcccatt ggagaaattg ctcggtttag
caccgagcaa 1441 ttctcagcac catactcaac cagtggcaca gtttcactca
ctcttggcct accacataac 1501 gaaaacctct caatgtctgc aacacaccac
agtttccttc caattccaac acaaaacatc 1561 caaattggaa gtgaaccaaa
tcatgagttt ggtagcttaa acacaccaac atcagctcac 1621 tcaacatcaa
gcgtctatga aaccttcaac attcagaaca gaaagaggtt cgccgcaccc 1681
ttgttaccag attttgttgc ctgatcacaa aaacaaaaac aggttttggc aacagacaaa
1741 cttctgtcgc taaacaagga catgatttag cgacagataa cttcagtcgc
taacttagcg 1801 actgaaaact tctgtcgcta agcatgaaca tgtattagcg
acatacagta tgcaactgta 1881 tgtcactaaa caagaacatg atgaattagt
gacggacaac ttctgtcgct aaacaacaaa 1921 aaaaaatcca tgttttagta
tattgtttct cattctatca tatcatggta gtgtaaagaa 1981 tcaagaaaca
agttttacat agtaacagtc tttatacatt ggagatgaag aaccatttaa 2041
gttcttcaaa atagatagat tttctaggtt acttctanaa gatatatata tggttgaggg
2101 tttgtatatt aaaaaaaaaa aaaaaaaa
[0066] The nucleic acid sequence corresponding to SEQ ID NO:11
encodes a BEL transcription factor isolated from Solanum tuberosum
identified herein as StBEL-29, which has a deduced amino acid
sequence corresponding to SEQ ID NO:12 as follows:
TABLE-US-00012 Gln Gly Leu Ser Leu Ser Leu Ser Ser Ser Gln Gln Pro
Gly Phe Gly 1 5 10 15 Asn Phe Thr Ala Ala Arg Glu Leu Val Ser Ser
Pro Ser Gly Ser Ala 20 25 30 Ser Ala Ser Gly Ile Gln Gln Gln Gln
Gln Gln Gln Gln Ser Ile Ser 35 40 45 Ser Val Pro Leu Ser Ser Lys
Tyr Met Lys Ala Ala Gln Glu Leu Leu 50 55 60 Asp Glu Val Val Asn
Val Gly Lys Ser Met Lys Ser Thr Asn Ser Thr 65 70 75 80 Asp Val Val
Val Asn Asn Asp Val Lys Lys Ser Lys Asn Met Gly Asp 85 90 95 Met
Asp Gly Gln Leu Asp Gly Val Gly Ala Asp Lys Asp Gly Ala Pro 100 105
110 Thr Thr Glu Leu Ser Thr Gly Glu Arg Gln Glu Ile Gln Met Lys Lys
115 120 125 Ala Lys Leu Val Asn Met Leu Asp Glu Val Glu Gln Arg Tyr
Arg His 130 135 140 Tyr His His Gln Met Gln Ser Val Ile His Trp Leu
Glu Gln Ala Ala 145 150 155 160 Gly Ile Gly Ser Ala Lys Thr Tyr Thr
Ala Leu Ala Leu Gln Thr Ile 165 170 175 Ser Lys Gln Phe Arg Cys Leu
Lys Asp Ala Ile Ile Gly Gln Ile Arg 180 185 190 Ser Ala Ser Gln Thr
Leu Gly Glu Glu Asp Ser Leu Gly Gly Lys Ile 195 200 205 Glu Gly Ser
Arg Leu Lys Phe Val Asp Asn Gln Leu Arg Gln Gln Arg 210 215 220 Ala
Leu Gln Gln Leu Gly Met Ile Gln His Asn Ala Trp Arg Pro Gln 225 230
235 240 Arg Gly Leu Pro Glu Arg Ala Val Ser Val Leu Arg Ala Trp Leu
Phe 245 250 255 Glu His Phe Leu His Pro Tyr Pro Lys Asp Ser Asp Lys
Met Met Leu 260 265 270 Ala Lys Gln Thr Gly Leu Thr Arg Ser Gln Val
Ser Asn Trp Phe Ile 275 280 285 Asn Ala Arg Val Arg Leu Trp Lys Pro
Met Val Glu Glu Met Tyr Leu 290 295 300 Glu Glu Ile Lys Glu His Glu
Gln Asn Gly Leu Gly Gln Glu Lys Thr 305 310 315 320 Ser Lys Leu Gly
Glu Gln Asn Glu Asp Ser Thr Thr Ser Arg Ser Ile 325 330 335 Ala Thr
Gln Asp Lys Ser Pro Gly Ser Asp Ser Gln Asn Lys Ser Phe 340 345 350
Val Ser Lys Gln Asp Asn His Leu Pro Gln His Asn Pro Ala Ser Pro 355
360 365 Met Pro Asp Val Gln Arg His Phe His Thr Pro Ile Gly Met Thr
Ile 370 375 380 Arg Asn Gln Ser Ala Gly Phe Asn Leu Ile Gly Ser Pro
Glu Ile Glu 385 390 395 400 Ser Ile Asn Ile Thr Gln Gly Ser Pro Lys
Lys Pro Arg Asn Asn Glu 405 410 415 Met Leu His Ser Pro Asn Ser Ile
Pro Ser Ile Asn Met Asp Val Lys 420 425 430 Pro Asn Glu Glu Gln Met
Ser Met Lys Phe Gly Asp Asp Arg Gln Asp 435 440 445 Arg Asp Gly Phe
Ser Leu Met Gly Gly Pro Met Asn Phe Met Gly Gly 450 455 460 Phe Gly
Ala Tyr Pro Ile Gly Glu Ile Ala Arg Phe Ser Thr Glu Gln 465 470 475
480 Phe Ser Ala Pro Tyr Ser Thr Ser Gly Thr Val Ser Leu Thr Leu Gly
485 490 495 Leu Pro His Asn Glu Asn Leu Ser Met Ser Ala Thr His His
Ser Phe 500 505 510 Leu Pro Ile Pro Thr Gln Asn Ile Gln Ile Gly Ser
Glu Pro Asn His 515 520 525 Glu Phe Gly Ser Leu Asn Thr Pro Thr Ser
Ala His Ser Thr Ser Ser 530 535 540 Val Tyr Glu Thr Phe Asn Ile Gln
Asn Arg Lys Arg Phe Ala Ala Pro 545 550 555 560 Leu Leu Pro Asp Phe
Val Ala 565
The BEL transcription factor has a molecular mass of approximately
56.2 kDa. StBEL-29, isolated from Solanum tuberosum, has a single
open reading frame ("ORF") of 1704 bp, extending between
nucleotides 1-1704.
[0067] In a seventh embodiment, the BEL transcription factor from
Solanum tuberosum is identified herein as StBEL-30 and is encoded
by a nucleic acid molecule having a nucleotide sequence of SEQ ID
NO:13 as follows:
TABLE-US-00013 1 atctccaagt aaaaaggtta ttgagaaaag taacacagat
ggcgacttat tttcctagtc 61 caaacaatca aagagatgct gatcagacat
ttcaatattt taggcaatct ttgcctgagt 121 cttattcaga agcttcaaat
gctccagaaa acatgatggt attcatgaac tattcttctt 181 ctggggcata
ttcagatatg ttgacgggta cttcccaaca acaacacaac tgcatcgata 241
tcccatctat aggagccacg cctttcaaca catcccaaca agaaatattg tcaaatcttg
301 gaggatcgca gatggggatt caggattttt cttcatggag agatagcaga
aatgagatgc 361 tagctgataa tgtctttcaa gttgcacaaa atgtgcaggg
tcaaggatta tccctcagtc 421 ttggctccaa tataccatct ggaattggaa
tttcacatgt ccaatctcag aatcctaacc 481 aaggtggcgg ttttaacatg
tcctttggag atggtgataa ttcccaacca aaagaacaaa 541 gaaatgcaga
ttattttcct ccggataatc ctggaaggga cttggatgct atgaaagggt 601
ataattctcc atatggtacg tcgagtattg caaggaccat tcccagctcg aagtatttga
661 aagcagctca atatttgctt gatgaggttg ttagtgtcag aaaggccatc
aaggagcaaa 721 attctaagaa agagttgaca aaggattcca gagagtctga
tgtggactcg aaaaatatat 781 catcagatac tcctgcaaat gggggttcaa
atcctcatga gtccaaaaac aaccaaagtg 841 aactttcacc taccgagaag
caagaagtgc agaacaaact ggccaaactt ctgtcaatgc 901 tggatgagat
tgatagaagg tacagacaat attatcatca gatgcaaata gtggtttcat 961
catttgatgt ggtagctgga gaaggagcag ctaaaccata cacagctctt gctctccaga
1021 caatttcccg acacttccgt tgcttgcgtg atgcaatctg cgatcagatt
cgagcatcac 1081 gaagaagtct tggagagcaa gatgcttcag aaaacagcaa
agcgattgga atatcacgcc 1141 tgcgttttgt ggatcatcat attagacage
agagagccct gcagcagctt ggtatgatgc 1201 aacaacatgc ctggaggcct
cagaggggat tgcctgaaag ctctgtttca gttttgcgtg 1261 cttggctctt
tgagcacttt cttcatccct acccgaaaga ttctgacaaa attatgctag 1321
caaggcaaac tggcttaacg agaagtcagg tatcaaattg gttcataaat gcacgggtgc
1381 gtctttggaa acccatggtt gaggaaatgt acaaagaaga ggctggtgat
gctaaaatag 1441 actcaaattc ttcatcggat gttgccccca gacttgcaac
aaaagactca aaagttgaag 1501 aaagaggaga attgcaccag aatgcagctt
cagaatttga gcagtacaat agtggccaaa 1561 tcctggagtc aaaatctaac
catgaagctg atgtagaaat ggagggagca agtaatgcag 1621 aaactcaaag
tcaatctgga atggaaaacc aaacaggcga acccctgcct gctatggata 1681
attgcaccct ttttcaggac gcatttgttc aaagcaacga tagattctca gaatttggta
1741 gttttggaag tggaaatgta ctacccaatg gagtttcact tacattgggg
ctgcagcaag 1801 gtgaaggaag caacctacct atgtccatcg aaactcacgt
tagttatgta ccattaaggg 1861 cagatgacat gtatagtaca gcacctacta
ctatggtccc tgaaacagca gaattcaact 1921 gcttggattc tgggaatagg
cagcaaccat tttggctcct accatctgct acatgatttt 1981 gtatgtgttg
tagaattaaa ctgcaagttt tgagtacatc aacattcatc ttcaaaaaaa 2041
aaaaaaaaaa aaaaaaaaaa aaaaa
[0068] The nucleic acid sequence corresponding to SEQ ID NO:13
encodes a BEL transcription factor isolated from Solanum tuberosum
identified herein as StBEL-30, which has a deduced amino acid
sequence corresponding to SEQ ID NO:14 as follows:
TABLE-US-00014 Met Ala Thr Tyr Phe Pro Ser Pro Asn Asn Gln Arg Asp
Ala Asp Gln 1 5 10 15 Thr Phe Gln Tyr Phe Arg Gln Ser Leu Pro Gln
Ser Tyr Ser Gln Ala 20 25 30 Ser Asn Ala Pro Gln Asn Met Met Val
Phe Met Asn Tyr Ser Ser Ser 35 40 45 Gly Ala Tyr Ser Asp Met Leu
Thr Gly Thr Ser Gln Gln Gln His Asn 50 55 60 Cys Ile Asp Ile Pro
Ser Ile Gly Ala Thr Pro Phe Asn Thr Ser Gln 65 70 75 80 Gln Glu Ile
Leu Ser Asn Leu Gly Gly Ser Gln Met Gly Ile Gln Asp 85 90 95 Phe
Ser Ser Trp Arg Asp Ser Arg Asn Gln Met Leu Ala Asp Asn Val 100 105
110 Phe Gln Val Ala Gln Asn Val Gln Gly Gln Gly Leu Ser Leu Ser Leu
115 120 125 Gly Ser Asn Ile Pro Ser Gly Ile Gly Ile Ser His Val Gln
Ser Gln 130 135 140 Asn Pro Asn Gln Gly Gly Gly Phe Asn Met Ser Phe
Gly Asp Gly Asp 145 150 155 160 Asn Ser Gln Pro Lys Glu Gln Arg Asn
Ala Asp Tyr Phe Pro Pro Asp 165 170 175 Asn Pro Gly Arg Asp Leu Asp
Ala Met Lys Gly Tyr Asn Ser Pro Tyr 180 185 190 Gly Thr Ser Ser Ile
Ala Arg Thr Ile Pro Ser Ser Lys Tyr Leu Lys 195 200 205 Ala Ala Gln
Tyr Leu Leu Asp Glu Val Val Ser Val Arg Lys Ala Ile 210 215 220 Lys
Glu Gln Asn Ser Lys Lys Glu Leu Thr Lys Asp Ser Arg Glu Ser 225 230
235 240 Asp Val Asp Ser Lys Asn Ile Ser Ser Asp Thr Pro Ala Asn Gly
Gly 245 250 255 Ser Asn Pro His Gln Ser Lys Asn Asn Gln Ser Glu Leu
Ser Pro Thr 260 265 270 Gln Lys Gln Glu Val Gln Asn Lys Leu Ala Lys
Leu Leu Ser Met Leu 275 280 285 Asp Glu Ile Asp Arg Arg Tyr Arg Gln
Tyr Tyr His Gln Met Gln Ile 290 295 300 Val Val Ser Ser Phe Asp Val
Val Ala Gly Glu Gly Ala Ala Lys Pro 305 310 315 320 Tyr Thr Ala Leu
Ala Leu Gln Thr Ile Ser Arg His Phe Arg Cys Leu 325 330 335 Arg Asp
Ala Ile Cys Asp Gln Ile Arg Ala Ser Arg Arg Ser Leu Gly 340 345 350
Glu Gln Asp Ala Ser Glu Asn Ser Lys Ala Ile Gly Ile Ser Arg Leu 355
360 365 Arg Phe Val Asp His His Ile Arg Gln Gln Arg Ala Leu Gln Gln
Leu 370 375 380 Gly Met Met Gln Gln His Ala Trp Arg Pro Gln Arg Gly
Leu Pro Glu 385 390 395 400 Ser Ser Val Ser Val Leu Arg Ala Trp Leu
Phe Glu His Phe Leu His 405 410 415 Pro Tyr Pro Lys Asp Ser Asp Lys
Ile Met Leu Ala Arg Gln Thr Gly 420 425 430 Leu Thr Arg Ser Gln Val
Ser Asn Trp Phe Ile Asn Ala Arg Val Arg 435 440 445 Leu Trp Lys Pro
Met Val Glu Glu Met Tyr Lys Glu Glu Ala Gly Asp 450 455 460 Ala Lys
Ile Asp Ser Asn Ser Ser Ser Asp Val Ala Pro Arg Leu Ala 465 470 475
480 Thr Lys Asp Ser Lys Val Glu Glu Arg Gly Glu Leu His Gln Asn Ala
485 490 495 Ala Ser Glu Phe Glu Gln Tyr Asn Ser Gly Gln Ile Leu Glu
Ser Lys 500 505 510 Ser Asn His Glu Ala Asp Val Glu Met Glu Gly Ala
Ser Asn Ala Glu 515 520 525 Thr Gln Ser Gln Ser Gly Met Glu Asn Gln
Thr Gly Glu Pro Leu Pro 530 535 540 Ala Met Asp Asn Cys Thr Leu Phe
Gln Asp Ala Phe Val Gln Ser Asn 545 550 555 560 Asp Arg Phe Ser Glu
Phe Gly Ser Phe Gly Ser Gly Asn Val Leu Pro 565 570 575 Asn Gly Val
Ser Leu Thr Leu Gly Leu Gln Gln Gly Glu Gly Ser Asn 580 585 590 Leu
Pro Met Ser Ile Glu Thr His Val Ser Tyr Val Pro Leu Arg Ala 595 600
605 Asp Asp Met Tyr Ser Thr Ala Pro Thr Thr Met Val Pro Glu Thr Ala
610 615 620 Glu Phe Asn Cys Leu Asp Ser Gly Asn Arg Gln Gln Pro Phe
Trp Leu 625 630 635 Leu Pro Ser Ala Thr 645
The BEL transcription factor has a molecular mass of approximately
71 kDa. StBEL-30, isolated from Solanum tuberosum, has a single
open reading frame ("ORF") of 1938 bp, extending between
nucleotides 39-1976.
[0069] Fragments of the above BEL transcription factors are
encompassed by the present invention.
[0070] Suitable fragments can be produced by several means. In one
method, subclones of the genes encoding the BEL transcription
factors of the present invention are produced by conventional
molecular genetic manipulation by subcloning gene fragments. The
subclones then are expressed in vitro or in vivo in bacterial cells
to yield a smaller protein or peptide.
[0071] In another approach, based on knowledge of the primary
structure of the protein, fragments of a BEL transcription factor
encoding gene may be synthesized by using the PCR technique
together with specific sets of primers chosen to represent
particular portions of the protein. These then would be cloned into
an appropriate vector for increased expression of a truncated
peptide or protein.
[0072] Chemical synthesis can also be used to make suitable
fragments. Such a synthesis is carried out using known amino acid
sequences for a BEL transcription factor being produced.
Alternatively, subjecting a full length BEL transcription factor to
high temperatures and pressures will produce fragments. These
fragments can then be separated by conventional procedures (e.g.,
chromatography, SDS-PAGE).
[0073] Another example of suitable fragments of the nucleic acids
of the present invention are fragments of the genes which have been
identified as conserved ("con") regions of the proteins, or
alternatively, those portions of nucleotide sequences that have
been identified as variable ("var") regions. Conserved regions in
accordance with the present invention include the homeodomain
region (including the proline-tyrosine-proline loop between helices
I and II), the amino-terminal SKY box, the BELL domain, and the
carboxy-terminal VSLTLGL-box (SEQ ID NO:15), as described in
Examples 20-32, below. Thus, one embodiment of the present
invention relates to an isolated nucleic acid molecule encoding a
protein having at least 85%, preferably 90%, similarity to the
homeodomain region, the amino-terminal SKY box, the BELL domain,
and the carboxy-terminal VSLTLGL-box (SEQ ID NO:15) in either SEQ
ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ
ID NO:12, or SEQ ID NO:14 by basic BLAST using default parameters
analysis. Sequences identified using DNAStar Mega alignment program
as either variable or conserved in a gene can be amplified using
standard PCR methods using forward and reverse primers designed to
amplify the region of choice and which include a restriction enzyme
sequence to allow ligation of the PCR product into a vector of
choice. Combinations of amplified conserved and variable region
sequences can be ligated into a single vector to create a
"cassette" which contains a plurality of DNA molecules in one
vector.
[0074] Mutations or variants of the above polypeptides or proteins
are encompassed by the present invention. Variants may be made by,
for example, the deletion or addition of amino acids that have
minimal influence on the properties, secondary structure, and
hydropathic nature of a polypeptide or protein. For example, a
polypeptide may be conjugated to a signal (or leader) sequence at
the N-terminal end of the protein which co-translationally or
post-translationally directs transfer of the protein. The
polypeptide may also be conjugated to a linker or other sequence
for ease of synthesis, purification, or identification of the
polypeptide.
[0075] Also suitable as an isolated nucleic acid molecule according
to the present invention is a nucleic acid molecule having a
nucleotide sequence that is at least 55% similar, preferably at
least 80% similar, and most preferably, at least 90% similar, to
the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:13 by basic
BLAST using default parameters analysis.
[0076] Suitable nucleic acid molecules are those that hybridize to
a nucleic acid molecule comprising a nucleotide sequence of SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, or SEQ ID NO:13 under stringent conditions. For the purposes
of defining the level of stringency, reference can conveniently be
made to Sambrook et al., Molecular Cloning: a Laboratory Manual,
2.sup.nd Edition, Cold Spring Harbor, N.Y., Cold Spring Harbor
Laboratory Press, at 11.45 (1989). An example of low stringency
conditions is 4-6.times.SSC/0.1-0.5% w/v SDS at 37'-45.degree. C.
for 2-3 hours. Depending on the source and concentration of the
nucleic acid involved in the hybridization, alternative conditions
of stringency may be employed such as medium stringent conditions.
Examples of medium stringent conditions include 1-4.times.SSC/0.25%
w/v SDS at .gtoreq.45.degree. C. for 2-3 hours. An example of high
stringency conditions includes 0.1-1.times.SSC/0.1% w/v SDS at
60.degree. C. for 1-3 hours. The skilled artisan is aware of
various parameters which may be altered during hybridization and
washing and which will either maintain or change the stringency
conditions. Other examples of high stringency conditions include:
4-5.times.SSC/0.1% w/v SDS at 54.degree. C. for 1-3 hours and
4.times.SSC at 65.degree. C., followed by a washing in
0.1.times.SSC at 65.degree. C. for about one hour. Alternatively,
an exemplary stringent hybridization condition is in 50% formamide,
4.times.SSC, at 42.degree. C. Still another example of stringent
conditions include hybridization at 62.degree. C. in 6.times.SSC,
0.05.times.BLOTTO, and washing at 2.times.SSC, 0.1% SDS at
62.degree. C.
[0077] The precise conditions for any particular hybridization are
left to those skilled in the art because there are variables
involved in nucleic acid hybridizations beyond those of the
specific nucleic acid molecules to be hybridized that affect the
choice of hybridization conditions. These variables include: the
substrate used for nucleic acid hybridization (e.g., charged vs.
non-charged membrane); the detection method used (e.g., radioactive
vs. chemiluminescent); and the source and concentration of the
nucleic acid involved in the hybridization. All of these variables
are routinely taken into account by those skilled in the art prior
to undertaking a nucleic acid hybridization procedure.
[0078] A BEL transcription factor of the present invention is
preferably produced in purified form (e.g., at least about 80%,
more preferably 90% pure) by conventional techniques. For example,
a BEL transcription factor of the present invention may be secreted
into the growth medium of recombinant host cells. To isolate the
BEL transcription factor, a protocol involving a host cell such as
Escherichia coli may be used, in which protocol the E. coli host
cell carrying a recombinant plasmid is propagated, homogenized, and
the homogenate is centrifuged to remove bacterial debris. The
supernatant is then subjected to sequential ammonium sulfate
precipitation. The fraction containing the BEL transcription factor
of the present invention is subjected to gel filtration in an
appropriately sized dextran or polyacrylamide column to separate
the proteins or polypeptides. If necessary, the protein fraction
may be further purified by high performance liquid chromatography
("HPLC").
[0079] The present invention relates to a DNA construct that
contains a DNA molecule encoding for a BEL transcription factor.
This involves incorporating one or more of the nucleic acid
molecules of the present invention, or a suitable portion thereof,
into host cells using conventional recombinant DNA technology.
Generally, this involves inserting the nucleic acid molecule into
an expression system to which the nucleic acid molecule is
heterologous (i.e. not normally present). The expression system
contains the necessary elements for the transcription and
translation of the inserted protein-coding sequences.
[0080] The present invention also relates to an expression vector
containing a nucleic acid molecule encoding a BEL transcription
factor of the present invention. The nucleic acid molecules of the
present invention may be inserted into any of the many available
expression vectors and cell systems using reagents that are well
known in the art. In preparing a DNA vector for expression, the
various DNA sequences may normally be inserted or substituted into
a bacterial plasmid. Any convenient plasmid may be employed, which
will be characterized by having a bacterial replication system, a
marker which allows for selection in a bacterium, and generally one
or more unique, conveniently located restriction sites. Numerous
plasmids, referred to as transformation vectors, are available for
transformation. The selection of a vector will depend on the
preferred transformation technique and target cells for
transfection.
[0081] Suitable vectors include, but are not limited to, the
following viral vectors such as lambda vector system gt11, gt
WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325,
pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290,
pKC37, pKC101, SV 40, pBluescript II SK+/- or KS+/- (see
"Stratagene Cloning Systems" Catalog (1993) from Stratagene, La
Jolla, Calif., which is hereby incorporated by reference in its
entirety), pQE, pIH821, pGEX, pET series (see F. W. Studier et.
al., "Use of T7 RNA Polymerase to Direct Expression of Cloned
Genes," Gene Expression Technology vol. 185 (1990), which is hereby
incorporated by reference in its entirety), pCB201, and any
derivatives thereof. Any appropriate vectors now known or later
described for genetic transformation are suitable for use with the
present invention. Recombinant molecules can be introduced into
cells via transformation, particularly transduction, conjugation,
mobilization, or electroporation. The DNA sequences are cloned into
the vector using standard cloning procedures in the art, as
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor Press, NY (1989), and
Ausubel, F. M. et al. (1989) Current Protocols in Molecular
Biology, John Wiley & Sons, New York, N.Y., which are hereby
incorporated by reference in their entirety.
[0082] U.S. Pat. No. 4,237,224 issued to Cohen and Boyer, which is
hereby incorporated by reference in its entirety, describes the
production of expression systems in the form of recombinant
plasmids using restriction enzyme cleavage and ligation with DNA
ligase. These recombinant plasmids are then introduced by means of
transformation and replicated in unicellular cultures including
prokaryotic organisms and eukaryotic cells grown in tissue
culture.
[0083] A variety of host-vector systems may be utilized to express
the protein-encoding sequence(s). Primarily, the vector system must
be compatible with the host cell used. Host-vector systems include
but are not limited to the following: bacteria transformed with
bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such
as yeast containing yeast vectors; mammalian cell systems infected
with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell
systems infected with virus (e.g., baculovirus); and plant cells
infected by bacteria. The expression elements of these vectors vary
in their strength and specificities. Depending upon the host-vector
system utilized, any one of a number of suitable transcription and
translation elements can be used.
[0084] Thus, certain "control elements" or "regulatory sequences"
are also incorporated into the plasmid-vector constructs of the
present invention. These include non-transcribed regions of the
vector and 5' and 3' untranslated regions, which interact with host
cellular proteins to carry out transcription and translation. Such
elements may vary in their strength and specificity. Depending on
the vector system and host utilized, any number of suitable
transcription and/or translation elements, including constitutive,
inducible, and repressible promoters, as well as minimal 5'
promoter elements may be used. A constitutive promoter is a
promoter that directs expression of a gene throughout the
development and life of an organism. An inducible promoter is a
promoter that is capable of directly or indirectly activating
transcription of one or more DNA sequences or genes in response to
an inducer. In the absence of an inducer, the DNA sequences or
genes will not be transcribed or will only be minimally
transcribed.
[0085] The DNA sequences of eukaryotic promoters differ from those
of prokaryotic promoters. Furthermore, eukaryotic promoters and
accompanying genetic signals may not be recognized in or may not
function in a prokaryotic system, and, further, prokaryotic
promoters are not recognized and do not function in eukaryotic
cells.
[0086] Promotors vary in their "strength" (i.e. their ability to
promote transcription). For the purposes of expressing a cloned
gene, it is desirable to use strong promoters in order to obtain a
high level of transcription and, hence, expression of the gene.
Depending upon the host cell system utilized, any one of a number
of suitable promoters may be used. For instance, when cloning in E.
coli, its bacteriophages, or plasmids, promotors such as the T7
phage promoter, lac promotor, trp promotor, recA promotor,
ribosomal RNA promotor, the P.sub.R and P.sub.L promoters of
coliphage lambda and others, including but not limited, to lacUV5,
ompF, bla, lpp, and the like, may be used to direct high levels of
transcription of adjacent DNA segments. Additionally, a hybrid
trp-lacUV5 (tac) promotor or other E. coli promoters produced by
recombinant DNA or other synthetic DNA techniques may be used to
provide for transcription of the inserted gene.
[0087] Other examples of some constitutive promoters that are
widely used for inducing expression of transgenes include the
nopoline synthase (NOS) gene promoter, from Agrobacterium
tumefaciens, (U.S. Pat. No. 5,034,322 issued to Rogers et al.,
which is hereby incorporated by reference in its entirety), the
cauliflower mosaic virus (CaMV) 35S and 19S promoters (U.S. Pat.
No. 5,352,605 issued to Fraley et al., which is hereby incorporated
by reference in its entirety), the enhanced CaMV35S promoter ("enh
CaMV35S"), the figwort mosaic virus full-length transcript promoter
("FMV35S"), those derived from any of the several actin genes,
which are known to be expressed in most cells types (U.S. Pat. No.
6,002,068 issued to Privalle et al., which is hereby incorporated
by reference in its entirety), and the ubiquitin promoter, which is
a gene product known to accumulate in many cell types. Examples of
constitutive promoters for use in mammalian cells include the RSV
promoter derived from Rous sarcoma virus, the CMV promoter derived
from cytomegalovirus, Wactin and other actin promoters, and the
EF1.alpha. promoter derived from the cellular elongation factor
1.alpha. gene.
[0088] Bacterial host cell strains and expression vectors may be
chosen which inhibit the action of the promoter unless specifically
induced. In certain operations, the addition of specific inducers
is necessary for efficient transcription of the inserted nucleic
acid. For example, the lac operon is induced by the addition of
lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of
other operons, such as trp, pro, etc., are under different
controls.
[0089] Other examples of some inducible promoters, induced, for
examples by a chemical agent, such as a metabolite, growth
regulator, herbicide or phenolic compound, or a physiological
stress/physical means, such as cold, heat, salt, toxins, or through
the action of a pathogen or disease agent such as a virus or
fungus, include a glucocorticoid-inducible promoter (Schena et al.,
Proc. Natl. Acad. Sci. 88:10421-5 (1991), which is hereby
incorporated by reference in its entirety), the heat shock promoter
("Hsp"), IPTG or tetracycline ("Tet on" system), the
metallothionine promoter, which is activated by heavy metal ions,
and hormone-responsive promoters, which are activated by treatment
of certain hormones. A host cell containing an inducible promoter
may be exposed to an inducer by externally applying the inducer to
the cell. In addition, "tissue-specific" promoters can be used,
which are promoters that function in a tissue specific manner to
regulate the gene of interest within selected tissues of the host.
Examples of such tissue specific promoters include seed, flower, or
root specific promoters as are well known in the field (e.g., U.S.
Pat. No. 5,750,385 to Shewmaker et al., which is hereby
incorporated by reference in its entirety). Promoters of the
nucleic acid construct of the present invention may be either
homologous (derived from the same species as the host cell) or
heterologous (derived from a different species than the host
cell).
[0090] Specific initiation signals are also required for efficient
gene transcription and translation in prokaryotic cells. These
transcription and translation initiation signals may vary in
"strength" as measured by the quantity of gene specific messenger
RNA and protein synthesized, respectively. The DNA expression
vector, which contains a promoter, may also contain any combination
of various "strong" transcription and/or translation initiation
signals. For instance, efficient translation in E. coli requires an
SD sequence about 7-9 bases 5' to the initiation codon ("ATG") to
provide a ribosome binding site. Thus, any SD-ATG combination that
can be utilized by host cell ribosomes may be employed. Such
combinations include but are not limited to the SD-ATG combination
from the cro gene or the N gene of coliphage lambda, or from the E.
coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG
combination produced by recombinant DNA or other techniques
involving incorporation of synthetic nucleotides may be used.
[0091] The constructs of the present invention also include an
operable 3' regulatory region, selected from among those which are
capable of providing correct transcription termination and
polyadenylation of mRNA for expression in the host cell of choice,
operably linked to a DNA molecule which encodes for a protein of
choice. A number of 3' regulatory regions are known in the art.
Virtually any 3' regulatory region known to be operable in the host
cell of choice would suffice for proper expression of the coding
sequence of the nucleic acid of the present invention.
[0092] In one aspect of the present invention, the nucleic acid
molecule of the present invention is incorporated into an
appropriate vector in the sense direction, such that the open
reading frame is properly oriented for the expression of the
encoded protein under control of a promoter of choice. This
involves the inclusion of the appropriate regulatory elements into
the DNA-vector construct. These include non-translated regions of
the vector, useful promoters, and 5' and 3' untranslated regions
which interact with host cellular proteins to carry out
transcription and translation. Such elements may vary in their
strength and specificity. Depending on the vector system and host
utilized, any number of suitable transcription and translation
elements, including constitutive and inducible promoters, may be
used.
[0093] A nucleic acid molecule of the preset invention, promoter of
choice, an appropriate 3' regulatory region, and, if desired, a
reporter gene, can be incorporated into a vector-expression system
to contain a nucleic acid of the present invention, or a suitable
fragment thereof, using standard cloning techniques as described in
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Press, NY (1989), and Ausubel et al.
(1989) Current Protocols in Molecular Biology, John Wiley &
Sons, New York, N.Y., which are hereby incorporated by reference in
their entirety. The transcriptional and translational elements are
operably linked to the nucleic acid molecule of the present
invention or a fragment thereof, meaning that the resulting vector
expresses the BEL transcription factor when placed in a suitable
host cell.
[0094] Once an isolated DNA molecule encoding a BEL transcription
factor has been cloned into an expression vector, it is ready to be
incorporated into a host cell. Such incorporation can be carried
out by the various forms of transformation noted above, depending
upon the vector/host cell system. Recombinant molecules can be
introduced into cells via transformation, particularly
transduction, conjugation, mobilization, or electroporation. The
nucleic acid sequences are cloned into the host cell using standard
cloning procedures known in the art, as described by Sambrook et
al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold
Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is
hereby incorporated by reference in its entirety. Suitable host
cells include, but are not limited to, bacteria, virus, yeast,
mammalian cells, insect, plant, and the like.
[0095] Thus, the present invention also relates to a host cell
incorporating one or more of the isolated nucleic acid molecules of
the present invention. In one embodiment, the isolated nucleic acid
molecule is heterologous to the host cell. Such incorporation can
be carried out by the various forms of transformation noted above,
depending upon the vector/host system, and using the various host
cells described above.
[0096] Methods of transformation may result in transient or stable
expression of the DNA under control of the promoter. Preferably,
the nucleic acid of the present invention is stably inserted into
the genome of the host cell as a result of the transformation,
although transient expression can serve an important purpose.
[0097] One approach to transforming host cells with a nucleic acid
molecule of the present invention is particle bombardment (also
known as biolistic transformation) of the host cell. This can be
accomplished in one of several ways. The first involves propelling
inert or biologically active particles at cells. This technique is
disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792,
all to Sanford et al., which are hereby incorporated by reference
in their entirety. Generally, this procedure involves propelling
inert or biologically active particles at the cells under
conditions effective to penetrate the outer surface of the cell and
to be incorporated within the interior thereof. When inert
particles are utilized, the vector can be introduced into the cell
by coating the particles with the vector containing the
heterologous DNA. Alternatively, the target cell can be surrounded
by the vector so that the vector is carried into the cell by the
wake of the particle. Biologically active particles (e.g., dried
bacterial cells containing the vector and heterologous DNA) can
also be propelled into plant cells. Other variations of particle
bombardment, now known or hereafter developed, can also be
used.
[0098] Transient expression in protoplasts allows quantitative
studies of gene expression, because the population of cells is very
high (on the order of 10.sup.6). To deliver DNA inside protoplasts,
several methodologies have been proposed, but the most common are
electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA
82:5824-5828 (1985), which is hereby incorporated by reference in
its entirety) and polyethylene glycol (PEG) mediated DNA uptake
(Krens et al., Nature 296:72-74 (1982), which is hereby
incorporated by reference in its entirety). During electroporation,
the DNA is introduced into the cell by means of a reversible change
in the permeability of the cell membrane due to exposure to an
electric field. PEG transformation introduces the DNA by changing
the elasticity of the membranes. Unlike electroporation, PEG
transformation does not require any special equipment and
transformation efficiencies can be equally high. Another
appropriate method of introducing the nucleic acid molecule of the
present invention into a host cell is fusion of protoplasts with
other entities, either minicells, cells, lysosomes, or other
fusible lipid-surfaced bodies that contain the chimeric gene
(Fraley, et al., Proc. Natl. Acad. Sci. USA 76:3348-52 (1979),
which is hereby incorporated by reference in its entirety).
[0099] Stable transformants are preferable for the methods of the
present invention. An appropriate method of stably introducing the
nucleic acid molecule into plant cells is to infect a plant cell
with Agrobacterium tumefaciens or Agrobacterium rhizogenes
previously transformed with a DNA construct of the present
invention. Under appropriate conditions known in the art, the
transformed plant cells are grown to form shoots or roots, and
develop further into plants.
[0100] Plant tissues suitable for transformation include without
limitation, floral buds, leaf tissue, root tissue, meristems,
zygotic and somatic embryos, megaspores, callus, protoplasts,
tassels, pollen, embryos, anthers, and the like. The means of
transformation chosen is that most suited to the tissue to be
transformed.
[0101] Suitable plants include dicots and monocots. Monocots
suitable for the present invention include Gramineae (e.g., grass,
corn, grains, bamboo, sugar cane), Liliaceae (e.g., onion, garlic,
asparagus, tulips, hyacinths, day lily, and aloes), Iridaceae
(e.g., iris, gladioli, freesia, crocus, and watsonia), and
Orchidacea (e.g., orchid). Examples of dicots suitable for the
present invention include Salicaceae (e.g., willow, and poplar),
Ranunculaceae (e.g., Delphinium, Paeonia, Ranunculus, Anemone,
Clematis, columbine, and marsh marigold), Magnoliaceae (e.g., tulip
tree and Magnolia), Cruciferae (e.g., mustards, cabbage,
cauliflower, broccoli, brussel sprouts, kale, kohlrabi, turnip, and
radish), Rosaceae (e.g., strawberry, blackberry, peach, apple,
pear, quince, cherry, almond, plum, apricot, and rose), Leguminosae
(e.g., pea, bean, peanut, alfalfa, clover, vetch, redbud, broom,
wisteria, lupine, black locust, and acacia), Malvaceae (e.g.,
cotton, okra, and mallow), Umbelliferae (e.g., carrot, parsley,
parsnips, and hemlock), Labiatae (e.g., mint, peppermints,
spearmint, thyme, sage, and lavender), Solanaceae (e.g., potato,
tomato, pepper, eggplant, tobacco, henbane, atropa, physalis,
datura, and Petunia), Cucurbitaceae (e.g., melon, squash, pumpkin,
and cucumber), Compositae (e.g., sunflower, endive, artichoke,
lettuce, safflower, aster, marigold, dandelions, sage brush, Dalia,
Chrysanthemum, and Zinna), and Rubiaceae (e.g., coffee).
[0102] After transformation, the transformed plant cells can be
selected and regenerated. Preferably, transformed cells are first
identified using a selection marker simultaneously introduced into
the host cells along with the DNA construct of the present
invention. Suitable selection markers include, without limitation,
markers encoding for antibiotic resistance, such as the nptII gene
which confers kanamycin resistance (Fraley, et al., Proc. Natl.
Acad. Sci. USA 80:4803-4807 (1983), which is hereby incorporated by
reference in its entirety), and the genes which confer resistance
to gentamycin, G418, hygromycin, streptomycin, spectinomycin,
tetracycline, chloramphenicol, and the like. Any known
antibiotic-resistance marker can be used to transform and select
transformed host cells in accordance with the present invention.
Cells or tissues are grown on a selection medium containing the
appropriate antibiotic, whereby generally only those transformants
expressing the antibiotic resistance marker continue to grow. Other
types of markers are also suitable for inclusion in the expression
cassette of the present invention. For example, a gene encoding for
herbicide tolerance, such as tolerance to sulfonylurea is useful,
or the dhfr gene, which confers resistance to methotrexate
(Bourouis et al., EMBO J. 2:1099-1104 (1983), which is hereby
incorporated by reference in its entirety). Similarly, "reporter
genes," which encode for enzymes providing for production of a
compound identifiable are suitable. The most widely used reporter
gene for gene fusion experiments has been uidA, a gene from
Escherichia coli that encodes the .beta.-glucuronidase protein,
also known as GUS (Jefferson et al., EMBO J. 6:3901-3907 (1987),
which is hereby incorporated by reference in its entirety).
Similarly, enzymes providing for production of a compound
identifiable by luminescence, such as luciferase, are useful. The
selection marker employed will depend on the target species; for
certain target species, different antibiotics, herbicide, or
biosynthesis selection markers are preferred.
[0103] Once a recombinant plant cell or tissue has been obtained,
it is possible to regenerate a full-grown plant therefrom. It is
known that practically all plants can be regenerated from cultured
cells or tissues. Means for regeneration vary from species to
species of plants, but generally a suspension of transformed
protoplasts or a petri plate containing transformed explants is
first provided. Callus tissue is formed and shoots may be induced
from callus and subsequently rooted. Alternatively, embryo
formation can be induced in the callus tissue. These embryos
germinate as natural embryos to form plants. The culture media will
generally contain various amino acids and hormones, such as auxin
and cytokinins. It is also advantageous to add glutamic acid and
proline to the medium, especially for such species as corn and
alfalfa. Efficient regeneration will depend on the medium, on the
genotype, and on the history of the culture. If these three
variables are controlled, then regeneration is usually reproducible
and repeatable.
[0104] Plant regeneration from cultured protoplasts is described in
Evans, et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan
Publishing Co., New York, 1983); and Vasil I. R. (ed.), Cell
Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando,
Vol. 1, 1984, and Vol. III (1986), which are hereby incorporated by
reference in their entirety.
[0105] After the DNA construct is stably incorporated in transgenic
plants, it can be transferred to other plants by sexual crossing or
by preparing cultivars. With respect to sexual crossing, any of a
number of standard breeding techniques can be used depending upon
the species to be crossed. Cultivars can be propagated in accord
with common agricultural procedures known to those in the field.
Alternatively, transgenic seeds or propagules (e.g., cuttings) are
recovered from the transgenic plants. The seeds can then be planted
in the soil and cultivated using conventional procedures to produce
transgenic plants.
[0106] The present invention is also directed to a method for
enhancing tuber development in a plant. This method includes
transforming a tuberous plant with a first DNA construct including
a first nucleic acid molecule encoding a BEL transcription factor
or a KNOX transcription factor, and a first operably linked
promoter and first 3' regulatory region, whereby tuber development
in the plant is enhanced.
[0107] Suitable BEL transcription factors include BEL transcription
factors from potato, as described above. Other suitable BEL
transcription factors include, but are not limited to, those from
tobacco, tomato (see, e.g., GenBank Accession Nos. AF375964,
AF375965, and AF375966), Arabidopsis, rice, barley, apple, and bago
(Gnetum gnemon).
[0108] As used herein, a KNOX transcription factor is encoded by a
Knotted-like homeobox (knox) gene and includes a KNOX domain. KNOX
transcription factors regulate growth, in particular, leaf
architecture and meristem growth. KNOX transcription factors have
been isolated from several plant species (reviewed in Reiser et
al., "Knots in the Family Tree: Evolutionary Relationships and
Functions of knox Homeobox Genes," Plant Mol. Biol. 42:151-166
(2000), which is hereby incorporated by reference in its entirety)
and can be divided into two classes based on expression patterns
and sequence similarity (Kerstetter et al., "Sequence Analysis and
Expression Patterns Divide the Maize knotted1-like Homeobox Genes
into Two Classes," Plant Cell 6:1877-1887 (1994), which is hereby
incorporated by reference in its entirety). Class I knox genes have
high similarity to the maize knotted1 (kn1) homeodomain and
generally have a meristem-specific mRNA expression pattern. Class
II knox genes usually have a more widespread expression pattern.
Knox genes are members of the three amino acid loop extension
(TALE) superclass of homeobox genes (Burglin, "Analysis of TALE
Superclass Homeobox Genes (MEIS, PBC, KNOX, Iroquois, TGIF) Reveals
a Novel Domain Conserved Between Plants and Animals," Nucleic Acids
Res 25:4173-4180 (1997), which is hereby incorporated by reference
in its entirety). Knox genes share conserved regions outside of the
homeodomain including the MEINOX and ELK domains.
[0109] Suitable KNOX transcription factors include, but are not
limited to, POTH1, POTH15, POTH2, HO9, NTH Types (1, 9, 15, 20, 22)
(Nishamura et al., Plant J. 18:337-347 (1999), which is hereby
incorporated by reference in its entirety), those from Arabidopsis,
maize, barley, tobacco, tomato, pea, cabbage, Ipomoea, Helianthus,
Medicago, and Dendrobium.
[0110] In one embodiment, the KNOX transcription factor is POTH1
and is encoded by a nucleic acid molecule having a nucleotide
sequence of SEQ ID NO:16 as follows:
TABLE-US-00015 1 gagtttctct cccttttaaa aaagaaaaaa aaaacacaac
acccacttca aatatcaaac 61 aaatttctca tttgattatt tctaagtgat
ttacactact ttgtattttt gtttgttttt 121 ttttagatat atatatggat
gatgaaatgt atggttttca ttcaacaaga gacgattacg 181 cggataaagc
tttgatgtca ccggagaatt tgatgatgca aactgagtac aacaatttcc 241
acaactatac caactcgtcc atcttgactt ctaatccgat gatgtttgga tccgatgata
301 ttcaattatc atcggaacaa actaattctt tcagtactat gactcttcaa
aataatgata 361 atatttatca aattagaagt ggaaattgtg gcggaggcag
tggcagtggt ggtagcagta 421 aggatcataa tgataataac aataataatg
aagattatga tgaagatggt tcaaatgtta 481 tcaaggctaa aatcgtctca
catccttatt atcctaaatt actcaacgct tatattgatt 541 gccaaaaggt
tggagcacca gcgggtatag taaatctgct ggaagaaata aggcaacaaa 601
ctgattttcg taaaccaaac gctacttcta tatgtatagg agctgatcct gaacttgatg
661 agtttatgga aacgtattgt gatatattgt tgaagtataa gtccgatctg
tctaggcctt 721 ttgatgaagc aacaacgttc ctcaacaaga ttgaaatgca
actaggtaat ctttgcaaag 781 atgatggtgg tgtatcatca gatgaggagt
taagttgtgg tgaggcagat gcatcaatga 841 gaagtgagga taatgaactc
aaagatagac tcctacgtaa gtttggaagt catttaagta 901 gtctaaagtt
ggaattttca aagaaaaaga agaaagggaa gctaccaaaa gaggcaaggc 961
aaatgttact tgcatggtgg gatgatcact ttagatggcc ttaccctacg gaggctgata
1021 agaattcact agcagaatca acaggacttg atccaaagca gatcaacaat
tggtttataa 1081 atcaaaggaa gagacattgg aaaccatcag agaatatgca
gttagctgtt atggataatc 1141 taagctctca gttcttctca tcagatgatt
gagtttgaat ggaaattgtg aaaatactgc 1201 tcttcatttc tctttttatt
atatataata tataaatagt atatttttgg gaaagaaaga 1261 agttatttta
ttaatcaaaa tctctataaa taatggtaga gattaattaa tgttgaattc 1321
ttcttgatca tgtaaatatt caatctagct aattgtcaaa attaatgctt acctaaaaaa
1381 aaa
The cDNA (Genbank Accession # U65648) includes an open reading
frame of 1035 nt coding for a 345-residue protein estimated to have
a mass of 37.95 kDa having an amino acid sequence corresponding to
SEQ ID NO:17 as follows:
TABLE-US-00016 Met Asp Asp Glu Met Tyr Gly Phe His Ser Thr Arg Asp
Asp Tyr Ala 1 5 10 15 Asp Lys Ala Leu Met Ser Pro Glu Asn Leu Met
Met Gln Thr Gln Tyr 20 25 30 Asn Asn Phe His Asn Tyr Thr Asn Ser
Ser Ile Leu Thr Ser Asn Pro 35 40 45 Met Met Phe Gly Ser Asp Asp
Ile Gln Leu Ser Ser Glu Gln Thr Asn 50 55 60 Ser Phe Ser Thr Met
Thr Leu Gln Asn Asn Asp Asn Ile Tyr Gln Ile 65 70 75 80 Arg Ser Gly
Asn Cys Gly Gly Gly Ser Gly Ser Gly Gly Ser Ser Lys 85 90 95 Asp
His Asn Asp Asn Asn Asn Asn Asn Glu Asp Tyr Asp Glu Asp Gly 100 105
110 Ser Asn Val Ile Lys Ala Lys Ile Val Ser His Pro Tyr Tyr Pro Lys
115 120 125 Leu Leu Asn Ala Tyr Ile Asp Cys Gln Lys Val Gly Ala Pro
Ala Gly 130 135 140 Ile Val Asn Leu Leu Glu Glu Ile Arg Gln Gln Thr
Asp Phe Arg Lys 145 150 155 160 Pro Asn Ala Thr Ser Ile Cys Ile Gly
Ala Asp Pro Glu Leu Asp Glu 165 170 175 Phe Met Glu Thr Tyr Cys Asp
Ile Leu Leu Lys Tyr Lys Ser Asp Leu 180 185 190 Ser Arg Pro Phe Asp
Glu Ala Thr Thr Phe Leu Asn Lys Ile Glu Met 195 200 205 Gln Leu Gly
Asn Leu Cys Lys Asp Asp Gly Gly Val Ser Ser Asp Glu 210 215 220 Glu
Leu Ser Cys Gly Glu Ala Asp Ala Ser Met Arg Ser Glu Asp Asn 225 230
235 240 Glu Leu Lys Asp Arg Leu Leu Arg Lys Phe Gly Ser His Leu Ser
Ser 245 250 255 Leu Lys Leu Glu Phe Ser Lys Lys Lys Lys Lys Gly Lys
Leu Pro Lys 260 265 270 Glu Ala Arg Gln Met Leu Leu Ala Trp Trp Asp
Asp His Phe Arg Trp 275 280 285 Pro Tyr Pro Thr Glu Ala Asp Lys Asn
Ser Leu Ala Glu Ser Thr Gly 290 295 300 Leu Asp Pro Lys Gln Ile Asn
Asn Trp Phe Ile Asn Gln Arg Lys Arg 305 310 315 320 His Trp Lys Pro
Ser Glu Asn Met Gln Leu Ala Val Met Asp Asn Leu 325 330 335 Ser Ser
Gln Phe Phe Ser Ser Asp Asp 340 345
[0111] In accordance with the present invention, the BEL or KNOX
transcription factor may be expressed throughout the plant to
achieve enhanced tuber development (see Examples below).
Alternatively, the BEL or KNOX transcription factor may be
expressed in an organ-specific manner. This is beneficial when, for
example with POTH1, expression throughout the plant results in
dwarf transgenic plants with altered leaf morphology. In these
circumstances, specific expression in the stolon, for example, may
be desirable.
[0112] In one embodiment of this method of the present invention,
the tuberous plant is transformed with one or more DNA constructs
which include nucleic acid molecules encoding both a BEL
transcription factor and a KNOX transcription factor.
Alternatively, a plant expressing one or more of a BEL
transcription factor or a KNOX transcription factor may be
transformed with a DNA construct including a nucleic acid molecule
encoding only one of a BEL transcription factor or a KNOX
transcription factor.
[0113] Tuberous plants suitable for use in this method of the
present invention include potato, dahlia, caladium, Jerusalem
artichoke (Helianthus tuberosus), yam (Dioscorea alta), sweet
potato (Impomoea batatus), cassava (Manihot esculenta), tuberous
begonia, cyclamen, and other solanum species (e.g., wild
potato).
[0114] Another aspect of the present invention relates to a method
of enhancing growth in a plant. This method includes transforming a
plant with a DNA construct including a nucleic acid molecule
encoding a BEL transcription factor from Solanum tuberosum and an
operably linked promoter and 3' regulatory region, whereby growth
in the plant is enhanced.
[0115] Suitable plants which may be transformed in this method of
the present invention include Gramineae (e.g., grass, corn, grains,
bamboo, sugar cane), Liliaceae (e.g., onion, garlic, asparagus,
tulips, hyacinths, day lily, and aloes), Iridaceae (e.g., iris,
gladioli, freesia, crocus, and watsonia), Orchidacea (e.g.,
orchid), Salicaceae (e.g., willow, and poplar), Ranunculaceae
(e.g., Delphinium, Paeonia, Ranunculus, Anemone, Clematis,
columbine, and marsh marigold), Magnoliaceae (e.g., tulip tree and
Magnolia), Cruciferae (e.g., mustards, cabbage, cauliflower,
broccoli, brussel sprouts, kale, kohlrabi, turnip, and radish),
Rosaceae (e.g., strawberry, blackberry, peach, apple, pear, quince,
cherry, almond, plum, apricot, and rose), Leguminosae (e.g., pea,
bean, peanut, alfalfa, clover, vetch, redbud, broom, wisteria,
lupine, black locust, and acacia), Malvaceae (e.g., cotton, okra,
and mallow), Umbelliferae (e.g., carrot, parsley, parsnips, and
hemlock), Labiatae (e.g., mint, peppermints, spearmint, thyme,
sage, and lavender), Solanaceae (e.g., potato, tomato, pepper,
eggplant, tobacco, henbane, atropa, physalis, datura, and Petunia),
Cucurbitaceae (e.g., melon, squash, pumpkin, and cucumber),
Compositae (e.g., sunflower, endive, artichoke, lettuce, safflower,
aster, marigold, dandelions, sage brush, Dalia, Chrysanthemum, and
Zinna), and Rubiaceae (e.g., coffee). In one particular embodiment,
the plant transformed is a solanaceous species.
[0116] Yet another embodiment of the present invention relates to a
method of regulating flowering in a plant. This method includes
transforming a plant with a DNA construct including a nucleic acid
molecule encoding a BEL transcription factor from Solanum tuberosum
and an operably linked promoter and 3' regulatory region, whereby
flowering in the plant is regulated.
[0117] Suitable plants in accordance with this method of the
present invention are described above.
[0118] The BEL transcription factors from Solanum tuberosum of the
present invention appear to play a diverse role in plant growth by
regulating the development of both reproductive and vegetative
meristems. Accordingly, they can be used in the methods for
enhancing growth or regulating flowering of the present invention.
In particular, the BEL transcription factors of the present
invention are involved in regulating photoperiodic responses in
potato (tuberization), and BEL transcription factors have
previously been identified as contributing to flower development
(Muller et al., "In vitro Interactions Between Barley TALE
Homeodomain Proteins Suggest a Role for Protein-Protein
Associations in the Regulation of Knox Gene Function," Plant J.
27:13-23 (2001); Mondrusan et al., "Homeotic Transformation of
Ovules into Carpel-Like Structures in Arabidopsis," Plant Cell
6:333-349 (1994); Reiser et al., "The BELL1 Gene Encodes a
Homeodomain Protein Involved in Patterns Formation in the
Arabidopsis Ovule Primordium," Cell 83:735-742 (1995), which are
hereby incorporated by reference in their entirety) and are present
in numerous photoperiodic flowering species (e.g., rice, tobacco,
morning glory, Arabidopsis), thus it appears that they contribute
to regulating flower induction in many plants.
EXAMPLES
Example 1
Amplification of Potato Homeobox Fragment for Use as Probe
[0119] Two primers, Primer 1 (5'-AAGAAGAAGAAGAAAGGGAA) (SEQ ID
NO:18) and Primer 2 (5'-ATGAACCAGTTGTTGAT) (SEQ ID NO:19) were
designed based on comparison of the homeobox regions of five class
I homeobox genes (KN1, KNAT1, KNAT2, OSH1, and SBH1) to correspond
to the most highly conserved portions of the homeobox, and were
synthesized at the DNA Synthesis Facility at Iowa State University.
Template DNA was prepared from a mass in vivo excision of a 4-day
axillary bud tuber .lamda.ZAP.RTM.II cDNA library (Stratagene, La
Jolla, Calif.) from potato cv. Superior. The potato homeobox
fragment was amplified using an annealing temperature of 45.degree.
C. and cloned into the pCR2.1 vector of the TA Cloning.RTM. Kit
(Invitrogen, Carlsbad, Calif.).
Example 2
Library Screening and Sequence Analysis
[0120] The early tuberization stage library was constructed as
described in Kang et al., "A Novel MADS-box Gene of Potato (Solanum
tuberosum L.) Expressed During the Early Stages of Tuberization,"
Plant Mol. Biol. 31: 379-386 (1996), which is hereby incorporated
by reference in its entirety. Screening of 400,000 pfu was
accomplished using 100 ng of .sup.32P-labeled PCR-generated probe
in 50% formamide (50% deionized formamide, 6.times.SSC,
3.4.times.Denhardt's solution, 25 mM sodium phosphate buffer, pH
7.0, 120 .mu.g/ml denatured salmon sperm DNA, 0.4% SDS) at
42.degree. C. for 48 hours. Membranes were washed with
2.times.SSC/0.1% SDS, at 25.degree. C. for 5 minutes; then twice
with 2.times.SSPE/0.1% SDS, at 65.degree. C. for 20 minutes.
[0121] POTH1 was sequenced at the Nucleic Acid Sequencing Facility
at Iowa State University. Sequence analyses performed included
BLAST (Altschul et al., "Basic Local Alignment Search Tool," J.
Mol. Biol. 215:403-410 (1990), which is hereby incorporated by
reference in its entirety) and GAP [Genetics Computer Group (GCG),
Madison, Wis.].
Example 3
RNA Isolation and Northern Blot Analysis
[0122] Total RNA was isolated (Dix et al., "In vivo Transcriptional
Products of the Chloroplast DNA of Euglena gracilis," Curr. Genet.
7:265-273 (1983), which is hereby incorporated by reference in its
entirety) from potato (Solanum tuberosum L.) plants grown in the
greenhouse at 20 to 25.degree. C. under 16 hours of light. Total
RNA was enriched for poly (A)+ RNA by separation over an oligo-dT
column and northern gel electrophoresis was performed using methyl
mercury as a denaturant. Ethidium bromide staining under UV light
was used to ascertain equal gel loading and efficient transfer to
nylon membranes. The Genius.TM. nonradioactive nucleic acid
labeling and detection system (Roche Biochemicals, Indianapolis,
Ind.) was used. Fifteen ng/ml of digoxygenin-UTP-labeled antisense
RNA probe in 50% formamide was hybridized to filters at 55.degree.
C. overnight. Membranes were washed twice for 5 minutes in
2.times.SSC, 0.1% SDS at 25.degree. C., and then washed twice for
15 minutes in 0.1.times.SSC, 0.1% SDS at 68.degree. C. The
membranes were then incubated 30 minutes in blocking
solution:maleic acid buffer pH 7.5 (1:10), 30 minutes in
anti-digoxygenin-alkaline-phosphatase conjugate:maleic acid buffer
(1:10,000), washed twice for 15 minutes in maleic acid buffer, and
equilibrated 5 minutes in detection buffer before addition of
disodium
3-[4-methoxyspiro{1,2-dioxetane-3,2'-[5'-chloro]tricyclo[3.3.1.1.sup.3,7]-
decan}-4-yl]phenyl phosphate (CSPD) substrate solution. Membranes
were exposed to film for 30 to 45 minutes at 25.degree. C.
Example 4
In situ Hybridization Analysis
[0123] Preparation of tissue samples and in situ hybridizations
were performed as described in Canas et al., "Nuclear Localization
of the Petunia MADS Box Protein FBP1," Plant J. 6:597-604 (1994),
which is hereby incorporated by reference in its entirety.
Digoxygenin-UTP-labeled RNA probes, both sense and antisense, were
transcribed with RNA polymerases according to instructions (Roche
Biochemicals, Indianapolis, Ind.), and hydrolyzed using 0.2 M
sodium carbonate and 0.2 M sodium bicarbonate at 65.degree. C. for
51 minutes. Unincorporated nucleotides were removed over a Sephadex
G-50 column.
[0124] For immunological detection, the slides were incubated in
buffer 1 (1% blocking solution, 100 mM Tris pH 7.5, 150 mM NaCl)
for one hour, then equilibrated with buffer 2 (100 mM Tris pH 7.5,
150 mM NaCl, 0.5% BSA, and 0.3% Triton X-100). Tissue sections were
then incubated with anti-digoxygenin-alkaline-phosphatase conjugate
diluted 1:1000 in buffer 2 in a humidified box for two hours, then
washed three times for 20 minutes in 100 mM Tris pH 7.5, 150 mM
NaCl. The tissue sections were equilibrated in buffer 3 (100 mM
Tris pH 9.5, 100 mM NaCl, 50 mM MgCl.sub.2) for 10 minutes, then
incubated in 3.2 .mu.g/ml 5-bromo-4-chloro-3-indolyl-phosphate
(BCIP):6.6 .mu.g/ml nitro-blue tetrazolium salt (NBT) in buffer 3
in a humidified box for 13 hours (above-ground tissues) or 7 hours
(underground tissues). Accumulation of POTH1 mRNA was visualized as
an orange/brown stain under dark field illumination. Sections were
viewed and photodocumented using the dark field mode on the Leitz
Orthoplan light microscope.
Example 5
35S-POTH1 Transformation of Potato Plants
[0125] The full length POTH1 cDNA was cloned into the binary
vector, pCB201 (Xiang et al., "A Mini Binary Vector Series for
Plant Transformation," Plant Mol. Biol. 40:711-718 (1999), which is
hereby incorporated by reference in its entirety) between the CaMV
35S promoter and the nos terminator. Two potato cultivars, Solanum
tuberosum ssp. andigena and cv. FL-1607, were transformed by the
Agrobacterium tumefaciens (strain GV2260) mediated leaf-disk
transformation method (Liu et al., "Transformation of Solanum
Brevidens Using Agrobacterium Tumefaciens," Plant Cell Reports
15:196-199 (1995), which is hereby incorporated by reference in its
entirety). A total of thirty independent transgenic lines from
andigena and twenty independent transgenic lines from `FL-1607`
were screened for insertion of the transgene and accumulation of
POTH1 mRNA. Five independent transgenic lines of S. tuberosum spp.
andigena and 4 lines of S. tuberosum cv. FL-1607 that showed high
levels of POTH1 mRNA accumulation were selected for further
analysis. Untransformed tissue culture plants were used as
controls.
Example 6
Nucleic Acid Hybridizations
[0126] Genomic DNA was isolated using the cetyltrimethylammonium
bromide (CTAB) mini-plant DNA extraction method (Doyle et al., "A
Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf
Tissue," Phytochem. Bull. 19:11-15 (1987), which is hereby
incorporated by reference in its entirety). DNA (10 .mu.g) was
digested with Hind III or Xba I (Promega, Madison, Wis.), and gel
electrophoresis was performed. DNA was denatured and blotted
according to the methods described by Kolomiets et al., "A Leaf
Lipoxygenase of Potato Induced Specifically by Pathogen Infection,"
Plant Physiol. 124:1121-1130 (2000), which is hereby incorporated
by reference in its entirety. Total RNA was isolated with TriPure
Isolation Reagent (Roche Biochemicals, Indianapolis, Ind.) and gel
electrophoresis was performed using 10 mM methyl mercury (II)
hydroxide as a denaturant. For hybridization with STGA20ox1, shoot
tip samples were collected at the same time of day to avoid
variations due to diurnal regulation. Probes were labeled with
[.alpha.-.sup.32P]dCTP (RadPrime DNA Labeling System, Gibco BRL,
Gaithersburg, Md.). POTH1 probes were generated by using the 730 nt
EcoR I fragment of POTH1 from the pCR2.1 vector (Invitrogen,
Carlsbad, Calif.) with the ELK and homeodomains deleted. The 1.5 kb
EcoR I-Xho I fragment of StGA20ox1 cDNA (Carrera et al., "Feedback
Control and Diurnal Regulation of Gibberellin 20-oxidase Transcript
Levels in Potato," Plant Physiol. 119:765-773 (1999), which is
hereby incorporated by reference in its entirety) was provided by
Salome Prat (Barcelona, Spain). All membranes were hybridized at
42.degree. C. for 70 hours in 50% formamide. The membranes were
rinsed in 2.times.SSC/0.1% SDS, at 25.degree. C., followed by
1.times.SSC/0.1% SDS for 0-20 minutes at 65.degree. C., then
0.1.times.SSC/0.1% SDS for 20-30 minutes at 65.degree. C. Film was
exposed for 4 to 7 days.
Example 7
Light Microscopy
[0127] Leaf tissue was fixed in 2% glutaraldehyde, 2%
paraformaldehyde in 0.1M sodium phosphate buffer pH 7.0 at
4.degree. C. for 72 hours, dehydrated in a graded ethanol series,
and embedded in LR White resin (Electron Microscopy Sciences, Ft.
Washington, Pa.). One .mu.m thick sections were cut on an
ultramicrotome (Reichert/Leica, Deerfield, Ill.) and stained with
1% toludine blue. Sections were viewed and photodocumented using
bright field microscopy.
Example 8
GA Analysis
[0128] Three replicates of shoot tips down to the sixth expanded
leaf (10 g each), were harvested in liquid nitrogen and frozen at
-80.degree. C. The tissue was ground with 80% methanol (MeOH) and
incubated at 4.degree. C. overnight. [.sup.2H.sub.2]-GA internal
standards were added in the following amounts in ng/g fwt:
GA.sub.1: 1, GA.sub.8: 10, GA.sub.19: 10, GA.sub.20: 20, and
GA.sub.53: 5. The extract was filtered through Highflo Supercel and
washed with 80% MeOH. After evaporation of the MeOH in vacuo, 0.5 M
Na.sub.2HPO.sub.4 was added to bring the pH to about 8.5, followed
by addition of 20 mL of hexane. The flask was mixed well and the
hexanes were evaporated off in vacuo. The solution was than
acidified to pH 3-3.5 with glacial CH.sub.3COOH (acetic acid) and
incubated for 15 minutes. The sample was then filtered through
polyvinylpolypyrrolidone (PVPP) and washed with 0.2% acetic acid.
The eluate was loaded onto a prepared Baker SPE (C.sub.18)
cartridge and washed with 0.2% acetic acid. The sample was eluted
off the column with 7 mL of 80% MeOH, evaporated to dryness, and
dissolved in 1 mL 100% MeOH. The MeOH-insoluble precipitate was
removed by centrifugation and the supernatant was evaporated to
dryness, redissolved in 0.8 mL 0.2% acetic acid, and filtered
through a 45 .mu.m filter. A one mL loop was used to load the
sample onto the C.sub.18 HPLC column (Econosphere: Phenomenex,
Torrance, Calif.) run with the following 0.2% acetic acid to
acetonitrile gradient: 5%-20% over 2 minutes; 20-35% over 15
minutes; 35-75% over 15 minutes. Fractions for the following GAs
were taken as follows: 10-14.3 minutes for GA.sub.8; 15.3-17.45
minutes for GA.sub.1; 23-27 minutes for GA.sub.19 and GA.sub.20;
27-29.3 minutes for GA.sub.53. Fractions were collected separately
and methylated with diazomethane in ether. Samples were dried,
redissolved in 1 mL ethyl acetate, and partitioned against water.
The aqueous phase was partitioned against another 1 mL of ethyl
acetate and the ethyl acetate fractions were combined. The samples
were dried and placed under high vacuum over P.sub.2O.sub.5. The
samples were dissolved in 2 .mu.L dry pyridine and 10 .mu.L BSTFA
[bis(trimethylsilyl)trifluoro-acetamide] with 1% TMCS
(trimethylchlorosilane) (Sylon BFT: Pierce, Rockford, Ill.) and
heated at 80.degree. C. for 20 minutes. Samples were analyzed by
GC-SIM on a GC-MS (HewlettPackard 5890 GC+5970B MS) with a 15m
Zebron ZB1 column (Phenomenex, Torrance, Calif.). The carrier gas,
He, was set at a flow rate of approximately 35 cm/sec. The initial
column temperature was 60.degree. C. for one minute and then
increased at a rate of 30.degree. C./minute to 240.degree. C., and
then to 290.degree. C. at a rate of 4.degree. C./minute. The
injector temperature was 225.degree. C. and the temperature of the
detector was 300.degree. C. Concentrations of GA.sub.53, GA.sub.19,
GA.sub.20, GA.sub.1, and GA.sub.8 were determined by calculating
the area of the peaks, 448/450, 434/436, 418/420, 506/508, and
594/596, respectively, at the correct Kovats retention indices.
Reference spectra were obtained from Gaskin et al., "GC-MS of the
Gibberellins and Related Compounds: Methodology and a Library of
Spectra," Bristol UK: Cantock's Enterprises (1991), which is hereby
incorporated by reference in its entirety. Cross-ion corrections
were calculated according to the following formula where: R.sub.1=%
endogenous ion in final; R.sub.2=% heavy ion in final; A.sub.1=%
endogenous ion in natural unlabelled sample; A.sub.2=% heavy ion in
natural unlabelled sample; B=heavy isotope internal standard.
Amount of natural compound ( A ) = [ R 1 ] [ R 2 .times. A 1 - R 1
.times. A 2 ] .times. Amount of B added ##EQU00001##
Example 9
In Vitro Tuberization
[0129] Cuttings of transgenic and control plants were placed in
Murashige-Skoog (MS) media plus 6% sucrose (Konstantinova et al.,
"Photoperiodic Control of Tuber Formation in Potato Solanum
Tuberosum ssp. Andigena in vivo and in vitro," Russian J. Plant
Physiol. 46:763-766 (1999), which is hereby incorporated by
reference in its entirety). After 2 weeks under long days (16 hours
of light, 8 hours of dark) to promote rooting, plants were cultured
separately under either long or short day (8 hours of light, 16
hours of dark) conditions. Plants were examined for tuber activity
(percentage of plants that produced either swollen stolons or
tubers) and the number of tubers were counted.
Example 10
Results: Isolation and Characterization of POTH1
[0130] An early stage tuber cDNA library (Kang et al., "Nucleotide
Sequences of Novel Potato (Solanum tuberosum L.) MADS-box cDNAs and
Their Expression in Vegetative Organs," Gene 166:329-330 (1995),
which is hereby incorporated by reference in its entirety) from
Solanum tuberosum `Superior` was screened for members of the
homeobox gene family. PCR primers were designed from the consensus
sequence of the homeoboxes of the class I genes kn1 from maize
(Vollbrecht et al., "The Developmental Gene Knotted-1 is a Member
of a Maize Homeobox Gene Family," Nature 350:241-243 (1991), which
is hereby incorporated by reference in its entirety), KNAT1 and
KNAT2 from Arabidopsis (Lincoln et al., "A Knotted1-like Homeobox
Gene in Arabidopsis is Expressed in the Vegetative Meristem and
Dramatically Alters Leaf Morphology When Overexpressed in
Transgenic Plants," Plant Cell 6:1859-1876 (1994), which is hereby
incorporated by reference in its entirety), OSH1 from rice
(Matsuoka et al., "Expression of a Rice Homeobox Gene Causes
Altered Morphology of Transgenic Plants," Plant Cell 5:1039-1048
(1993), which is hereby incorporated by reference in its entirety),
and SBH1 from soybean (Ma et al., "Identification of a
Homeobox-Containing Gene With Enhanced Expression During Soybean
(Glycine max L.) Somatic Embryo Development," Plant Mol. Biol.
24:465-473 (1994), which is hereby incorporated by reference in its
entirety). A mass excision of the tuber cDNA library was performed,
and this DNA was used as the PCR template. A band corresponding to
the expected size of 158 nt was purified, cloned, and sequenced.
This potato homeobox fragment was 87% identical to the conserved
positions of the consensus sequence created from the five class I
genes, and was used as a probe to screen the cDNA library. Library
screening resulted in the isolation of a truncated, 1053-nt
homeobox cDNA from the library, which was used as a probe to screen
the library again. Three clones were isolated, and the full-length
1383-nt potato homeobox cDNA, POTH1, was selected for further
study. The cDNA (Genbank Accession # U65648) includes an open
reading frame of 1035 nt coding for a 345-residue protein estimated
to have a mass of 37.95 kDa. It contains a 134-nt 5'-untranslated
region, and a 216-nt 3'-untranslated region, including the poly-A
tail. The coding sequence of the protein includes the 97-aa MEINOX
domain, the 22-aa ELK domain, and the 64-aa homeodomain.
[0131] To identify proteins with similarity to POTH1, a BLAST
analysis (Altschul et al., "Basic Local Alignment Search Tool," J.
Mol. Biol. 215:403-410 (1990), which is hereby incorporated by
reference in its entirety), was performed on the protein sequence
and GAP analysis [Wisconsin Package Version 9.1, Genetics Computer
Group (GCG), Madison, Wis.] was used to determine percent
similarity. POTH1 shares 86% similarity with the homeodomain of
KN1, classifying it as a class I homeobox protein (Kerstetter et
al., "Sequence Analysis and Expression Patterns Divide the Maize
Knotted1-like Homeobox Genes Into Two Classes," Plant Cell
6:1877-1887 (1994), which is hereby incorporated by reference in
its entirety). However, over the entire protein sequence, POTH1
shares only 51% similarity with KN1. The five proteins with the
most similarity to POTH1 include TKN3 from tomato (U76408), NTH22
of tobacco (Nishimura et al., "The Expression of Tobacco
Knotted1-type Class 1 Homeobox Genes Correspond to Regions
Predicted by the Cytohistological Zonation Model," Plant J. 18:
337-347 (1999), which is hereby incorporated by reference in its
entirety), PKN2 of Ipomoea nil (AB016000), KNAT2 of Arabidopsis
(Lincoln et al., "A Knotted1-like Homeobox Gene in Arabidopsis is
Expressed in the Vegetative Meristem and Dramatically Alters Leaf
Morphology When Overexpressed in Transgenic Plants," Plant Cell
6:1859-1876 (1994), which is hereby incorporated by reference in
its entirety) and NTH15 of tobacco (Tamaoki et al., "Ectopic
Expression of a Tobacco Homeobox Gene, NTH15, Dramatically Alters
Leaf Morphology and Hormone Levels in Transgenic Tobacco," Plant
Cell Physiol. 38:917-927 (1997), which is hereby incorporated by
reference in its entirety) with 94, 88, 73, 69, and 56% similarity
overall, respectively. As expected, relatively high levels of
conservation were observed in the homeodomains (97 to 83%) and in
the MEINOX domains (95 to 63%) of this group.
Example 11
Results: Southern Analysis
[0132] To study the complexity of the POTH1 gene family in the
tetraploid potato genome, Southern analysis was performed. Genomic
DNA from both S. tuberosum cv. FL-1607 and spp. andigena was
digested with Hind III and Xba I. For both species, only two bands
hybridized to a gene-specific probe for POTH1 (FIG. 1), indicating
that POTH1 is a member of a small gene family. A Hind III site is
located within the cDNA sequence of POTH1.
Example 12
Results: Accumulation of POTH1 mRNA
[0133] Northern blot analysis was used to determine the pattern of
POTH1 mRNA accumulation in various organs of potato (FIG. 2).
Poly(A)+ enriched RNA samples were hybridized with a
digoxygenin-UTP labeled 780-nt RNA antisense probe with the
conserved ELK region, homeobox region, and poly-A tail deleted. A
single band, approximately 1.3 kb in length, representing POTH1
mRNA, was present in RNA extracted from stem, root, inflorescence,
shoot apex, and swollen stolon apex (FIG. 2, lanes 2, 3, 4, 6, and
7, respectively). POTH1 transcripts were not detected in either
mature leaf or mature tuber RNA (FIG. 2, lanes 1 and 5). Equal
loading and the quality of the RNA loaded were ascertained via
ethidium bromide staining. This autoradiograph was representative
of several replicate hybridization blots.
[0134] To determine more precisely the location of POTH1 mRNA
accumulation, in situ hybridization was performed on vegetative
meristems of potato (FIG. 3). The potato SAM is comprised of two
tunica layers, which divide anticlinally to produce the epidermis
and contribute to lateral organs such as leaves, and three corpus
layers, which divide both periclinally and anticlinally to
contribute to lateral organ and stem development (Esau, "The Stem:
Primary State of Growth. In Wiley, eds., Anatomy of Seed Plants,
2nd Edition New York: pp. 243-294 (1977); Sussex, "Morphogenesis in
Solanum Tuberosum L.: Apical Structure and Developmental Pattern of
the Juvenile Shoot," Phytomorphology 5:253-273 (1955), which are
hereby incorporated by reference in their entirety). POTH1 mRNA
accumulates in the two tunica and three corpus layers of the SAM,
the leaf primordia, the procambium, and the lamina of young leaves
(FIG. 3A). Lower levels of POTH1 transcript can also be detected in
the developing leaflets of an older leaf (FIG. 3A, OL). A slightly
lower level of POTH1 transcript can be detected in the central zone
of the SAM, where initials divide less rapidly than adjacent
cells.
[0135] Potato plants produce underground stems that grow
horizontally, called stolons (Jackson, "Multiple Signaling Pathways
Control Tuber Induction in Potato," Plant Physiol. 119:1-8 (1999),
which is hereby incorporated by reference in its entirety). Under
optimum conditions, the subapical region of the stolon tip will
begin to swell and eventually develop into a tuber. A nontuberizing
stolon will elongate with most of its growth occurring in the
tunica and corpus layers. The greatest concentration of POTH1
signal can be detected in the apical meristem of the elongating
stolon (FIG. 3B). Expression levels are also high in the lamina of
the youngest leaf, the procambium, and the perimedullary parenchyma
associated with the vascular tissue (FIG. 3B). Differentiation of
the procambium into mature vascular tissue is marked by the
appearance of xylem elements (Esau, "The Stem: Primary State of
Growth. In Wiley, eds., Anatomy of Seed Plants, 2nd Edition New
York: pp. 243-294 (1977), which is hereby incorporated by reference
in its entirety), and POTH1 transcript accumulates in this
differentiated tissue as well (FIG. 3B). No signal is detected in
an elongating stolon tip hybridized with a sense POTH1 probe (FIG.
3C).
[0136] The apex of a tuberizing stolon, visibly swollen in FIG. 3D,
continues to accumulate POTH1 mRNA in the apical meristem, the
procambium, the lamina of new leaves, and the perimedullary
parenchyma, but the signal is less intense than in the elongating
stolon apical meristem (FIG. 3B). In the subapical portion of the
swollen stolon tip (FIG. 3E), where rapid radial expansion is
occurring (Xu et al., "Cell Division and Cell Enlargement During
Potato Tuber Formation," J Exp. Bot. 49:573-582 (1998), which is
hereby incorporated by reference in its entirety), POTH1 signal is
detected, especially in the perimedullary parenchyma, associated
with the vascular tissue. There is some signal as well in the pith
and inner cortex (FIG. 3E). FIG. 3F is the sense probe control
corresponding to the section in FIG. 3E. Similar results were
observed with sense probe controls in each section examined. The
data presented in FIG. 3 is representative of several independent
replications. Because FIGS. 3A-D are longitudinal sections through
various apices at the same magnification, the location of labeled
tissues is similar from one apex to the next.
Example 13
Results: The Overexpression of POTH1 in Transgenic Potato
Plants
[0137] To determine the effect of POTH1 overexpression on the
development of potato, the full-length POTH1 sequence was placed
under the control of the CaMV 35S promoter in the binary vector,
pCB201 (Xiang et al., "A Mini Binary Vector Series for Plant
Transformation," Plant Mol. Biol. 40:711-718 (1999), which is
hereby incorporated by reference in its entirety). To examine the
role of POTH1 in tuberization, two cultivars of potato (Solanum
tuberosum cv. FL-1607 and S. tuberosum ssp. andigena) were selected
for transformation. Andigena plants are photoperiod sensitive,
tuberizing only under short-day conditions (Carrera et al.,
"Changes in GA 20-oxidase Gene Expression Strongly Affect Stem
Length, Tuber Induction and Tuber Yield of Potato Plants," Plant J.
22:1-10 (2000), which is hereby incorporated by reference in its
entirety), whereas `FL-1607` plants tuberize under both long- and
short-day photoperiods. A total of thirty independent transgenic
lines from andigena and twenty independent transgenic lines from
`FL-1607` were generated and screened for increased POTH1 mRNA
expression. Among 10 sense lines of andigena and 15 lines of
`FL-1607` that showed high levels of POTH1 mRNA accumulation, five
independent transgenic lines of andigena and 4 lines of `FL-1607`
were chosen for further analysis. An aberrant phenotype was
observed only in those lines with detectable levels of POTH1 mRNA
from total RNA samples. Two transgenic lines, andigena lines 15 and
18 had the highest levels of POTH1 mRNA accumulation (FIG. 4A),
whereas andigena lines 11, 20, and 29 had intermediate levels of
POTH1 mRNA (FIG. 4A). Similar high levels of POTH1 accumulation
were observed in `FL-1607` overexpression lines that exhibited
mutant phenotypes. Equivalent loading of RNA samples was verified
by using an 18S rRNA probe from wheat (FIG. 4B).
Example 14
Results: Phenotype of POTH1 Overexpression Lines
[0138] Overexpression of POTH1 resulted in a phenotype
characterized by a reduction in plant height and leaf size (FIGS.
4C-F). Lines with the most abundant POTH1 RNA levels had the
greatest reduction in overall height. The height of potato subsp.
andigena lines 15 and 18 was reduced by at least 64% compared with
wild-type plants (FIG. 4C). Transgenic lines with an intermediate
phenotype (andigena lines 20, 29, and 11) showed a 20 to 25%
reduction in plant height (FIG. 4C). The decrease in plant height
was due to a corresponding decrease in internode elongation (FIG.
4D). The average internode length of the severe mutant, andigena
line 15, was 4.0 mm compared to 16 mm for wild-type andigena
plants. The same pattern was observed for petiole and leaflet
length (FIGS. 4E and 4F) with the severe phenotypes displaying the
greatest reduction in size. Among the five sense lines, petiole
length was reduced by 70 to 96%, whereas leaflet length was reduced
by 29 to 87% compared to wild-type. The sixth expanded leaf from
the shoot apex was used to measure petiole and terminal leaflet
length. Similar results were seen for `FL-1607` overexpression
lines.
[0139] Transgenic plants that overexpressed POTH1 also exhibited
malformed leaves. The overall size of the leaflets was greatly
reduced and they were rounded, curved, and wrinkled (FIG. 5A-B).
Wild-type leaflets have an ovate form and display pinnate venation
with a prominent mid-vein (FIG. 5B, left). In the overexpression
mutants, the midvein is less prominent and the most severe
phenotypes exhibited a `mouse-ear` leaf phenotype (FIGS. 5B-D). The
leaflets are heart-shaped with a shortened mid-vein. In addition,
there has been a switch from pinnate to palmate venation (FIG. 5B).
The phyllotaxy is not altered in overexpression lines, although,
compared with wild-type plants (FIG. 5C), the leaves are clustered
closer to the stem due to shortened petioles (FIG. 5D). In tomato,
the dominant mutations, Mouse-ear (Me) and Curl (Cu), were caused
by a change in the spatial and temporal expression of the tomato
knox gene TKn2/LeT6 (Parnis et al., "The Dominant Developmental
Mutants of Tomato, Mouse-ear and Curl, are Associated With Distinct
Modes of Abnormal Transcriptional Regulation of a Knotted Gene,"
Plant Cell 9:2143-2158 (1997); Chen et al., "A Gene Fusion at a
Homeobox Locus: Alterations in Leaf Shape and Implications for
Morphological Evolution," Plant Cell 9:1289-1304 (1997), which are
hereby incorporated by reference in their entirety). Overexpression
of kn1 (Hareven et al., "The Making of a Compound Leaf: Genetic
Manipulation of Leaf Architecture in Tomato," Cell 84:735-744
(1996), which is hereby incorporated by reference in its entirety)
in tomato caused up to a six-fold increase in the level of leaf
compoundness resulting in a leaf bearing 700-2000 leaflets. Such a
marked increase in the level of compoundness was not observed in
POTH1 overexpression lines. Increased proliferation of leaflets
from sense lines, however, was common (compare wild-type and line
19 leaflets in FIG. 5E).
[0140] To determine whether POTH1 overexpression affected the leaf
at the cellular level, leaf cross-sections of the severe mutant,
potato subsp. andigena line 15, were examined. Wild-type leaves
consist of a palisade parenchyma layer on the adaxial side and a
spongy parenchyma layer on the abaxial side (FIG. 5F). The cells of
the palisade layer are aligned in a vertical orientation and are
tightly packed, whereas the spongy parenchyma cells are more
loosely arranged (FIG. 5F). In leaves of potato subsp. andigena
line 15, the palisade parenchyma layer is absent and the spongy
parenchyma cells are more closely packed (FIG. 5H). Overall cell
size in the leaves of andigena line 15 is reduced by about one
half.
[0141] Many of the traits of the phenotypes observed in POTH1
overexpression lines were similar to GA-deficient mutants. These
similarities included decreased plant height, decreased internode
length, and darker green coloration of the leaves (van den Berg et
al., "Morphology and [.sup.14C]Gibberellin A.sub.12 Metabolism in
Wild-Type and Dwarf Solanum Tuberosum ssp. Andigena Grown Under
Long and Short Photoperiods," J. Plant Physiol. 146:467-473 (1995),
which is hereby incorporated by reference in its entirety). Because
of this, exogenous GA.sub.3 was applied to determine whether the
overexpression lines were responsive to GA treatment. The shoot
apex of overexpression lines was sprayed to runoff with 10 .mu.M
GA.sub.3 in 0.002% (v/v) ethanol or with 0.002% (v/v) ethanol
alone. Application of GA.sub.3 not only caused plants with a severe
phenotype to increase in height, but also partially rescued the
leaf morphology of both severe and intermediate phenotypes.
Palisade and spongy parenchyma organization is partially rescued in
leaves from line 15 treated with GA.sub.3 (FIG. 5G). The compound
leaf structure of the of the potato subsp. andigena wild-type leaf
is shown in FIG. 5I. The GA.sub.3-treated leaf (FIG. 5J) of the
severe mutant, line 15, is more similar in morphology to the
wild-type leaf (FIG. 5K). Leaflets are more ovate in form rather
than the typical mouse-ear shape. Wild-type leaves have a prominent
mid-vein (FIG. 5L), whereas the mid-vein (FIG. 5M, arrow) is more
prominent in the mutant GA.sub.3-treated leaf than in the mutant
untreated leaf (FIG. 5N). The compound leaf structure of the
`FL-1607` wild-type leaf is shown in FIG. 5O. The GA.sub.3-treated
leaf (FIG. 5P) of the severe mutant, `FL-1607` line 5, is more
similar in morphology to the wild-type leaf than to the mutant
control leaf (FIG. 5Q). Leaflets are more ovate in form rather than
the typical `mouse-ear` shape. The mid-vein (arrow) is more
prominent in the GA.sub.3-treated leaf (FIG. 5P) than in the mutant
leaf (FIG. 5Q).
[0142] To determine whether GA biosynthesis was disrupted in POTH1
overexpression lines, levels of intermediates in the GA
biosynthesis pathway in potato (van den Berg et al., "Metabolism of
Gibberellin A12 and A12-aldehyde and the Identification of
Endogenous Gibberellins in Potato (Solanum tuberosum ssp. andigena)
Shoots," J. Plant Physiol. 146:459-466 (1995), which is hereby
incorporated by reference in its entirety) were measured. Levels of
the intermediates GA.sub.53 and GA.sub.19 increased in POTH1
overexpression lines, whereas GA.sub.1 and GA.sub.8 levels
decreased (FIG. 6). In potato subsp. andigena lines 29 and 20,
GA.sub.53 and GA.sub.19 levels increased approximately 2-fold
compared with wild-type lines (FIG. 6). The levels of GA.sub.1 and
GA.sub.8 present in potato subsp. andigena overexpression lines
were approximately one-half that of wild-type levels (FIG. 6).
Accumulation of GA.sub.53 and GA.sub.19 with a concomitant decrease
in GA.sub.1 and GA.sub.8 indicates that the GA biosynthetic pathway
is blocked at the oxidation of GA.sub.19 to GA.sub.20, leading to a
decrease in the levels of bioactive GA.sub.1. Similar patterns of
accumulation for GA intermediates were also observed for potato
subsp. andigena sense line 15 (in andigena line 15, GA.sub.53 and
GA.sub.19 levels increased 4.8.times. and 2.1.times., respectively,
compared to wild-type).
[0143] Overexpression lines were deficient in bioactive GAs, but
were responsive to the exogenous application of GA.sub.3. This
indicates that GA biosynthesis is inhibited in the overexpression
lines. In addition, accumulation of GA.sub.53 and GA.sub.19, with a
decrease in GA.sub.20, GA.sub.1, and GA.sub.8 (FIG. 6), indicates
that the activity of the biosynthetic gene, GA 20-oxidase, may be
suppressed. GA 20-oxidase catalyzes the oxidation of carbon 20 of
GA.sub.53 to GA.sub.44 to GA.sub.19 to GA.sub.20. The enzyme GA
3-oxidase then converts GA.sub.20 to the active GA.sub.1 (Hedden et
al., "Gibberellin Biosynthesis: Enzymes, Genes and Their
Regulation," Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:431-460
(1997), which is hereby incorporated by reference in its entirety).
To determine whether POTH1 overexpression causes a change in GA
20-oxidase mRNA levels, RNA blot analysis was performed using one
of the potato genes encoding GA 20-oxidase, StGA20ox1, as a probe
(Carrera et al., "Feedback Control and Diurnal Regulation of
Gibberellin 20-oxidase Transcript Levels in Potato," Plant Physiol.
119:765-773 (1999), which is hereby incorporated by reference in
its entirety). In the overexpression lines, StGA20ox1 mRNA levels
were reduced substantially compared to levels in wild-type lines
(FIG. 7).
[0144] GA is involved in regulating cell growth in a tuberizing
stolon (Xu et al., "The Role of Gibberellin, Abscisic Acid, and
Sucrose in the Regulation of Potato Tuber Formation in vitro,"
Plant Physiol. 117:575-584 (1998), which is hereby incorporated by
reference in its entirety) and in contributing to the control of
the photoperiodic response of tuber formation (Martinez-Garcia et
al., "The Interaction of Gibberellins and Photoperiod in the
Control of Potato Tuberization," J. Plant Growth Regul. 20:377-386
(2001), which is hereby incorporated by reference in its entirety).
Because levels of active GAs were reduced in transgenic plants, an
in vitro tuberization assay (Konstantinova et al., "Photoperiodic
Control of Tuber Formation in Potato Solanum Tuberosum ssp.
Andigena in vivo and in vitro," Russian J. Plant Physiol.
46:763-766 (1999), which is hereby incorporated by reference in its
entirety) was used to determine the effect of POTH1 overexpression
on tuberization. After 2 weeks under a 16 hour light/8 hour dark
photoperiod to induce rooting, plants were cultured on 6% (w/v)
sucrose under either an 8 hour light/16 hour dark (inductive) or 16
hour light/8 hour dark (noninductive) photoperiod. After 10 days,
the overexpression lines had 60 to 82% and 19 to 68% tuber activity
under short and long days, respectively, compared to 0% activity
for wild-type plants (Table 1).
TABLE-US-00017 TABLE 1 In vitro tuberization of POTH1
overexpression lines. S. tuberosum spp. andigena transgenics were
placed on Murashige-Skoog media supplemented with 6% sucrose under
either short-day (SD) or long-day (LD) conditions. At least 12
plants per line were monitored for total number of tubers that
formed and tuber activity (percentage of plants that produced
either swollen stolons or tubers). Numbers in parentheses are the
average number of tubers produced per plant. # tubers
(tubers/plant) % tuber activity line 14 d SD 14 d LD line 10 d SD
10 d LD control 1 (.08) 1 (.06) control 0 0 1200-29 21 (1.4) 14
(.88) 1200-29 60 40 1200-11 13 (.72) 22 (1.2) 1200-11 78 68 1200-15
17 (1.5) 2 (.12) 1200-15 82 19 1200-18 12 (.86) 8 (.57) 1200-18 79
43 line 21 d SD 21 d LD control (0.66) (0.43) 1200-29 (1.70) (1.25)
1200-11 (0.88) (1.30) 1200-15 (2.30) (0.38) 1200-18 (1.50)
(0.86)
Tuber activity was calculated as the percentage of plants that
formed either a swollen stolon or a tuber. At 14 days,
overexpression lines produced an average of 0.7 to 1.5 tubers per
plant under short days, whereas wild-type plants produced an
average of 0.08 tubers per plant (Table 1). Similar results were
observed under long days and after 21 days in culture (Table 1).
Overall, the POTH1 overexpression lines could produce more tubers
in less time than controls and apparently, also overcome the
negative effects of a long-day photoperiod on tuber formation. The
potato cv FL-1607 overexpression lines also exhibited increased
tuber activity under both photoperiods.
Example 15
Discussion: POTH1 has a Widespread mRNA Expression Pattern
[0145] Isolated from an early stage tuber cDNA library, POTH1 is a
homeobox gene belonging to the knox gene family. It contains the
conserved homeodomain, ELK, and MEINOX domains. The homeodomain
contains the invariant residues, PYP, between helices 1 and 2,
making it a member of the TALE superclass. Because of its close
sequence match with the KN1 homeodomain, POTH1 is classified as a
knox class I homeobox gene.
[0146] Even though POTH1 is classified as a class I knox gene, it
has a more widespread mRNA expression pattern than other class I
genes. POTH1 is expressed in actively growing organs, but not in
mature leaves or tubers. Unlike the mRNA expression pattern of kn1
which is limited to corpus cells of the apical meristem (Jackson et
al., "Expression of Maize KNOTTED1 Related Homeobox Genes in the
Shoot Apical Meristem Predicts Patterns of Morphogenesis in the
Vegetative Shoot," Development 120:405-413 (1994), which is hereby
incorporated by reference in its entirety), in situ hybridization
showed that POTH1 mRNA accumulates in the meristematic and
indeterminate cells of the SAM, determinate leaf primordia, the
expanding lamina of new leaves, and developing leaflets of older
leaves. The expression pattern of POTH1 mRNA in the unswollen
stolon is similar to that seen in the shoot apical meristem. Signal
was highest in undetermined, meristematic cells, but was also
detected in the lamina of young leaves and the vascular tissue of
the stem. Once tuberization has been initiated, the signal becomes
less intense at the stolon apex, but is present in the vascular
tissue in the subapical portion of the stolon. At this stage of
tuberization, elongation of the meristem has stopped, and rapid,
radial expansion occurs in the subapical region (Reeve et al.,
"Anatomy and Compositional Variation Within Potatoes I.
Developmental Histology of the Tuber," Amer. Pot. J. 46:361-373
(1969), which is hereby incorporated by reference in its
entirety).
[0147] Most class I knox genes have a more limited pattern of mRNA
expression, restricted to undifferentiated cells of the meristem
(Reiser et al., "Knots in the Family Tree: Evolutionary
Relationships and Functions of Knox Homeobox Genes," Plant Mol.
Biol. 42:151-166 (2000), which is hereby incorporated by reference
in its entirety). Members of the tobacco knox family have distinct
expression patterns within the SAM. NTH15 and NTH1 are expressed
throughout the corpus, NTH20 is expressed in the peripherary zone,
and NTH9 is expressed in the rib zone of the SAM (Nishimura et al.,
"The Expression of Tobacco Knotted1-type Class1 Homeobox Genes
Correspond to Regions Predicted by the Cytohistological Zonation
Model," Plant J. 18: 337-347 (1999), which is hereby incorporated
by reference in its entirety). The tomato knox class I genes, TKn1
and TKn2/LeT6, have a expression pattern similar to POTH1 with
transcripts detectable in meristematic and differentiated cells.
Expression of TKn2/LeT6 was detected in the corpus of the meristem,
developing leaf primordia, leaflet primordia and margins, and the
vascular cells of the leaf (Chen et al., "A Gene Fusion at a
Homeobox Locus: Alterations in Leaf Shape and Implications for
Morphological Evolution," Plant Cell 9:1289-1304 (1997); Janssen et
al., "Overexpression of a Homeobox Gene, LeT6, Reveals
Indeterminate Features in the Tomato Compound Leaf," Plant Physiol.
117: 771-786 (1998), which are hereby incorporated by reference in
their entirety). This expanded expression pattern in tomato has
been attributed to the differences in compound leaf development
compared to simple leaf development and the expansion of
undifferentiated tissues to include developing leaflets. Potato is
unique because it forms compound leaves from the vegetative shoot
apical meristem above ground, but forms simple, scale leaves from
the stolon meristem below ground (Sussex, "Morphogenesis in Solanum
Tuberosum L.: Apical Structure and Developmental Pattern of the
Juvenile Shoot," Phytomorphology 5:253-273 (1955), which is hereby
incorporated by reference in its entirety). Expression of POTH1 is
detected in young leaves that arise from both the shoot apical and
stolon meristems. This indicates that POTH1 mRNA expression alone
is not the determining factor for the development of compound
leaves in potato. In the shoot or stolon meristem, the activity of
POTH1 may be regulated differently through interaction with partner
proteins specific for shoot or stolon meristem development.
Example 16
Discussion: Phenotype of POTH1 Overexpression Transgenic Lines
[0148] Overexpression of POTH1 resulted in altered leaf morphology,
dwarfism, and increased rates of in vitro tuberization. Leaves were
small, wrinkled, and curved. Both severe and intermediate
phenotypes were characterized by a `mouse-ear` leaf phenotype.
Leaves were heart-shaped with a decreased midvein and palmate
venation. The petioles were reduced in length resulting in leaves
clustering closer to the stems. Overexpression lines exhibited
dwarfism as a result of reduced internode length. The severity of
the phenotype was correlated with the greatest levels of POTH1
sense transcript accumulation. Cross-sections of leaves revealed
that the mesophyll cell organization was disrupted with the
palisade parenchyma layer missing in POTH1 overexpression lines.
The tightly packed cells were about half the size of the wild-type
cells. A similar disruption in leaf parenchyma cell layers was
observed in sense mutants of KNAT1 and KNAT2 (Chuck et al., "KNAT1
Induces Lobed Leaves With Ectopic Meristems When Overexpressed in
Arabidopsis," Plant Cell 8:1277-1289 (1996); Frugis et al.,
"Overexpression of KNAT1 in Lettuce Shifts Leaf Determinate Growth
to a Shoot-like Indeterminate Growth Associated With an
Accumulation of Isopentenyl-type Cytokinins," Plant Physiol.
126:1370-1380 (2001); Pautot et al., "KNAT2: Evidence for a Link
Between Knotted-like Genes and Carpel Development," Plant Cell
13:1719-1734 (2001), which are hereby incorporated by reference in
their entirety). Because class I knox genes are implicated in
maintaining the undifferentiated state of cells (Chan et al.,
"Homeoboxes in Plant Development," Biochim. Biophys. Acta 1442:1-19
(1998), which is hereby incorporated by reference in its entirety),
disruption in leaf architecture is likely a result of a defect in
the normal differentiation program.
[0149] Based on overexpression phenotypes, POTH1 and NTH22 of
tobacco (Nishimura et al., "Over-Expression of Tobacco
Knotted1-type Class1 Homeobox Genes Alter Various Leaf Morphology,"
Plant Cell Physiol. 41:583-590 (2000), which is hereby incorporated
by reference in its entirety) appear to have similar functions that
overlap, but are distinct from, the class I knox genes, kn1, NTH15,
OSH1, and KNAT1. Like overexpression of POTH1 in potato and NTH22
in tobacco, overexpression of kn1, NTH15, OSH1, KNAT1 in tobacco or
Arabidopsis (Sinha et al., "Overexpression of the Maize Homeo Box
Gene, KNOTTED-1, Causes a Switch From Determinate to Indeterminate
Cell Fates," Genes Dev. 7:787-795 (1993); Sato et al., "Abnormal
Cell Divisions in Leaf Primordia Caused by the Expression of the
Rice Homeobox Gene OSH1 Lead to Altered Morphology of Leaves in
Transgenic Tobacco," Mol. Gen. Genet. 251:13-22 (1996); Tamaoki et
al., "Ectopic Expression of a Tobacco Homeobox Gene, NTH15,
Dramatically Alters Leaf Morphology and Hormone Levels in
Transgenic Tobacco," Plant Cell Physiol. 38:917-927 (1997); Chuck
et al., "KNAT1 Induces Lobed Leaves With Ectopic Meristems When
Overexpressed in Arabidopsis," Plant Cell 8:1277-1289 (1996);
Lincoln et al., "A Knotted1-like Homeobox Gene in Arabidopsis is
Expressed in the Vegetative Meristem and Dramatically Alters Leaf
Morphology When Overexpressed in Transgenic Plants," Plant Cell
6:1859-1876 (1994), which are hereby incorporated by reference in
their entirety) resulted in dwarfism, decreased internode
elongation, shortened petioles, and small deformed leaves.
Additional phenotypes, including ectopic meristem formation, loss
of apical dominance, and delayed senescence, however, were not
observed in POTH1 or NTH22 overexpression transgenic lines. Whereas
there seems to be some redundancy in function between different
members of the knox gene family, (for example, regulation of GA
biosynthesis), POTH1 is not likely to have an identical function to
kn1, NTH15, or OSH1. Rather, these genes are likely to have
different subsets of target genes, which is reflected in their
differences in homeodomain sequence (83 to 86% match to POTH1's
homeodomain, compared to a 98% match for NTH22).
Example 17
Discussion: Ectopic Expression of POTH1 Results in GA
Deficiency
[0150] Similar to the knox genes NTH15 of tobacco and OSH1 of rice,
the results above indicate that POTH1 is a negative regulator of GA
biosynthesis. POTH1 overexpression transgenic lines share many
phenotypes with GA-deficient mutants including dwarfism, decreased
internode elongation, and darker leaf coloration (van den Berg et
al., "Morphology and [.sup.14C]Gibberellin A.sub.12 Metabolism in
Wild-Type and Dwarf Solanum Tuberosum ssp. Andigena Grown Under
Long and Short Photoperiods," J. Plant Physiol. 146:467-473 (1995),
which is hereby incorporated by reference in its entirety).
Exogenous application of GA.sub.3 partially rescued the aberrant
leaf phenotype indicating that overexpression lines were responsive
to GA. Levels of the bioactive GA, GA.sub.1, were reduced in
overexpression lines, whereas intermediates prior to GA.sub.20 in
the pathway accumulated. Additionally, the mRNA levels of a key GA
biosynthetic enzyme, GA 20-oxidase1, were reduced in overexpression
lines. When NTH15 and OSH1 were overexpressed in tobacco, the
levels of the hormones, auxin, cytokinin, abscisic acid, and GA
were altered. GA.sub.1 levels were reduced to 1.4% and 0.4-3.5% of
controls in intermediate 35S-NTH15 and severe or mild 35S-OSH1
transgenics, respectively (Kusaba et al., "Alteration of Hormone
Levels in Transgenic Tobacco Plants Overexpressing the Rice
Homeobox Gene OSH1," Plant Physiol. 116:471-476 (1998); Tamaoki et
al., "Ectopic Expression of a Tobacco Homeobox Gene, NTH15,
Dramatically Alters Leaf Morphology and Hormone Levels in
Transgenic Tobacco," Plant Cell Physiol. 38:917-927 (1997), which
are hereby incorporated by reference in their entirety). In
tobacco, NTH15 affects plant growth by negatively regulating GA
levels by suppressing the transcription of the tobacco GA
20-oxidase gene through a direct interaction with regulatory
elements (Sakamoto et al., "KNOX Homeodomain Protein Directly
Suppresses the Expression of a Gibberellin Biosynthetic Gene in the
Tobacco Shoot Apical Meristem," Genes Dev. 15:581-590 (2001), which
is hereby incorporated by reference in its entirety).
[0151] POTH1 overexpression lines exhibited an increase in both the
rate of tuberization and the total number of tubers formed under
both short- and long-day photoperiods. These sense lines appear to
have the capacity to overcome the negative effects of a long-day
photoperiod on tuberization in vitro. Enhanced tuberization can be
partially attributed to the decrease in GA.sub.1 levels caused by
POTH1 suppression of GA 20-oxidase1. Pytochrome B (PHYB) and GAs
are involved in inhibiting tuberization under long-day
photoperiods. A long-day photoperiod is sensed by the leaves and an
inhibitory signal mediated by PHYB is transmitted from the leaves
to the stolons to inhibit tuberization (Jackson, "Multiple
Signaling Pathways Control Tuber Induction in Potato," Plant
Physiol. 119:1-8 (1999), which is hereby incorporated by reference
in its entirety). GA activity is regulated by light, decreasing
under short-day photoperiods (Railton et al., "Effects of Daylength
on Endogenous Gibberellins in Leaves of Solanum Andigena I. Changes
in Levels of Free Acidic Gibberellin-like Substances," Physiol.
Plant. 28:88-94 (1973), which is hereby incorporated by reference
in its entirety) and is involved in the photoperiodic control of
stolon growth. High levels of GA in the stolon tip favor elongation
of stolon meristems, whereas decreasing levels are required for
initiation of tuberization (Xu et al., "The Role of Gibberellin,
Abscisic Acid, and Sucrose in the Regulation of Potato Tuber
Formation in vitro," Plant Physiol. 117:575-584 (1998), which is
hereby incorporated by reference in its entirety). GA 20-oxidase is
a key enzyme in the GA biosynthetic pathway. In potato, the GA
20-oxidase genes are regulated by GA, feedback inhibition, blue
light, and PHYB (Jackson et al., "Regulation of Transcript Levels
of a Potato Gibberellin 20-Oxidase Gene by Light and Phytochrome
B," Plant Physiol. 124:423-430 (2000), which is hereby incorporated
by reference in its entirety). Whereas PHYB antisense plants were
able to form tubers under both long- and short-day photoperiods
(Jackson et al., "Phytochrome B Mediates the Photoperiodic Control
of Tuber Formation in Potato," Plant J. 9:159-166 (1996), which is
hereby incorporated by reference in its entirety), transgenic
antisense andigena plants with suppressed levels of GA 20-oxidase1
(StGA20ox1) were not able to overcome the negative effects of
photoperiod on tuberization in soil-grown plants (Carrera et al.,
"Changes in GA 20-oxidase Gene Expression Strongly Affect Stem
Length, Tuber Induction and Tuber Yield of Potato Plants," Plant J.
22:1-10 (2000), which is hereby incorporated by reference in its
entirety). While the experiments described above involved an in
vitro assay rather than soil grown plants, Konstantinova et al.,
"Photoperiodic Control of Tuber Formation in Potato Solanum
Tuberosum ssp. Andigena in vivo and in vitro," Russian J. Plant
Physiol. 46:763-766 (1999), which is hereby incorporated by
reference in its entirety, demonstrated that an in vitro assay for
tuber formation is a reliable method for ascertaining the effect of
photoperiod on tuberization in a photoperiod responsive cultivar.
While it is possible that GA levels are not reduced sufficiently in
antisense GA 20-oxidase1 plants, an additional signal may be
involved in the long-day-photoperiod inhibition of tuberization.
This indicates that in addition to reducing GA levels, POTH1
overexpression may enhance tuberization under long days by
overcoming the effects of other negative regulators.
Example 18
Discussion: Regulation of POTH1 Activity During Development
[0152] Overexpression of POTH1 potentially regulates development in
the SAM and in underground stolons through a reduction in bioactive
GA levels in vegetative meristems. Whereas GA levels are high in
the elongating unswollen stolon and decrease in swollen stolons (Xu
et al., "The Role of Gibberellin, Abscisic Acid, and Sucrose in the
Regulation of Potato Tuber Formation in vitro," Plant Physiol.
117:575-584 (1998), which is hereby incorporated by reference in
its entirety), POTH1 mRNA accumulates in both unswollen and swollen
stolons. If POTH1 is a negative regulator of GA synthesis, how can
its expression mediate a decrease in GA levels in the swollen
stolon leading to tuberization, but not in the elongating unswollen
stolon tip? With other TFs, an interaction with a partner protein
can regulate development by affecting the binding of the
homeodomain(s) to the DNA of a target gene. In Antirrhinum, for
example, formation of a ternary complex consisting of the MADS box
proteins, SQUA, DEF, and GLO, greatly increases DNA binding
compared to SQUA homodimers or DEF/GLO heterodimers alone
(Egea-Cortines et al., "Ternary Complex Formation Between the
MADS-box Proteins SQUAMOSA, DEFICIENS and GLOBOSA is Involved in
the Control of Floral Architecture in Antirrhinum majus," EMBO J.
18:5370-5379 (1999), which is hereby incorporated by reference in
its entirety). The interaction of HOX proteins with PBC proteins in
animals modulates the affinity of the HOX proteins for specific DNA
binding sites (Chang et al., "Meis Proteins are Major in vivo DNA
Binding Partners for Wild-Type but not Chimeric Pbx Proteins," Mol.
Cell. Biol. 17:5679-5687 (1997), which is hereby incorporated by
reference in its entirety). HOX homodimers have different DNA
binding sites than HOX-PBC heterodimers (Mann et al., "Extra
Specificity From Extradenticle: the Partnership Between HOX and
PBX/EXD Homeodomain Proteins," Trends Genet. 12:258-262 (1996),
which is hereby incorporated by reference in its entirety)
indicating that the target gene (and function) is dependent on
protein-protein interactions. Additionally, HOX-PBC complexes can
be activators or repressors of transcription depending on the
cell-type and the presence of a third interacting partner (Saleh et
al., "Cell Signaling Switches HOX-PBX Complexes From Repressors to
Activators of Transcription Mediated by Histone Deacetylases and
Histone Acetyltransferases," Mol. Cell. Biol. 20:8623-8633 (2000),
which is hereby incorporated by reference in its entirety). With
the formation of different combinations of heterodimers and ternary
complexes, the potential to regulate growth by interacting with
different target genes is greatly increased.
[0153] It is clear that the interaction of KNOX proteins with other
proteins is an important mechanism for regulating development.
Protein-protein interactions between BEL-type TFs and KNOX proteins
have been reported in barley (Muller et al., "In vitro Interactions
Between Barley TALE Homeodomain Proteins Suggest a Role for
Protein-protein Associations in the Regulation of Knox Gene
Function," Plant J. 27:13-23 (2001), which is hereby incorporated
by reference in its entirety) and Arabidopsis (Bellaoui et al.,
"The Arabidopsis BELL1 and KNOX TALE Homeodomain Proteins Interact
Through a Domain Conserved Between Plants and Animals," Plant Cell
13:2455-2470 (2001), which is hereby incorporated by reference in
its entirety). Homodimerization of KNOX proteins of barley (Muller
et al., "In vitro Interactions Between Barley TALE Homeodomain
Proteins Suggest a Role for Protein-protein Associations in the
Regulation of Knox Gene Function," Plant J. 27:13-23 (2001), which
is hereby incorporated by reference in its entirety) and rice
(Nagasaki et al., "Functional Analysis of the Conserved Domains of
a Rice KNOX Homeodomain Protein, OSH15," Plant Cell 13:2085-2098
(2001), which is hereby incorporated by reference in its entirety)
has also been demonstrated. Sakamoto et al., "The Conserved KNOX
Domain Mediates Specificity of Tobacco KNOTTED1-type Homeodomain
Proteins," Plant Cell 11: 1419-1431 (1999), which is hereby
incorporated by reference in its entirety, showed by expressing
chimeric proteins in transgenic tobacco plants that the region of
the MEINOX domain (designated KNOX2) involved in protein
interaction was more important than the homeodomain in determining
the severity of the mutant phenotype. By using a yeast two-hybrid
library screen, as described in Examples 20-32, below, seven unique
proteins were isolated from potato stolons that interact with
POTH1. These seven proteins are homeobox genes of the BEL1-like
family and members of the TALE superclass. Whereas POTH1 has a
widespread mRNA expression pattern, the seven potato BELs have a
differential pattern of expression. It is possible that POTH1
interacts with one BEL protein to negatively regulate GA levels in
the tuberizing stolon, but interacts with a different BEL partner
in the elongating stolon or SAM. Overexpression of one of the
POTH1-interacting proteins, StBEL-05, enhances tuberization under
both long- and short-day photoperiods; but unlike POTH1
overexpression, leaf morphology is not altered (see below). In a
tandem complex with a specific BEL partner, POTH1 could activate
transcription of a set of target genes in one organ or set of cells
and with another partner suppress those same genes in a different
organ.
Example 19
Overexpression of POTH1 Negatively Regulates GA Levels and Affects
Vegetative Morphology
[0154] To further examine the function of POTH1, transformed potato
plants (Solanum tuberosum spp. andigena) that overexpressed POTH1
mRNA were analyzed. For these experiments, the full-length cDNA
sequence of POTH1 in a sense orientation driven by the CaMV-35S
promoter in the binary vector, pCB201 (Xiang et al., "A Mini Binary
Vector Series for Plant Transformation," Plant Mol. Biol.
40:711-718 (1999), which is hereby incorporated by reference in its
entirety) was used. The accumulation of the POTH1 mRNA was tightly
correlated with a change in phenotype. These overexpressing lines
were characterized by distorted, smaller leaves, and dwarfism (FIG.
8). The mutant leaf traits are designated "mouse-ear" or "curled"
phenotype as reported previously in other knox mutants (Parnis et
al., "The Dominant Developmental Mutants of Tomato, Mouse-Ear and
Curl, Are Associated with Distinct Modes of Abnormal
Transcriptional regulation of a knotted Gene," Plant Cell
9:2143-2158 (1997); Tamaoki et al., "Ectopic Expression of a
Tobacco Homeobox Gene, NTH15, Dramatically Alters Leaf Morphology
and Hormone Levels in transgenic Tobacco," Plant Cell Physiol.
38:917-927 (1997), which are hereby incorporated by reference in
their entirety). Application of GA.sub.3 produced a partial
reversal of the leaf phenotype and completely rescued the dwarf
phenotype (see above).
[0155] Because of the similarity of this POTH1 phenotype to those
reported in tobacco (Tanaka-Ueguchi et al., "Overexpression of a
Tobacco Homeobox Gene, NTH15, Decreases the Expression of a
Gibberellin Biosynthetic Gene Encoding GA 20-oxidase," Plant J.
15:391-400 (1998); Tamaoki et al., "Transgenic Tobacco
Over-Expressing a Homeobox Gene Shows a Developmental Interaction
Between Leaf Morphogenesis and Phyllotaxy," Plant Cell Physiol.
40:657-557 (1999), which are hereby incorporated by reference in
their entirety), the effect of GA 20-oxidase mRNA accumulation in
these POTH1 overexpressers was examined. GA 20-oxidase is a key
biosynthetic enzyme in the GA pathway, catalyzing the conversion of
GA.sub.53 to GA.sub.20 via GA.sub.44 and GA.sub.19 (Hedden et al.,
"Gibberellin Biosynthesis: Enzymes, Genes and Their Regulation,"
Ann. Rev. Plant Physiol. Plant Mol. Biol. 48:431-460 (1997), which
is hereby incorporated by reference in its entirety). Using a probe
for the potato GA 20-oxidase1 gene (Carrera et al., "Feedback
Control and Diurnal Regulation of Gibberellin 20-oxidase Transcript
Level in Potato," Plant Physiol. 119:765-773 (1999), which is
hereby incorporated by reference in its entirety), a reduction in
GA 20-oxidase1 mRNA in shoots of the most severe mutant phenotypes
was observed (FIG. 8). Both internode length and overall plant
height were reduced approximately threefold in these mutant plants
relative to controls. In addition, in a biochemical analysis
performed in collaboration with Dr. Peter Davies, Cornell
University, the levels of GA.sub.53 and GA.sub.19 increased,
whereas the levels of GA.sub.20 and GA.sub.1 decreased in shoot
tips of these plants. These results indicate that POTH1 is a
negative regulator of GA biosynthesis and that it plays a role in
controlling vegetative pattern formation.
Example 20
Two-Hybrid Selection and Deletion Analysis
[0156] The Matchmaker two-hybrid system (Clontech, CA) was used for
the yeast two-hybrid screen. Yeast transformation and plasmid
rescue into DH5-.alpha. E. coli cells were according to the
manufacturer's instructions. Full-length POTH1 was cloned into the
pBridge (Clontech, CA) vector and used as bait to screen the potato
(S. tuberosum Desiree) stolon cDNA library in pAD-GAL4-2.1
(Stratagene, CA). Positive interactions were confirmed by
cotransforming yeast strain AH109 with each purified pAD plasmid
and pBridge: POTH1 and plating on -leucine/-tryptophan
(transformation control) and
-leucine/-tryptophan/-histidine/-adenine (selection) nutrient
medium. Induction of the AH109 reporter gene, lacZ, was measured
with a yeast .beta.-galactosidase assay kit (Pierce Chemicals).
.beta.-galactosidase activity (FIG. 9B) was determined from a known
density of yeast cells and calculated as 1000.times.OD.sub.420/time
of color reaction (minutes).times.volume of yeast culture
(ml).times.OD.sub.600.
[0157] The StBEL-05 deletion constructs were amplified by PCR, then
cloned into the vector, pGAD, in-frame with the GAL4 activation
domain. POTH1 deletion constructs were amplified by PCR, and cloned
into pBridge (Clontech) in-frame with the GAL4 binding domain.
Sequencing of selected cDNAs and constructs was performed at the
Iowa State University DNA Facility. For deletion analysis, modified
constructs of POTH1 were cloned into the pBridge vector for fusion
with the DNA-binding domain of GAL4 (FIG. 10A). For StBEL-05,
constructs were cloned into the pGAD vector for fusion with the
activating domain of GAL4 (FIG. 10B). Deletion constructs were made
from both the amino and carboxy termini. These mutants were then
tested for interaction in the yeast two-hybrid system by
cotransforming into yeast strain AH109 with the corresponding
full-length partner (StBEL-05 in pGAL4 or POTH1 in pBridge). All
constructs were sequenced to verify that they were in-frame.
Positive interactions were verified for lacZ induction by using a
.beta.-galactosidase assay (Pierce Chemical Company). For POTH1,
seven deletion constructs were tested (FIG. 10A). For the BEL TFs,
a fusion construct of StBEL-05 (653 aa of StBEL-05 sequence) and
nine deletion constructs were tested (FIG. 10B).
[0158] GenBank accession numbers for StBEL-05, -11, -13, -14, -22,
-29, and -30 are AF406697, AF406698, AF406699, AF406700, AF406701,
AF406702, AF406703, respectively.
Example 21
In Vitro Binding Assay
[0159] In vitro binding experiments were performed as described by
Ni et al., "PIF3, a Phytochrome-Interacting Factor Necessary for
Normal Photoinduced Signal Transduction, is a Novel Basic
Helix-Loop-Helix Protein," Cell 95:657-667 (1998), which is hereby
incorporated by reference in its entirety. The full-length sequence
for POTH1 was cloned into a pET17b/GAD fusion cassette and
transcribed under the control of the T7 promoter. The BEL cDNAs
were cloned into pGEM11Z vectors and were transcribed under the
control of the T7 promoter. .sup.35S-methionine labeled bait and
prey proteins were synthesized using the TnT in vitro
transcription-translation kit (Promega) according to the
manufacturer's protocols. Each 50 .mu.l TnT reaction contained 2.0
.mu.g of template plasmid DNA and 20 .mu.mol (20 .mu.Ci) of labeled
.sup.35S-methionine. The POTH1:GAD/BEL complex was
immunoprecipitated with anti-GAD antibodies (Santa Cruz
Biotechnology, CA). The proteins from the pellet (one-half the
fraction) and for the prey (one-fourth of the reaction volume) were
resolved on a 10% SDS-PAGE gel and visualized by
autoradiography.
Example 22
Hybridization Blot Analysis
[0160] Total RNA was extracted from various organs of Solanum
tuberosum ssp. andigena plants grown under a long-day photoperiod
by using TRI REAGENT.RTM. according to the manufacturer's manual
(Molecular Research Center, Inc., Cincinnati, Ohio). Swollen
stolons (newly formed tubers) and tubers were harvested from
short-day plants. For FIG. 11B, RNA was extracted from leaves and
stolons that were harvested from the photoperiod-responsive species
Solanum tuberosum ssp. andigena grown under a short-day
photoperiod. Total RNA was size-fractionated via electrophoresis
through a 1.4% agarose gel that contained 5.0 mM methyl-mercury
hydroxide and transferred onto a MagnaGraph nylon membrane (Micron
Separations Inc., Westboro, Mass.). Hybridization and washing
conditions were the same as described by Kolomiets et al.,
"Lipoxygenase is Involved in the Control of Potato Tuber
Development," Plant Cell 13:613-626 (2001), which his hereby
incorporated by reference in its entirety. For autoradiography,
membranes were exposed to X-ray film with intensifying screens for
three to six days at -80.degree. C. A 1.2 kb wheat 18S ribosomal
RNA probe was used to confirm uniform loading of RNA for the blots
in FIG. 11A. Blots presented are representative examples of at
least two independent experiments.
Example 23
Plant Transformation
[0161] Transformation and regeneration of plants was undertaken on
leaf sections from Solanum tuberosum ssp. andigena line 7540 as
described by Liu et al., "Transformation of Solanum brevidens Using
Agrobacterium tumefaciens," Plant Cell Reports 15:196-199 (1995),
which is hereby incorporated by reference in its entirety. These
autotetraploid andigena plants, strictly photoperiodic for
tuberization, were obtained from the Institut fur Pflanzenbau und
Pflanzenzuchtung, Braunchsweig, Germany. The sense constructs were
made from a 2.0 kb fragment from the StBEL-05 cDNA and cloned into
the binary vector pCB201 (Xiang et al., "A Mini Binary Vector
Series for Plant Transformation," Plant Mol Biol 40:711-718 (1999),
which is hereby incorporated by reference in its entirety) driven
by the constitutive CaMV-35S promoter. Constructs were checked by
using PCR with clone-specific primers. Positive recombinants were
transferred to the Agrobacterium tumefaciens strain GV2260 by using
the procedure of direct transformation (An et al., Binary vectors.
in Plant Mol. Biol. Manual, pp. A3:1-19, Kluwer Academic, Belgium
(1988), which is hereby incorporated by reference in its entirety).
Control plants in the tuberization study were andigena plants
regenerated in vitro. Functional transformants were identified by
PCR analysis of genomic DNA and by detection of the accumulation of
sense transcripts of StBEL-05 in shoot tip samples. From among
these positives, the seven independent transformants (lines 7, 11,
12, 14, 16, 19, and 20 for StBEL-05) used in this study were
selected on the basis of abundant accumulation of sense mRNA in
shoot tips. Quantitative analysis of cytokinins was performed by
using liquid chromatography as described above. Three replicate 200
mg (fresh wt) samples of shoot tips down to the fourth visible
expanded leaf were collected, frozen in liquid nitrogen,
lyophilized, and analyzed.
Example 24
Evaluation of Tuber Formation
[0162] For in vitro tuberization, cultured transgenic plants were
grown on a Murashige and Skoog medium with 6.0% sucrose under a
long-day photoperiod (16 hours of light, 8 hours of dark) in a
growth chamber for two weeks and then transferred to a short-day
photoperiod (8 hours of light, 16 hours of dark) in the same growth
chamber. For tuber induction, plants were evaluated daily for tuber
formation. Soil-grown plants were grown in 10-cm pots under long
days (16 hours of light, 8 hours of dark) in the greenhouse
supplemented with high pressure sodium HID lamps until they reached
the 16-leaf stage and then transferred to short days in the growth
chamber. After 14 days under short days, plants were evaluated for
tuber formation.
Example 25
Results: Isolation of Potato KNOX Interactive Proteins
[0163] Making use of the two-hybrid selection system in yeast,
approximately 10.sup.6 transformants from a stolon cDNA library of
potato were screened using POTH1 in the GAL4-binding domain vector,
pBridge (Clontech), as bait. Thirty-eight positive clones that grew
on selective media were identified. Of the 38 that were sequenced,
23 clones could be grouped into seven unique genes encoding
different members of the TALE superclass of transcription factors
(Chan et al., "Homeoboxes in Plant Development," Biochim Biophys
Acta 1442:1-19 (1998), which is hereby incorporated by reference in
its entirety). All seven, designated StBEL-05, -11, -13, -14, -22,
-29, and -30 (GenBank accession numbers AF406697, AF406698,
AF406699, AF406700, AF406701, AF406702, AF406703, respectively)
showed selective interaction when tested in the yeast system both
for nutritional markers and for lacZ activation (FIGS. 9A and 9B).
Interaction occurred also when the prey cDNAs were cloned into
pBridge and transformed with POTH1 in a GAL4-activation domain
vector. As a test for autoactivation, the pAD transformants (5, 11,
13, 14, 22, 29, 30) did not grow on -histidine, -adenine, and
-leucine medium and the pBD transformant did not grow on
-histidine, -tryptophan, and -adenine medium. In vitro binding
experiments verified the results of the two-hybrid selection. POTH1
pulled down three representative StBEL proteins with divergent
sequence similarity in the BELL domain (5, 13, and 30) and
synthesized by in vitro transcription/translation in
immunoprecipitation assays (FIG. 9C).
Example 26
Results: The Proteins that Interact with the Potato KNOX Protein
are Members of the BEL Family of Transcription Factors
[0164] A phylogenetic analysis of the sequences of the seven
interacting proteins identified them as members of the BEL1-like
family of transcription factors (FIG. 12). These seven can be
organized into four subgroups based on amino acid sequence
similarity. Three clones (StBEL-05, -11, and -29) had 60-69%
similarity to each other overall and two other clones had a 78%
match (StBEL-13 and -22). These two groups range in similarity to
the others from 45-53% and a third (StBEL-30) has about 51%
similarity to the others. The sequence similarity of StBEL-14 to
the other six ranged from 45 to 56%. The amino acid sequence of
StBEL-05 has overall 56% similarity to BLH1 of Arabidopsis that
interacts with KNAT1 (GenBank accession number AAK43836), StBEL-13
matches an apple BEL (Dong et al., "MDH1: an Apple Homeobox Gene
Belonging to the BEL1 Family," Plant Mol Biol 42:623-633 (2000),
which is hereby incorporated by reference in its entirety, GenBank
accession number AAF43095) at 74% similarity, and StBEL-30 matches
another Arabidopsis BEL (GenBank accession number T05281) at 59%
similarity. The close match of all seven to the conserved
homeodomain and the presence of the proline-tyrosine-proline
(P-Y-P) loop between helices I and II (FIG. 13A) distinguish these
novel proteins as BEL types in the TALE superclass (Burglin,
"Analysis of TALE Superclass Homeobox Genes (MEIS, PBC, KNOX,
Iroquois, TGIF) Reveals a Novel Domain Conserved Between Plants and
Animals," Nucleic Acids Res 25:4173-4180 (1997), which is hereby
incorporated by reference in its entirety). The homeodomain region
is nearly identical among these seven (FIG. 13A, encompassing
helices I, II, and III). Other regions of conserved sequence
identity are shown schematically in FIG. 13A. These include the
amino-terminal SKY box consisting of 20 aa (from ser-207 to lys-226
in StBEL-05), the 120-aa domain starting at leu-272 of the StBEL-05
sequence, and the carboxy-terminal VSLTLGL-box (SEQ ID NO:15)
beginning at val-620. Three .alpha.-helices were predicted from the
conserved 120-aa region of the BEL protein StBEL-05 (underlined
sequence of FIG. 13B). Among the seven BELs, the percent similarity
of the amino acid sequence in this conserved 120-aa domain ranged
from 58 to 90%. Bellaoui et al., "The Arabidopsis BELL1 and KNOX
TALE Homeodomain Proteins Interact Through a Domain Conserved
Between Plants and Animals," Plant Cell 13:2455-2470 (2001), which
is hereby incorporated by referenced in its entirety, referred to
this region as the BELL domain.
[0165] The deduced lengths of the seven original cDNAs are 688 aa
for StBEL-05, 535 aa for StBEL-11, 586 aa for StBEL-13, 589 aa for
StBEL-14, 620 aa for StBEL-22, 567 aa for StBEL-29, and 645 aa for
StBEL-30. Five-RACE was used to verify the full-length of StBEL-05,
-13, -14 and -30. For blot hybridizations, a representative clone
from each of the four subgroups (StBEL-05, -13, -14, and -30) was
used. Southern blot analysis revealed that these genes are unique
and belong to small gene subfamilies, based on the complexity of
bands detected by gene-specific probes from each of the cDNAs (FIG.
13C).
Example 27
Results: Patterns of mRNA Accumulation for the Potato BELs
[0166] The BEL1-like gene represented by StBEL-05 exhibited mRNA
accumulation in all organs examined, with the greatest levels in
leaves and stems (FIG. 11A). Transcript accumulation of StBEL-11
and StBEL-29 was similar to the pattern of StBEL-05. Transcripts
for StBEL-13 accumulated to the highest levels in the SAM and in
fully formed flowers but were barely detectable in other organs
(FIG. 11A). The autoradiographs for StBEL-13 were exposed two-times
longer than the other StBELs. For StBEL-14, transcripts were
detected in flowers, leaves, roots, and stolons. The greatest
accumulation of StBEL-30 was in flowers with detectable levels in
all organs examined. To examine more closely the dynamics of StBEL
expression during tuber induction, a temporal study was undertaken
for the accumulation of StBEL-05 transcripts in leaves and stolons
of the photoperiod-sensitive potato species S. tuberosum ssp.
andigena grown under short-day conditions. Steady-state levels of
StBEL-05 mRNA increased in both leaves and stolons after exposing
the plants to short-day (SD) conditions (FIG. 11B). Visible tuber
formation for the plants grown under SD conditions was observed
between 10 to 14 days. In this study, the accumulation of mRNA for
the BEL cDNA, StBEL-05, was linked to the induction of tuber
formation in the leaves and stolons of a potato species responsive
to a SD photoperiod. In addition, a temporal study was undertaken
for the accumulation of BEL transcripts in stolons of the
photoperiod-sensitive potato species S. demissum grown under
short-day conditions. The induction of StBEL-05, StBEL-14, and
StBEL-30 expression was first detected in stolons one day after
exposing the plants to short-day (SD) conditions (FIG. 11C). This
increase in RNA levels remained steady through 7 days. Transcripts
for StBEL-13 were not detected in stolons in any stage of
development (FIG. 11C). Visible tuber formation for the plants
grown under SD conditions was observed between 10 to 14 days. In
this study, the accumulation of mRNA for the BEL cDNAs, StBEL-05,
StBEL-14, and StBEL-30 was linked to the induction of tuber
formation in the stolons of a potato species responsive to a SD
photoperiod.
Example 28
Results: Determining the Protein Binding Regions in POTH1 and the
BEL-Like Proteins
[0167] Interaction with StBEL-05 was observed with all deletions
outside the KNOX domain, with pBHD2 (missing the amino-terminus and
the first 48 aa of the KNOX domain, FIG. 10A), with pBHD6 (missing
the carboxy terminus and 52 aa of the carboxy-end of the KNOX
domain), and with pBHD-9 (first amino-terminal 114 aa but no KNOX
domain sequence). No interaction was observed with pBHD3 (missing
all of the KNOX domain and the first 114 aa). Control experiments
identified the first 114 aa of the N-terminus (pBHD9) as a
transcriptional activator. This construct transformed alone into
AH109 exhibited nutrient selection on -histidine, -tryptophan, and
-adenine medium. Co-transformation of pBHD9 with an empty pGAD
cassette produced transformants capable of growth on -histidine,
-tryptophan, -adenine, and -leucine medium and induction of lacZ.
None of the other constructs containing this domain were capable of
growing on selection media without StBEL-05. Using the in vitro
binding protocol, both the pBHD6 construct, containing the
amino-terminal half of the KNOX domain, and the pBHD9 construct
were unable to pull-down StBEL-05. When the pBDH9 construct was
cloned into the pGAD vector, no interaction was observed with
StBEL-05 in pBridge.
[0168] Fusion constructs of StBEL-05 that dissected the 120-aa
domain (pAD5-2, -3, -4, -9, and -11) were tested because this is
one of the regions that is conserved in BEL TFs from other plant
species (Bellaoui et al., "The Arabidopsis BELL1 and KNOX TALE
Homeodomain Proteins Interact Through a Domain Conserved Between
Plants and Animals," Plant Cell 13:2455-2470 (2001), which is
hereby incorporated by reference in its entirety; FIG. 13B).
Interaction with POTH1 was observed with all constructs that had
deletions exclusively outside of the conserved 120-aa box (FIG.
10B). The only exception to this was with pAD5-9 that demonstrated
an interaction and included a 43-aa deletion from the carboxy end
of the 120-aa domain. Even with as little as a 27-aa deletion from
the amino end of the 120-aa domain, interaction did not occur (FIG.
13B, FIG. 10B, pAD5-2). Similar to the results of Bellaoui et al.,
"The Arabidopsis BELL1 and KNOX TALE Homeodomain Proteins Interact
Through a Domain Conserved Between Plants and Animals," Plant Cell
13:2455-2470 (2001), which is hereby incorporated by reference in
its entirety, deletion of the SKY box (construct pAD5-1) resulted
in a 55% decrease in the induction of the lacZ marker as measured
by .beta.-galactosidase activity relative to the full-length
construct, StBEL-05 (FIG. 10B).
Example 29
Results: Enhanced Tuber Formation in Transgenic Plants that
Overexpress the BEL cDNA, StBEL-05
[0169] To examine the function of the potato BELs, transformed
potato plants (Solanum tuberosum ssp. andigena) that over expressed
StBEL-05 from a constitutive promoter were analyzed. This BEL gene
was selected because of its moderate level of activity in stolons
and tubers and its increase in RNA levels in response to inductive
conditions for tuber formation (FIG. 11). For these experiments, a
2000-bp fragment of the coding sequence of StBEL-05 in a sense
orientation driven by the CaMV-35S promoter in the binary vector
pCB201 (Xiang et al., "A Mini Binary Vector Series for Plant
Transformation," Plant Mol Biol 40:711-718 (1999), which is hereby
incorporated by reference in its entirety) was used. Transformants
were identified by PCR analysis of genomic DNA and by detection of
the accumulation of sense transcripts of StBEL-05 in RNA samples
from vegetative meristems. From among approximately twenty-five
positives, four independent lines with the highest levels of
StBEL-05 mRNA accumulation (FIG. 14A) were selected for evaluation
of tuber formation in vitro under both inductive (SD) and
noninductive (LD) conditions. The highest expressers of StBEL-05
sense transcripts (lines 11, 12, 14, and 19) exhibited tuber
formation under LD conditions (FIG. 14B). Control plants (WT and
line 6) produced tubers only under SD conditions. The highest
overexpressers of StBEL-05 also produced more tubers than control
plants over the course of this experiment and were more responsive
to inductive conditions. After seven days under SD conditions, the
control plants had produced no tubers, whereas the overexpression
mutants (lines 11, 12, 14, and 19) had produced 10, 8, 15, and 4
tubers, respectively (FIG. 14B). After 14 days under SD, controls
had increased to 6 and 4 tubers, whereas the overexpression lines
had increased to 12, 14, 24, and 10 tubers, respectively. Tuber
yields (fr wt) also increased in overexpression lines 12, 14, and
19 (FIG. 14C). The greatest tuber production was exhibited by lines
12 and 14 with a five- and sixteenfold increase, respectively,
relative to wild-type plants (FIG. 14B, bottom panel). Tubers from
the overexpression lines grew larger than controls. Select tubers
from line 14 reached fresh weights of almost 700 mg, whereas the
largest control tuber reached only 140 mg.
[0170] With whole plants grown in soil under SD conditions for 14
days, StBEL-05 overexpression lines produced an average of three-
to fivefold more tubers per plant and more than a threefold greater
tuber yield per plant than controls (Table 2).
TABLE-US-00018 TABLE 2 Rate of tuberization for overexpression
lines of StBEL-05 under soil-grown, short-day conditions. Plants
were grown in 10- cm pots under long days (16 hours of light, 8
hours of dark) until they reached the 16-leaf stage and then
transferred to short days (8 hours of light, 16 hours of dark).
After 14 days under short days, four plants per independent line
were evaluated for tuber formation. Standard errors of the mean are
shown. Number tubers Tuber yield Plant line plant.sup.-1
plant.sup.-1 (g) Wild-type 2.2 .+-. 1.4 1.4 .+-. 0.9 StBEL5-12 8.0
.+-. 0.8 5.4 .+-. 1.3 StBEL5-14 8.3 .+-. 0.9 4.6 .+-. 1.3 StBEL5-19
11.5 .+-. 2.1 4.7 .+-. 1.4
Increased yields (as high as 50%) were maintained for these lines
even after six weeks of growth in soil. Seven overexpressing sense
lines (lines 7, 11, 12, 14, 16, 19, and 20) also exhibited tuber
activity (swollen stolons or tuber formation) on soil-grown plants
under LD greenhouse conditions. Five of these plants produced
tubers, whereas control plants exhibited no tuber activity. In
addition, the rate of tuberization for plants grown in vitro under
short-day conditions for 21 days is shown in Table 3, below.
TABLE-US-00019 TABLE 3 Rate of tuberization for overexpression
lines of StBEL-05. Plants were grown in vitro under short days in
media with 6% sucrose for 21 days and scored for tuber formation.
Twenty-five plants per independent line were evaluated, thirty-five
for controls. Number tubers Tuber yield Plant line plant.sup.-1
plant.sup.-1 (mg) Control 0.4 18 StBEL-05-12 0.9 95 StBEL-05-14 1.3
292 StBEL-05-19 0.9 50
Similar to POTH1 overexpressers (see above), these results show
that the accumulation of StBEL-05 mRNA is correlated with an
increased rate of tuber formation. Other than this enhanced
capacity for tuberization, the StBEL-05 overexpression lines in
Table 2 did not exhibit the phenotype characteristic of KNOX gene
overexpressers, including extreme dwarfism and abnormal leaf
morphology (FIG. 15). The abnormal phenotype of KNOX overexpressers
is mediated by changes in hormone levels, specifically, a reduction
in gibberellins and an increase in cytokinins (see above; Sato et
al., "Abnormal Cell Divisions in Leaf Primordia Caused by the
Expression of the Rice Homeobox Gene OSH1 Lead to Altered
Morphology of Leaves in Transgenic Tobacco," Mol Gen Genet.
251:13-22 (1996); Tamaoki et al., "Ectopic Expression of a Tobacco
Homeobox Gene, NTH15, Dramatically Alters Leaf Morphology and
Hormone Levels in Transgenic Tobacco," Plant Cell Physiol
38:917-927 (1997); Frugis et al., "Overexpression of KNAT1 in
Lettuce Shifts Leaf Determinate Growth to a Shoot-like
Indeterminate Growth Associated With an Accumulation of
Isopentenyl-type Cytokinins," Plant Physiol 126:1370-1380 (2001),
which are hereby incorporated by reference in their entirety). With
the exception of two StBEL-05 sense mutants (lines 11 and 20), the
leaf and stem growth of the StBEL-05 overexpression lines was
similar to wild-type plants (FIG. 15). All five StBEL-05 lines
exhibited an enhanced rate of growth comparable to control plants
(Table 4).
TABLE-US-00020 TABLE 4 Plant height (cm) and fresh weight (g) of
overexpression lines of StBEL-05 under soil-grown, long-day
conditions. Plants were grown in 10-cm pots under long days (16
hours of light, 8 hours of dark) and plant height was measured
after 10 and 45 days. Four plants per independent line were
evaluated for growth. Fresh weight of leaves and stems was measured
after 45 days. Standard errors of the mean are shown. Fresh weight
(g) Plant height (cm) of Plant Line at 10 d at 45 d stem and leaves
Wild type 5.3 .+-. 0.3 35.2 .+-. 2.2 18.0 .+-. 2.6 StBEL5-11 7.3
.+-. 0.4 31.9 .+-. 3.0 19.6 .+-. 1.3 StBEL5-20 6.3 .+-. 0.6 32.2
.+-. 2.0 10.8 .+-. 0.5 StBEL5-12 7.1 .+-. 0.7 44.9 .+-. 0.9 23.3
.+-. 1.2 StBEL5-14 6.2 .+-. 0.2 38.2 .+-. 1.2 29.2 .+-. 1.0
StBEL5-19 7.1 .+-. 0.5 48.7 .+-. 1.9 25.5 .+-. 3.5
The average height of line 19 plants was 13.5 cm greater than
control plants after 45 days. Fresh weights of leaves and stems of
lines 12, 14, and 19 were 29 to 62% greater than control plants.
Lines 11 and 20 exhibited a more rapid rate of growth early (10
days) and then growth rate dropped off by 45 days (Table 4).
Accumulation of StBEL-05 transgenic mRNA in line 20 was equivalent
to line 11. Three-month old plants from lines 11 and 20 exhibited a
slight reduction in leaf size and stem height as a result of
decreased apical dominance. To examine the mechanism for this
reduced leaf morphology, cytokinin analysis was performed on shoot
apices down to the fourth visible true leaf. Similar to POTH1
overexpressers, shoot tips of both StBEL-05 lines 11 and 20
exhibited a two- to fivefold increase in the bioactive forms of
cytokinin (Table 5).
TABLE-US-00021 TABLE 5 Cytokinin content (picomoles g fr wt.sup.-1)
in shoot tips of POTH1 and StBEL-05 overexpression lines grown
under long days(16 hours of light, 8 hours of dark) in the
greenhouse. Wild-type lines are non- transformed Solanum tuberosum
spp. andigena. Zeatin types include zeatin, zeatin riboside,
dihydrozeatin, and dihyrozeatin riboside. Isopentenyl types include
isopentenyl and isopentenyladenine. Standard error was calculated
on three replicates. Sample Zeatin types Isopentenyl types
Wild-type 10.5 .+-. 1.0 12.0 .+-. 1.5 POTH1-15 42.5 .+-. 15 35.5
.+-. 7.0 POTH1-29 34.0 .+-. 12 30.0 .+-. 6.0 StBEL5-11 55.5 .+-. 30
31.5 .+-. 11 StBEL5-20 30.5 .+-. 6.0 29.5 .+-. 6.5
The overall magnitude increases in the cytokinin types among the
four StBEL and POTH1 mutant lines were remarkably consistent.
[0171] POTH1 sense lines had increased levels of GA.sub.53 and
GA.sub.19 and decreased levels of GA.sub.20 and GA.sub.1 in shoot
tips, indicating a down-regulation of the biosynthetic enzyme GA
20-oxidase1 (see above). Using a probe for the potato GA
20-oxidase1 gene (Carrera et al., "Changes in GA 20-oxidase Genes
Expression Strongly Affect Stem Length, Tuber Induction and Tuber
Yield of Potato Plants," Plant J. 22:1-10 (2000), which is hereby
incorporated by reference in its entirety), a reduction in GA
20-oxidase1 mRNA in shoots of the most severe mutant phenotypes for
POTH1 sense lines was observed (see above, FIG. 15). To determine
the effect of overexpression of the POTH1 partner, StBEL-05, RNA
levels for GA 20-oxidase1 were examined in the stolons of StBEL-05
sense lines grown under long-day photoperiod conditions. All three
of the StBEL-05 lines examined (lines 11, 12, and 20) exhibited a
reduction in GA 20-oxidase1 mRNA in stolon tips comparable to
controls (FIG. 16). No such reduction in the levels of GA
20-oxidase1 mRNA was observed in shoot tips of StBEL-05 lines grown
under long days.
[0172] To determine the effect of upregulating StBEL-05 mRNA levels
on POTH1 RNA accumulation, northerns were performed on total RNA
extracted from StBEL-05 sense lines 12, 14, 19, and 20 using POTH1
as a probe. There were no changes in the levels of POTH1 mRNA in
both shoot tips and stolon tips of these StBEL-05 lines relative to
wild-type plants. These results indicate that the enhancement of
tuber formation in StBEL-05 overexpression lines is not mediated by
an indirect increase in POTH1 expression.
Example 30
Discussion: Seven BEL Proteins Interact with a KNOX Protein of
Potato
[0173] Using a yeast two-hybrid library screen, seven unique
proteins from potato stolons that interact with the knotted-like
protein, POTH1, were identified. Sequence analysis revealed that
these interacting proteins are from the BEL1-like family in the
TALE superclass of homeodomain proteins. These proteins have
conserved regions in common with other TALE proteins, including the
homeodomain (comprised of three .alpha.-helices) and the
proline-tyrosine-proline "TALE" (Burglin, "Analysis of TALE
Superclass Homeobox Genes (MEIS, PBC, KNOX, Iroquois, TGIF) Reveals
a Novel Domain Conserved Between Plants and Animals," Nucleic Acids
Res 25:4173-4180 (1997), which is hereby incorporated by reference
in its entirety). These sequences have been implicated in
DNA-binding and protein/protein interactions, respectively (Mann et
al., "Extra Specificity From extradenticle: the Partnership Between
HOX and PBX/EXD Homeodomain Proteins," Trends in Genet 12:258-262
(1996); Passner et al., "Structure of DNA-Bound
Ultrabithorax-Extradenticle Homeodomain Complex," Nature
397:714-719 (1999), which are hereby incorporated by reference in
their entirety). A second conserved region of 120 aa just upstream
from the homeodomain (designated the BELL domain by Bellaoui et
al., "The Arabidopsis BELL1 and KNOX TALE Homeodomain Proteins
Interact Through a Domain Conserved Between Plants and Animals,"
Plant Cell 13:2455-2470 (2001), which is hereby incorporated by
reference in its entirety) was identified among BEL proteins by
using a BLAST analysis (FIG. 13B, bold letters). Sequence analysis
of the predicted secondary structure of this domain reveals the
presence of three putative .alpha.-helices within the 120 residues
(FIG. 13B, underlined sequence). Not all BEL proteins conserve the
third helix, however, including the Arabidopsis BEL, ATH1
(Quaedvlieg et al., "The Homeobox Gene ATH1 of Arabidopsis is
Depressed in the Photomorphogenic Mutants cop1 and det1," Plant
Cell 7:117-129 (1995), which is hereby incorporated by reference in
its entirety) and the barley BEL, JUBEL2 (Muller et al., "In vitro
Interactions Between Barley TALE Homeodomain Proteins Suggest a
Role for Protein-protein Associations in the Regulation of Knox
Gene Function," Plant J 27:13-23 (2001), which is hereby
incorporated by reference in its entirety). Protein interaction
using the two-hybrid system demonstrated that the first 80 aa of
this domain (up to QVKAT of the STBEL-05 sequence and comprising
the first two predicted helices of this region) are necessary to
mediate interaction with POTH1 (interaction of construct pAD5-9
with POTH1). Deletion of as little as the first 20 aa of this
domain (comprising a major portion of the first predicted helix)
interfered with the interaction with POTH1 (FIGS. 13B and 10B,
construct pAD5-2). The results also showed that deletion of 43 aa
from the carboxy-end of the 120-aa domain (see FIG. 10B, construct
pAD5-9; comprising the third helical structure) did not affect
protein interaction. Deletion of the two carboxyl-terminal helices
in this region (construct pAD5-11) resulted in a loss of
interaction. It appears that all three helical structures
contribute to specific binding affinity for POTH1 but that only the
amino-terminal two-thirds of the 120-aa domain are necessary for
binding to occur. Muller et al., "In vitro Interactions Between
Barley TALE Homeodomain Proteins Suggest a Role for Protein-protein
Associations in the Regulation of Knox Gene Function," Plant J
27:13-23 (2001), which is hereby incorporated by reference in its
entirety, identified a coiled-coil region in a BEL protein of
barley that was involved in the interaction with KNOX proteins.
This coiled-coil domain overlaps with 48 of the 80 aa (and
comprising the first helix) identified as essential for interaction
to occur.
[0174] Sequence differences in this putative protein-binding region
appear to contribute to the regulation of POTH1 activity by
affecting binding affinity to a shared partner. In the interaction
between PIF3, a basic helix-loop-helix factor, and phytochrome A
and B, phytochrome B has tenfold greater binding affinity for the
PIF3 partner than phytochrome A (Zhu et al., "Phytochrome B Binds
With Greater Affinity Than Phytochrome A to the Basic
Helix-loop-helix Factor PIF3 in a Reaction Requiring the PAS Domain
of PIF3," Proc Natl Acad Sci USA 97:13419-13424 (2000), which is
hereby incorporated by reference in its entirety). A comparison of
this 120-aa domain in the potato BELs revealed that StBEL-05
(expressed ubiquitously) has a 58% similarity match to StBEL-13
(expressed predominately in the SAM and flower only) and that
StBEL-13 has a 63% match to StBEL-30 in this conserved region. Such
differences in sequence may mediate binding affinities to shared
partners and, coupled with expression patterns, could reflect
organ-specific differences in function.
[0175] Conservation in sequence among these seven proteins was also
identified in two short amino acid sequence motifs, one near the
carboxyl-end of the protein (VSLTLGL) (SEQ ID NO:15) and another
just upstream of the BELL domain (SKY box, FIG. 13A). Both of these
regions are conserved among other plant BELs. Protein interaction
studies showed that the VSLTLGL (SEQ ID NO:15) box is not involved
in protein interaction with POTH1 and its function remains unknown.
Consistent with Bellaoui et al., "The Arabidopsis BELL1 and KNOX
TALE Homeodomain Proteins Interact Through a Domain Conserved
Between Plants and Animals," Plant Cell 13:2455-2470 (2001), which
is hereby incorporated by reference in its entirety, it was
observed that, whereas binding occurred without the 229 aa at the
amino terminus of StBEL-05 (construct pAD5-1), this 229 aa sequence
alone, containing the SKY box, was sufficient to mediate an
interaction with POTH1 (and other class I KNOX proteins). This
229-aa sequence, however, did not interact with a class II KNOX
protein. Muller et al., "In vitro Interactions Between Barley TALE
Homeodomain Proteins Suggest a Role for Protein-protein
Associations in the Regulation of Knox Gene Function," Plant J
27:13-23 (2001), which is hereby incorporated by reference in its
entirety, identified the SKY-box sequence in the barley BEL protein
to be a part of the KNOX-interacting domain. Our deletion analysis
indicates that the SKY box enhances the binding affinity of
StBEL-05 to KNOX partners.
Example 31
Discussion: The Protein Binding Region of POTH1
[0176] In addition to the homeodomain, KNOX TFs also contain a
conserved region of approximately 100 aa, upstream from the
homeodomain, known as the KNOX (MEINOX) domain, and postulated to
be involved in protein/protein interaction (Burglin, "The PBC
Domain Contains a MEINOX Domain: Coevolution of Hox and TALE
Homeobox Genes," Dev Genes Evol 208:113-116 (1998), which is hereby
incorporated by reference in its entirety). Using deletion mutants
in the two-hybrid yeast system, regions of amino acid sequence in
the KNOX domain of the class I KNOX protein, POTH1, that are
involved in an interaction with the BEL TFs have been identified.
Binding to the BEL partner is mediated by the KNOX domain but is
not dependent on the presence of the first half of the 120 aa KNOX
region (FIG. 10A). Similar results were obtained by Muller et al.,
"In vitro Interactions Between Barley TALE Homeodomain Proteins
Suggest a Role for Protein-protein Associations in the Regulation
of Knox Gene Function," Plant J 27:13-23 (2001), which is hereby
incorporated by reference in its entirety. Sakamoto et al., "The
Conserved KNOX Domain Mediates Specificity of Tobacco KNOTTED1-type
Homeodomain Proteins," Plant Cell 11: 1419-1431 (1999), which is
hereby incorporated by reference in its entirety, showed by using
chimeric proteins that the second half of the KNOX domain
(designated KNOX2) of a tobacco KNOX protein (NTH15, with 63%
similarity to POTH1 in the KNOX region) was most important for
determining the severity of the mutant phenotype. Their results
indicated that this conserved domain was even more important in
determining the phenotype than the DNA-binding domain. The deletion
analysis for POTH1 in the present study combined with the results
of Sakamoto et al., "The Conserved KNOX Domain Mediates Specificity
of Tobacco KNOTTED1-type Homeodomain Proteins," Plant Cell
11:1419-1431 (1999), which is hereby incorporated by reference in
its entirety, indicate that the interaction of the BEL proteins
with the KNOX domain is a prominent control mechanism for mediating
KNOX activity and maintaining stable development of the vegetative
meristem. KNOX2 contains 18 aa that are predicted to form an
.alpha.-helix and are conserved among all tobacco and potato KNOX
proteins. POTH1 has a close sequence match to members of the family
of KNOX proteins of tobacco (Nishimura et al., Over-expression of
Tobacco Knotted1-type Class1 Homeobox Genes Alters Various Leaf
Morphology," Plant Cell Physiol 41:583-590 (2000), which is hereby
incorporated by reference in its entirety), with an overall
sequence similarity ranging from 60 to 73% and an even greater
match in the conserved KNOX and homeodomain regions. Using the
two-hybrid system, all seven BELs of potato interacted with four
other tobacco class I-type KNOX proteins. Unlike KNOX proteins of
barley (Muller et al., "In vitro Interactions Between Barley TALE
Homeodomain Proteins Suggest a Role for Protein-protein
Associations in the Regulation of Knox Gene Function," Plant J
27:13-23 (2001), which is hereby incorporated by reference in its
entirety) and rice (Nagasaki et al., "Functional Analysis of the
Conserved Domains of a Rice KNOX Homeodomain Protein, OSH15," Plant
Cell 13:2085-2098 (2001), which is hereby incorporated by reference
in its entirety), however, POTH1 did not form homodimers in vitro.
Structural similarities to the MEIS domain of animal homeodomain
proteins (Burglin, "The PBC Domain Contains a MEINOX Domain:
Coevolution of Hox and TALE Homeobox Genes," Dev Genes Evol
208:113-116 (1998), which is hereby incorporated by reference in
its entirety) suggest that sequences in the KNOX domain of plants
are important for interactions with other proteins (Sakamoto et
al., "The Conserved KNOX Domain Mediates Specificity of Tobacco
KNOTTED1-type Homeodomain Proteins," Plant Cell 11:1419-1431
(1999), which is hereby incorporated by reference in its entirety).
These results confirm the function of this domain in an interaction
with a BEL1-like protein of potato.
Example 32
Discussion: The Function of the BEL/POTH1 Interaction
[0177] Through both molecular and genetic analyses, the BEL
proteins are known to function in the development of ovules. Reiser
et al., "The BELL1 Gene Encodes a Homeodomain Protein Involved in
Pattern Formation in the Arabidopsis Ovule Primordium," Cell
83:735-742 (1995), which is hereby incorporated by reference in its
entirety, showed that BELL1 of Arabidopsis was involved in the
pattern formation of ovule primordium. More specifically, the
expression of NOZZLE (a nuclear protein and putative TF) and BELL
are spatially linked and interact with other transcription factors
to determine distal-proximal pattern formation during ovule
development (Balasubramanian et al., "NOZZLE Links Proximal-Distal
and Adaxial-Abaxial Pattern Formation During Oovule Development in
Arabidopsis thaliana," Development 129:4291-4300 (2002), which is
hereby incorporated by reference in its entirety). Both NOZZLE and
BELL are chalazal identity genes that share overlapping functions
(Balasubramanian et al., "NOZZLE Regulates Proximal-Distal
Formation, Cell Pproliferation and Early Sporogenesis During Oovule
Development in Arabidopsis thaliana," Development 127:4227-4238
(2000), which is hereby incorporated by reference in its entirety).
In bell mutants, the chalazal domain undergoes altered development
and growth of the integuments is replaced by irregular outgrowths
(Mondrusan et al., "Homeotic Transformation of Ovules into
Carpel-like Structures in Arabidopsis," Plant Cell 6:333-349
(1994), which is hereby incorporated by reference in its entirety).
Overexpression of an apple BEL gene (MDH1) in Arabidopsis produced
plants that were dwarf, had reduced fertility, and exhibited
changes in both carpel and fruit shape (Dong et al., "MDH1: an
Apple Homeobox Gene Belonging to the BEL1 Family," Plant Mol Biol
42:623-633 (2000), which is hereby incorporated by reference in its
entirety). Overall, these results support that BEL proteins
function in controlling the formation of carpellate tissues and
plant fertility. Overexpression of a cDNA of a barley BEL in
tobacco produced plants that were dwarf and exhibited malformed
leaves and reduced apical dominance (Muller et al., "In vitro
Interactions Between Barley TALE Homeodomain Proteins Suggest a
Role for Protein-protein Associations in the Regulation of Knox
Gene Function," Plant J 27:13-23 (2001), which is hereby
incorporated by reference in its entirety). This BEL1-like cDNA
isolated from floral meristems produced a sense phenotype similar
to a class I knox overexpresser (Chan et al., "Homeoboxes in Plant
Development," Biochim Biophys Acta 1442:1-19 (1998), which is
hereby incorporated by reference in its entirety). All seven of the
BEL TFs in this study were isolated from stolons, a vegetative
organ. Based on these results and the patterns of mRNA accumulation
in potato, it appears that the BEL1 TFs of potato play a diverse
role in plant growth by regulating the development of both
reproductive and vegetative meristems.
[0178] Because the BEL TFs of potato and POTH1 interact, the
function of one provides a clue to the function of the other. The
KNOX protein of tobacco, NTH15, affects plant growth by regulating
GA levels through a direct interaction with a specific motif in
regulatory sequences of the GA 20-oxidase1 gene, a key GA
biosynthetic enzyme (Sakamoto et al., KNOX Homeodomain Protein
Directly Suppresses the Expression of a Gibberellin Biosynthesis
Gene in the Tobacco Shoot Apical Meristem," Genes Dev 15:581-590
(2001), which is hereby incorporated by reference in its entirety).
NTH15 directly suppresses the expression of GA 20-oxidase1 within
specific cells of the SAM to maintain the indeterminate state of
corpus cells. The knotted1-like protein of potato, POTH1, is also
involved in the regulation of GA synthesis and acts as a
developmental switch during tuber formation. Transgenic plants that
overexpressed POTH1 had reduced levels of GA 20-oxidase1 mRNA,
altered levels of GA intermediates, and exhibited a phenotype that
could be partially rescued by GA.sub.3 treatment (see above). These
plants were dwarf and developed malformed leaves. Under both
short-day (inductive conditions) and long-day (noninductive)
photoperiods, POTH1 overexpressing lines produced more tubers than
controls (see above). These sense lines exhibited a capacity for
enhanced tuber formation. Lines that overexpressed StBEL-05
produced tubers even under LD in vitro conditions, whereas control
plants produced tubers only after 10 days of SD conditions.
Overall, the BEL sense lines produced more tubers at a faster rate
than controls even on soil-grown plants. After 14 days of SD
conditions, soil-grown StBEL-05 overexpressers exhibited a
threefold increase in tuber production relative to wild-type plants
(Table 2). Thus, both POTH1 and StBel-05 overexpression lines
produced more tubers at a faster rate than controls (see FIGS.
17A-D). In FIG. 17D, stolon tips excised from in vitro plantlets
overexpressing POTH1 that were not tuberizing were cultured. After
a 20-day incubation in the dark on 8% (w/v) sucrose, stolons from
all five POTH1 sense lines produced more tubers than wild-type
stolons. Line 11 exhibited almost a 10-fold increase in tuber yield
(262 mg stolon tip.sup.-1) after 35 days in culture compared with
wild-type plants (27 mg stolon tip.sup.-1).
[0179] All of the above results show that that the expression of
both POTH1 and its protein partner, STBEL-05, is associated with an
enhanced rate of tuber formation. In addition to enhanced tuber
production, select StBEL-05 lines exhibited increases in cytokinin
levels and a reduction in GA 20-oxidase1 mRNA similar to POTH1
overexpression lines. This increase in cytokinin levels could
explain the enhanced rate of growth for the StBEL-05 lines,
although excessive accumulation may have led to the reduction in
growth exhibited by mature plants of lines 11 and 20. GA is
involved in regulating cell growth in a tuberizing stolon (Xu et
al., "The Role of Gibberellin, Abscisic Acid, and Sucrose in the
Regulation of Potato Tuber Formation in vitro," Plant Physiol
117:575-584 (1998), which is hereby incorporated by reference in
its entirety) and in contributing to the control of the
photoperiodic response of tuber formation (Jackson et al., "Control
of Tuberisation in Potato by Gibberellins and Phytochrome," B.
Physiol Plant 98:407-412 (1996), Martinez-Garcia et al., "The
Interaction of Gibberellins and Photoperiod in the Control of
Potato Tuberization," J Plant Growth Regul 20:377-386 (2001), which
are hereby incorporated by reference in their entirety). Low levels
of GA in the stolon tip are correlated with tuber induction (Xu et
al., "The Role of Gibberellin, Abscisic Acid, and Sucrose in the
Regulation of Potato Tuber Formation in vitro," Plant Physiol
117:575-584 (1998), which is hereby incorporated by reference in
its entirety). Tuberization is also affected by cytokinin
accumulation, with high levels inhibiting and moderate levels
promoting tuber formation (Galis et al., "The Effect of an Elevated
Cytokinin Level Using the ipt Gene and N.sup.6-Benzyladenine on
Single Node and Intact Potato Plant Tuberization in vitro," J Plant
Growth Regul 14:143-150 (1995); Romanov et al., "Effect of
Indole-3-Acetic Acid and Kinetin on Tuberisation Parameters of
Different Cultivars and Transgenic Lines of Potato in vitro," Plant
Growth Reg 32:245-251 (2000), which are hereby incorporated by
reference in their entirety). Local accumulation of cytokinins in
axillary buds of transgenic tobacco produced truncated, tuberizing
lateral branches (Guivarch et al., "Local Expression of the ipt
Gene in Transgenic Tobacco (Nicotiana tabacum L. cv. SR1) Axillary
Buds Establishes a Role for Cytokinins in Tuberization and Sink
Formation," J Exp Bot 53:621-629 (2002), which is hereby
incorporated by reference in its entirety). Through an interaction
with POTH1, the BEL protein encoded by StBEL-05 may also function
to regulate hormone levels in stolons or leaves to favor the
formation of tubers.
[0180] The results set forth above indicate that the physical
interaction between the KNOX and BEL proteins provides a molecular
basis for regulating processes of growth in the potato and that
overexpression of each partner alone affects vegetative development
and enhances tuber formation.
Example 33
Both POTH1 and StBEL-05 Interact to Repress Transcriptional
Activity of the GA20 Oxidase1 Gene of Potato--Preliminary
Results
[0181] If POTH1 and StBEL physically interact and their
overexpression produces transgenic plants that exhibit similar
developmental pathways, it is reasonable to assume that they target
the same gene. Using gel mobility shift assays (FIG. 18), it is
shown that in tandem POTH1 and StBEL-05 bind to the P1 region of
the GA20 oxidase1 promoter. In tandem, StBEL-05 and POTH1 had a
greater binding affinity for the ga20ox1 promoter than either
alone. The StBEL-05-POTH1 heterodimer bound specifically to a
composite sequence TTGACTTGAC (SEQ ID NO: 20) containing two
adjacent TGAC cores in the P1 region. Using a transcription assay
with GUS reporter driven by the ga20ox1 promoter in tobacco
protoplasts, StBEL-05 and POTH1 alone suppressed the activity of
the ga20ox1 promoter by more than 50%, together about 80%. The
binding affinity of POTH1 and StBEL-05 represses the
transcriptional activity of the promoter (FIG. 19).
[0182] Consistent with the in vitro results of StBEL/POTH1
repression of the GA20 oxidase1 promoter/GUS marker (FIG. 19), GA20
oxidase1 mRNA levels are also reduced in stolons of the StBEL-05
sense lines grown under long days (FIG. 20). This reduction in mRNA
will lead to a reduction in bioactive GA and result in facilitating
tuber formation. StBEL-05 mRNA levels were found to increase in
both stolons and leaves of WT plants in response to the inductive
conditions of short days. These results are consistent with the
proposed role of GA in mediating photoperiodic responses in potato
(Martinez-Garcia et al., "The Interaction of Gibberellins and
Photoperiod in the Control of Potato Tuberization," J. Plant Growth
Regul. 20:377-386 (2002), which is hereby incorporated by reference
in its entirety).
[0183] These preliminary data show that POTH1 and StBEL-05 proteins
interact in vitro and that overexpression of each separately,
produces plants that are enhanced in their capacity to form tubers.
Both proteins interact to repress the transcriptional activity of a
key GA biosynthetic gene. Because expression of the BEL TEs appears
to be differential, the BELs appear to act in tandem with POTH1 (or
other KNOX proteins) to regulate growth differently in the various
organs or cells of the potato. A more detailed description of the
above experiments is provided in Examples 34-43, below.
Example 34
BEL and KNOX Interaction Mediates Transcriptional Activity of the
Potato ga20ox1 Promoter--Plant Materials
[0184] Tobacco `Petit Havana` plants were maintained in Murashige
and Skoog basal medium (1962) supplemented with 2% sucrose and
incubated at 25.degree. C., under 16 hour photoperiods for three to
four weeks.
Example 35
BEL and KNOX Interaction Mediates Transcriptional Activity of the
Potato ga20ox1 Promoter--Protein Expression and Purification in E.
coli
[0185] Glutathione S-transferase (GST) fusion constructs were
generated by introducing full-length cDNAs of StBEL-05 and POTH1 in
frame with GST into the pGEX-5X-2 expression vector (Roche,
Indianapolis, Ind.) and transformed into BL21 (DE3) E. coli cells
(Stratagene, La Jolla, Calif.). Cells were grown at 30.degree. C.
until the OD.sub.600 reached 0.6, induced with 1.0-mM
isopropyl-.beta.-D-thiogalactopyranoside, and cultured for 5 hours.
The manufacturer's protocol (Roche) was followed for cell lysis and
affinity purification by using glutathione sepharose 4B beads. The
GST portion of the fusion protein was cleaved by Factor Xa protease
(Promega, Madison, Wis.). Purified StBEL-05 and POTH1 protein were
frozen in liquid N.sub.2 and stored at -80.degree. C.
Example 36
BEL and KNOX Interaction Mediates Transcriptional Activity of the
Potato ga20ox1 Promoter--Gel Retardation Assay
[0186] The first intron with partial flanking exon sequence (450
bp) of potato ga20ox1 and its promoter (981 bp, provided by Dr.
Salome Prat, CSIC Cantoblanco Campus, Univ. of Madrid, Spain) were
used for gel mobility shift assays. Polymerase chain reaction (PCR)
was used to amplify three regions of the promoter: -981 to 636
(P1), -660 to 307 (P2), and -331 to 0 (P3). About a 25-bp overlap
was maintained between P1 and P2 or P2 and P3 in the chance that
the protein-binding site would span the overlapped region. The
first intron of this gene was amplified from potato genomic DNA by
using PCR and the oligos 5'-GGATCCTTGAAGTGGCTCTTCTCT-3' (SEQ ID
NO:21) and 5'-AATCTAGAGACACTCTCTTTTTCGT-3' (SEQ ID NO:22) as
primers. These primers were designed based on the site of the first
intron of the tobacco GA20 oxidase gene Ntc12. The four fragments
were purified on a 1.4% agarose gel and labeled with
.alpha..sup.32P-dATP using Klenow fragment. DNA-binding reactions
were set up on ice in 20 .mu.L containing 10-mM Tris-HCl (pH 7.5),
5% glycerol, 0.5-mM EDTA, 0.5-mM DTT, 0.05% NP-40, 50-mM NaCl,
50-mgL.sup.-1 poly (dG-dC).cndot.poly (dG-dC) (Amersham Pharmacia
Biotech, Piscataway, N.J.), 100-ng protein, and 1-fmol labeled DNA.
After incubation on ice for 30 minutes, the reactions were resolved
on a 6% native polyacrylamide gel in 1.times.TGE
(Tris-Glycine-EDTA) buffer. The gel was dried and exposed to X-ray
film.
[0187] In the competition assays, unlabeled double-stranded DNA
fragments (10.times., 25.times., 50.times., 100.times.) were
incubated with the recombinant protein before the addition of the
radioactive probe. The dissociation rates were determined by adding
500-fold more cold DNA fragments to the DNA-binding reactions that
were being incubated on ice, and loaded onto the running gel every
10 minutes. Mutated oligos for binding sites were synthesized by
the DNA Sequencing and Synthesis Facility, Iowa State University
(Ames, Iowa).
Example 37
BEL and KNOX Interaction Mediates Transcriptional Activity of the
Potato ga20ox1 Promoter--Transcription Assay
Generation of Reporters and Effectors
[0188] The cauliflower mosaic virus (CaMV) 35S promoter in pBI221
(Clontech, Palo Alto, Calif.) was replaced by an enhancer fragment
(-832 to -50) of the 35S promoter plus 980 bp of the ga20ox1
promoter to generate the pGAOP::.beta.-glucuronidase (GUS) reporter
construct. With this construct, the reporter GUS transcription
level is augmented but its transcription may still be affected by
the ga20ox1 promoter. A CaMV 35S promoter-driven luciferase (LUC)
construct 35S-LUC (obtained from Dr. Takahashi, Dept. of Biological
Sciences, Graduate School of Science, Univ. of Tokyo, Japan) was
used as an internal control. Effector constructs were also
generated by using pBI221 vector as a backbone, with the GUS gene
replaced by the full-length cDNAs of either StBEL-05 or POTH1,
downstream of the CaMV 35S promoter. Truncated cDNAs that encode
the N-terminal protein-binding domains of StBEL-05 or POTH1 were
used to generate the dominant negative constructs,
StBEL5.DELTA.C295 and POTH1.DELTA.C122, respectively. The reporter
construct with the mutated promoter was generated by site-directed
PCR mutagenesis with oligos 5'-CTATTTGACTTC*ACACGGTTATTT-3' (SEQ ID
NO:23) and 5'-AAATAACCGTGTG*AAGTCAAATAG-3' (SEQ ID NO:24).
Transfection Assay
[0189] Fully expanded leaves from three- to four-week-old tobacco
plants were excised and placed in K3 basal media (Kao et al.,
"Nutritional Requirements for Growth of Vicia hajastana Cells and
Protoplasts at a Very Low Density in Liquid Media," Planta
126:105-110 (1975), which is hereby incorporated by reference in
its entirety) supplemented with 0.4 M sucrose, 0.25% (w-v)
cellulases (Karlan Research Products, Santa Rosa, Calif.), and
0.05% (w-v) macerases (Calbiochem, La Jolla, Calif.) and incubated
for overnight at 28.degree. C. After incubation, the liberated
protoplasts were filtered through sterile cheesecloth into a
Babcock bottle, and centrifuged for 10 minutes at 1000 rpm.
Protoplasts were collected from the bottleneck area and washed once
in K3 media with 0.4 M sucrose and resuspended in K3 media
containing 0.4 M glucose to a final concentration of
4.times.10.sup.6 protoplasts per milliliter.
[0190] For each transfection analysis, 700 .mu.L of tobacco
protoplasts (prepared as described above) were mixed with 30 .mu.L
2 M KCl and plasmid DNA in an electroporation cuvette with 0.4-cm
electrode gap. The plasmid DNA was a mixture of 2 .mu.g of the
pGAOP::GUS reporter construct, 0.1 .mu.g of the 35S-LUC construct
as internal control, and a different combination of 2 .mu.g of each
effector plasmid. After electroporation (voltage=170 V,
capacitance=125 .mu.F, Gene Pulser Transfection Apparatus; Bio-Rad,
Hercules, Calif.), 4.0 mL of Murashige and Skoog (1962) basal media
was added, and the protoplasts were incubated in the dark at room
temperature for 40 to 48 hours before conducting GUS and LUC
activity assays. Transfections were performed three times for each
effector combination.
[0191] Luciferase assays were performed by injecting 100-.mu.L
luciferase substrate (Promega, Madison, Wis.) into 20 .mu.L of
extract and measuring the emitted photons for 15 seconds in a TD-20
luminometer (Turner Designs, Sunnyvale, Calif.). Fluorometric GUS
assays were performed as described (Jefferson, "Assaying Chimeric
Genes in Plants: The GUS Gene Fusion System," Plant Mol. Biol. Rep.
5:387-405 (1987), which is hereby incorporated by reference in its
entirety). A fluorescence multiwell plate reader, Fluoroskan II
(MTX labs, Vienna, Va.), was used to measure GUS activity at 365 nm
(excitation) and 455 nm (emission). Each sample was measured three
times for both LUC and GUS activity. Relative GUS-LUC activity was
calculated by dividing the ratio of GUS activity to LUC activity
from different effectors with the ratio from reporter plasmid
alone. Relative activities calculated from three transfection
replications were presented as a mean .+-.SE.
Example 38
Results: StBEL-05 and POTH1 Bind to the Regulatory Regions of
ga20ox1
[0192] Recombinant StBEL-05 protein expressed from E. coli retarded
the mobility of all three promoter sequences and the first intron
(FIGS. 18A and B). POTH1 only formed a complex with P1. StBEL-05
and POTH1 together produced a supershifted band with P1, which had
stronger signal intensity and migrated much slower than either the
StBEL-05-P1 or POTH1-P1 complexes (FIG. 18A). Competition assays
were performed with labeled P1 and unlabeled P1 or unlabeled P3.
With increased unlabeled P1, the P1-StBEL-05 complex quickly
disappeared (FIG. 21A). With unlabeled P3, however, even at a
concentration 100-fold more than labeled P1, the shifted band was
still present (FIG. 21A). Unlabeled P1 also reduced the P1-POTH1
complex formation, but unlabeled P3 had no effect on the P1-POTH1
complex (FIG. 21B).
[0193] Consistent with the increased signal intensity of the
StBEL-05-POTH1-P1 complex, the dissociation rate of this complex
was much slower than either the StBEL-05-P1 or POTH1-P1 complexes
(FIG. 22). Although StBEL-05 could bind to P2, P3, and the intron
fragments, there was no supershifted band formed when both StBEL-05
and POTH1 were incubated with these three DNA fragments (FIG. 18A).
These results indicate that both StBEL-05 and POTH1 are required
for binding to the P1 DNA fragment. Based on these results, at
least two TALE homeodomain binding sites may be present in P1. To
support this premise, excessive amounts of a truncated protein
containing only the HD portion of StBEL-05 produced a supershifted
band similar to the POTH1-StBEL-05-P1 complex. Apparently, there
were two binding sites recognized by StBEL-05 in P1. No
supershifted band was detected, however, when P1 was incubated with
excessive amounts of full-length StBEL-05 or POTH1. This indicates
that the two binding sites in P1 are in close proximity to one
other and that two full-length StBEL-05 molecules cannot bind to
both sites at the same time because of size constraints.
Example 39
Results: The StBEL-05-POTH1 Heterodimer Binds Specifically to the
TGA(C/G)(T/A)TGAC Site
[0194] Based on the Arabidopsis KNOX-BEL heterodimer binding site
TGACAG(G/C)T (SEQ ID NO:25) (Smith et al., "Selective Interaction
of Plant Homeodomain Proteins Mediates High DNA-Binding Affinity,"
Proc. Natl. Acad. Sci. 99:9579-9584 (2002), which is hereby
incorporated by reference in its entirety) and the TGAC binding
core confirmed for MEINOX proteins (Smith et al., "Selective
Interaction of Plant Homeodomain Proteins Mediates High DNA-Binding
Affinity," Proc. Natl. Acad. Sci. 99:9579-9584 (2002); Tejada et
al., "Determinants of the DNA-Binding Specificity of the Avian
Homeodomain Protein, AKR," DNA and Cell Biol. 18:791-804 (1999),
which are hereby incorporated by reference in their entirety, one
putative site, TTGACTTGAC (SEQ ID NO:20), in the potato ga20ox1
promoter P1 region was identified. Oligonucleotides with serial
point mutations across this site were used as probes in
gel-retardation assays in the presence of StBEL-05, POTH1, or both.
Point mutations across this site did not affect the binding of
either StBEL-05 or POTH1 alone, but most mutations in TGACTTGAC
(SEQ ID NO:26) abolished the binding by StBEL-05-POTH1 heterodimer.
Based on these results, it was deduced that the consensus sequence
of the StBEL-05-POTH1 heterodimer is TGA(C/G)(T/A)TGAC (SEQ ID
NO:27).
Example 40
Results: Repression of ga20ox1 Promoter Requires the Interaction of
StBEL-05 and POTH1
[0195] POTH1 encodes for a 345-residue protein estimated to have a
mass of 37.95 kDa. The coding sequence of the protein includes the
97-aa KNOX domain and the 64-aa homeodomain consisting of three
helices (FIG. 23A). The KNOX domain of POTH1 contains two conserved
regions, designated Knox I and II. StBEL-05 is 688 aa in length
with an estimated mass of 75.68 kDa. The coding sequence of
StBEL-05 contains the conserved sky box, BELL domain, homeodomain,
and the proline-tyrosine-proline (P-Y-P) loop between helices I and
II (FIG. 23B). The BELL domain is 120 aa in length and the HD of
StBEL-05 is 61 aa.
[0196] When co-transfected with effector p35S::StBEL5, p35S::POTH1,
or both (FIG. 24A), relative GUS-LUC activity of the pGAOP::GUS
reporter construct decreased by more than half (FIG. 24B). Neither
StBEL-05 nor POTH1 showed any effect on the activity of the CaMV
35S promoter (FIG. 24C). To eliminate the possibility that
endogenous BEL1-like or KNOX proteins cooperatively interact with
POTH1 or StBEL-05, respectively, truncated forms of StBEL-05 and
POTH1, StBEL5.DELTA.C295 and POTH1.DELTA.C122 (FIG. 25A), were
generated to use as dominant negatives in the transcription assays.
StBEL5.DELTA.C295 and POTH1.DELTA.C122 contain the intact
protein-binding domain, but lack the carboxy-terminal region
including the homeodomain. StBEL5.DELTA.C295 and POTH1.DELTA.C122
can interact with endogenous KNOX or BEL1-like proteins,
respectively. Such heterodimers are not functional due to the lack
of the homeodomain from the truncated proteins. In transcription
assays with pGAOP::GUS as reporter, StBEL5.DELTA.C295 had little
effect on the activity of the ga20ox1 promoter (FIG. 25B). When
co-transfected with StBEL-05, StBEL5.DELTA.C295 abolished almost
all of the repression activity of StBEL-05 (FIG. 25B).
POTH1.DELTA.C122 had a similar effect on the repression activity of
POTH1 (FIG. 25C).
Example 41
Results: The Binding Site in the ga20ox1 Promoter Acts as a
Cis-Element for the Repression by StBEL-05-POTH1 Heterodimer
[0197] To investigate whether the StBEL-05-POTH1 binding site
identified through EMSA studies functions as a cis-element, a
reporter construct with a point mutation in the binding site was
used for the transcription assay (FIG. 26A). Constructs containing
this single mutation exhibited no detectable repression of promoter
activity when co-transfected with either StBEL-05, POTH1, or both
(FIGS. 26B-C).
Example 42
Discussion: Cooperative Interaction Between StBEL-05 and POTH1
Mediates Binding Affinity for the ga20ox1 Promoter
[0198] To regulate target gene expression, a transcription factor
binds to the regulatory sequence of its target gene or interacts
with another protein that does. Gel-retardation assays showed that
both StBEL-05 and POTH1 bound to the promoter region of potato
ga20ox1 gene, and StBEL-05 could also bind with the first intron
sequence (FIGS. 18A-B). Unlabeled P3 competed with the StBEL-05-P1
complex, but not as effectively as unlabeled P1 (FIG. 21A), whereas
P3 had no competition effect with the POTH1-P1 complex (FIG. 21B).
These results indicated that the interaction between these two TALE
HD proteins and P1 was specific and that StBEL-05 bound to P1 more
strongly than to P3. It is highly likely then that P1 contains the
cis element that functions with this protein complex in planta. The
tobacco KNOX protein, NTH15, binds to both the promoter and the
first intron of GA20 oxidase, but with higher affinity to the first
intron (Sakamoto et al., "KNOX Homeodomain Protein Directly
Suppresses the Expression of a Gibberellin Biosynthetic Gene in the
Tobacco Shoot Apical Meristem," Genes & Dev. 15:581-590 (2001),
which is hereby incorporated by reference in its entirety). NTH15
is not the tobacco homolog of POTH1 and this may explain the
disparity in binding affinities. No BEL partners were tested for
binding with the tobacco KNOX protein or the GA20 oxidase
promoter.
[0199] Several consensus binding sites for KNOX proteins have been
identified from either target gene promoters or in vitro binding
site selection by using KNOX HD proteins from barley (Krusell et
al., "DNA Binding Sites Recognized in Vitro by a Knotted Class 1
Homeodomain Protein Encoded by the Hooded Gene, K, in Barley
(Hordeum vulgare)," FEBS Lett. 408:25-29 (1997), which is hereby
incorporated by reference in its entirety, tobacco (Sakamoto et
al., "KNOX Homeodomain Protein Directly Suppresses the Expression
of a Gibberellin Biosynthetic Gene in the Tobacco Shoot Apical
Meristem," Genes & Dev. 15:581-590 (2001), which is hereby
incorporated by reference in its entirety), and rice (Nagasaki et
al., "Functional Analysis of the Conserved Domains of a Rice KNOX
Homeodomain Protein, OSH15," Plant Cell 13:2085-2098 (2001), which
is hereby incorporated by reference in its entirety). Because the
homeodomains, especially the third .alpha.-helix in the HD region,
of these KNOX proteins are almost identical, the consensus
sequences recognized by them share a core TGTCAC motif (Nagasaki et
al., "Functional Analysis of the Conserved Domains of a Rice KNOX
Homeodomain Protein, OSH15," Plant Cell 13:2085-2098 (2001), which
is hereby incorporated by reference in its entirety). Two
interacting TALE proteins of vertebrates, Meis1 and Pbx1, dimerize
on the composite DNA sequence, TGATTGACAG (SEQ ID NO:28),
containing 5'-Pbx and 3'-Meis half sites (Chang et al., "Meis
Proteins are Major in Vivo DNA Binding Partners for Wild-Type But
Not Chimeric Pbx Proteins," Mol. Cell. Biol. 7:5679-5687 (1997),
which is hereby incorporated by reference in its entirety). Using
random oligonucleotide selection, the consensus sequence,
TGACAG(G/C)T (SEQ ID NO:25), was identified for the Arabidopsis
BEL-KNOX heterodimeric complex (Smith et al., "Selective
Interaction of Plant Homeodomain Proteins Mediates High DNA-Binding
Affinity," Proc. Natl. Acad. Sci. 99:9579-9584 (2002), which is
hereby incorporated by reference in its entirety). Because the
StBEL-05-POTH1-P1 complex requires both proteins to bind the target
DNA, and increased amounts of the StBEL-05 homeodomain lead to a
supershifted band, this indicates that there are two closely
located TALE homeodomain binding sites in the P1 region similar to
the two half binding sites for Meis1 and Pbx1 (Chang et al., "Meis
Proteins are Major in Vivo DNA Binding Partners for Wild-Type But
Not Chimeric Pbx Proteins," Mol. Cell. Biol. 7:5679-5687 (1997),
which is hereby incorporated by reference in its entirety). Based
on these results and comparisons to the known binding motifs, a
potential StBEL5-POTH1 binding site, TTGACTTGAC (SEQ ID NO:25), has
been identified in the P1 fragment. Gel-retardation assays
confirmed that this oligo was sufficient for binding to StBEL-05,
POTH1, and StBEL5-POTH1. Mutational gel-retardation analysis of
this BEL-KNOX binding site showed that the StBEL-05-POTH1
heterodimer recognizes the 9-bp sequence, TGA(C/G)(T/A)TGAC (SEQ ID
NO:27), containing two TGAC cores. StBEL-05 and POTH1 could bind to
either one of the TGAC cores, because serial mutations had no
effect on the DNA-binding ability of StBEL-05 or POTH1.
[0200] It has been a paradox for HD proteins regarding their high
level of functional specificity in directing developmental programs
and their high degree of redundancy in binding site specificity.
Besides the low affinity and high redundancy in binding sites, the
5-base consensus sequences recognized by HD proteins randomly show
up on average once every 1.0 kb in eukaryotic genomes (Mann et al.,
"Extra Specificity From Extradenticle: The Partnership Between Hox
and Exd-Pbx Homeodomain Proteins. Trends Genet. 12:258-262 (1996),
which is hereby incorporated by reference in its entirety).
Therefore, it is likely that interaction with other DNA-binding
transcription factors is necessary for HDs to affect binding
affinity and specificity. Monomeric HD proteins have modest
specificity for DNA binding, but their specificity is greatly
increased through cooperative binding with other DNA binding
partners (Mann et al., "Extra Specificity From Extradenticle: The
Partnership Between Hox and Exd-Pbx Homeodoamin Proteins. Trends
Genet. 12:258-262 (1996), which is hereby incorporated by reference
in its entirety). The gel-retardation assays also showed that
StBEL-05 and POTH1 in tandem formed a complex with P1 with greater
signal intensity than either POTH1-P1 or StBEL5-P 1 complexes (FIG.
18A), and that the StBEL-05-POTH1-DNA complex had a much slower
dissociation rate (FIG. 22). Both of these results indicate that
the BEL-KNOX heterodimer has an increased binding affinity for the
target site.
Example 43
Discussion: STBEL-05-POTH1 Heterodimer Mediates the Repression of
the ga20ox1 Promoter
[0201] The previous examples showed that both StBEL-05 and POTH1
overexpression mutants exhibited decreased ga20ox1 mRNA levels in
stolons and leaves, respectively (see Examples 1-32).
Gel-retardation assay results showed that these two transcription
factors bound to the promoter and the first intron of ga20ox1.
These results indicate that StBEL-05 and POTH1 directly represses
ga20ox1 transcription by binding to the promoter region. Results
from the transcription assay showed that either StBEL-05 or POTH1
alone could repress reporter gene activity by more than 50%. The
fact that neither POTH1 nor StBEL-05 affected CaMV 35S promoter
activity (FIG. 24C) confirmed that such repression was not due to
inhibition of the general transcription machinery. Direct
repression of GA20 oxidase gene transcription by the KNOX protein
NTH15 has also been reported in tobacco (Sakamoto et al., "KNOX
Homeodomain Protein Directly Suppresses the Expression of a
Gibberellin Biosynthetic Gene in the Tobacco Shoot Apical
Meristem," Genes & Dev. 15:581-590 (2001), which is hereby
incorporated by reference in its entirety).
[0202] Although either StBEL-05 or POTH1 could repress ga20ox1
promoter in the transcription assay, the KNOX-BEL heterodimers were
possibly still formed with endogenous partners to function in
tobacco protoplasts. There are three lines of evidence to support
this possibility. First, of the seven BEL proteins identified in
potato, all seven interacted with four tobacco KNOX proteins (see
above). Second, the protein binding domains of the tobacco KNOX
NTHs were most important in determining the severity of transgenic
plant phenotypes (Sakamoto et al., "The Conserved KNOX Domain
Mediates Specificity of Tobacco KNOTTED-1 type Homeodomain
Proteins. Plant Cell 11:1419-1431 (1999), which is hereby
incorporated by reference in its entirety), implying that
interaction with protein partners, most probably the BEL1-like
proteins, is essential for KNOX function. Third, the identification
of BEL-KNOX binding sites (Smith et al., "Selective Interaction Of
Plant Homeodomain Proteins Mediates High DNA-Binding Affinity.
Proc. Natl. Acad. Sci. 99:9579-9584 (2002), which is hereby
incorporated by reference in its entirety) and the StBEL-05-POTH1
binding site in this study, further implies that the BEL-KNOX dimer
is involved in the regulation of target genes. In the transcription
assays, constructs of the dominant negatives, StBEL5.DELTA.C295 or
POTH1.DELTA.C122, abolished the repression activity of StBEL-05 or
POTH1, respectively (FIG. 25). Therefore, StBEL-05 or POTH1 protein
alone is not sufficient for the repression of ga20ox1 promoter. The
BEL-KNOX heterodimeric complex is required for repression of
transcription to occur.
[0203] The results above showed that the mutated P1 binding site of
the ga20ox1 promoter did not respond to StBEL-05-POTH1-mediated
repression, indicating that this binding site functions as a
cis-element for the StBEL-05-POTH1 heterodimer. Based on the
results from gel-retardation analysis of serial mutations in this
site, the mutated promoter was capable of binding with StBEL-05 or
POTH1 separately, but not the StBEL-05-POTH1 heterodimer. This is
further evidence that it is the BEL-KNOX heterodimer and not the
individual BEL or KNOX proteins that affect repression. The
interaction of StBEL-05/POTH1 to affect transcription is summarized
in the model of FIG. 27. The partner proteins interact through
conserved protein binding domains. For StBEL-05, this includes the
two amino-terminal helices of the BELL domain and the sky box (Chen
et al., "Interacting Transcription Factors From the TALE Superclass
Regulate Tuber Formation," Plant Physiol. (in press) (2003), which
is hereby incorporated by reference in its entirety). For POTH1,
this includes the KNOX domain with Knox II playing the most
significant role (Sakamoto et al., "The Conserved KNOX Domain
Mediates Specificity of Tobacco KNOTTED-1 type Homeodomain
Proteins. Plant Cell 11:1419-1431 (1999), which is hereby
incorporated by reference in its entirety). The sky box contributes
to the tandem formation and interacts weakly with Knox I.
Interaction between the respective protein binding domains and the
spatial arrangement of the first two helices of the homeodomain
bring the third helices of both TFs together in a major groove of
the DNA helix. Specificity is then provided within the spatial
constraints of the three components (StBEL-05, POTH1, and the
helical groove) through recognition of the binding motif. In this
case, the BEL/KNOX complex may repress transcription by interfering
with the binding of critical components of the transcriptional
machinery. Other BEL/KNOX complexes may affect gene expression
differentially by recognizing other cis-elements as a result of
slight modifications in protein structure.
[0204] The results indicate that similar to HDs in animals,
collaboration of HD proteins to modulate the expression of target
genes also occurs in plants. The interaction of HD proteins not
only enhances their DNA-binding affinity, but also imparts another
level of regulation to these complexes in fine-tuning developmental
processes. It is very likely that the numerous potential BEL/KNOX
protein interactions participate in a comprehensive system of
regulation that coordinates plant growth.
[0205] Although preferred embodiments have been depicted and
described in detail herein, it will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions, and the like can be made without departing from the
spirit of the invention and these are therefore considered to be
within the scope of the invention as defined in the claims which
follow.
Sequence CWU 1
1
2812735DNASolanum tuberosum 1catgcagaga taaaaatata gatcagtctg
acaagaaggc aacttctcaa agcttagaga 60gctaccaccc gaagatagac agttagttac
atgtactgtt atagataaaa ggagaaatcc 120gaagaagaaa gaattttttt
tgcagatatg tactatcaag gaacctcgga taatactaat 180atacaagctg
atcatcaaca acgtcataat catgggaata gtaataataa taatattcag
240acactttatt tgatgaaccc taacaattat atgcaaggct acactacttc
tgacacacag 300cagcagcagc agttactttt cctgaattct tcaccagcag
caagcaacgc gctttgccat 360gcgaatatac aacacgcgcc gctgcaacag
cagcactttg tcggtgtgcc tcttccggca 420gtaagtttgc acgatcagat
caatcatcat ggacttttac agcgcatgtg gaacaaccaa 480gatcaatctc
agcaggtgat agtaccatcg tcgacggggg tttctgccac gtcatgtggc
540gggatcacca cggacttggc gtctcaattg gcgtttcaga ggccgattcc
gacaccacaa 600caccgacagc agcaacaaca gcaaggcggt ctatctctaa
gcctttctcc tcagctacaa 660cagcaaatta gtttcaataa caatatttca
tcctcatcac caaggacaaa taatgttact 720attaggggaa cattagatgg
aagttctagc aacatggttt taggctctaa gtatctgaaa 780gctgcacaag
agcttcttga tgaagttgtt aatattgttg gaaaaagcat caaaggagat
840gatcaaaaga aggataattc aatgaataaa gaatcaatgc ctttggctag
tgatgtcaac 900actaatagtt ctggtggtgg tgaaagtagc agcaggcaga
aaaatgaagt tgctgttgag 960cttacaactg ctcaaagaca agaacttcaa
atgaaaaaag ccaagcttct tgccatgctt 1020gaagaggtgg agcaaaggta
cagacagtac catcaccaaa tgcaaataat tgtattatca 1080tttgagcaag
tagcaggaat tggatcagcc aaatcataca ctcaattagc tttgcatgca
1140atttcgaagc aattcagatg cctaaaggat gcaattgctg agcaagtaaa
ggcgacgagc 1200aagagtttag gtgaagagga aggcttggga gggaaaatcg
aaggctcaag actcaaattt 1260gtggaccatc atctaaggca acaacgcgcg
ctgcaacaga taggaatgat gcaaccaaat 1320gcttggagac cccaaagagg
tttacctgaa agagctgtct ctgtccttcg tgcttggctt 1380ttcgagcatt
ttcttcatcc ttacccaaag gattcagaca aaatcatgct tgctaagcaa
1440acggggctaa caaggagcca ggtgtctaac tggttcataa atgctcgagt
tcgattatgg 1500aagccaatgg tagaagaaat gtacttggaa gaagtgaaga
atcaagaaca aaacagtact 1560aatacttcag gagataacaa aaacaaagag
accaatataa gtgctccaaa tgaagagaaa 1620catccaatta ttactagcag
cttattacaa gatggtatta ctactactca agcagaaatt 1680tctacctcaa
ctatttcaac ttcccctact gcaggtgctt cacttcatca tgctcacaat
1740ttctccttcc ttggttcatt caacatggat aatactacta ctactgttga
tcatattgaa 1800aacaacgcga aaaagcaaag aaatgacatg cacaagtttt
ctccaagtag tattctttca 1860tctgttgaca tggaagccaa agctagagaa
tcatcaaata aagggtttac taatccttta 1920atggcagcat acgcgatggg
agattttgga aggtttgatc ctcatgatca acaaatgacc 1980gcgaattttc
atggaaataa tggtgtctct cttactttag gacttcctcc ttctgaaaac
2040ctagccatgc cagtgagcca acaaaattac ctttctaatg acttgggaag
taggtctgaa 2100atggggagtc attacaatag aatgggatat gaaaacattg
attttcagag tgggaataag 2160cgatttccga ctcaactatt accagatttt
gttacaggta atctaggaac atgaatacca 2220gaaagtctcg tattgatagc
tgaaaagata aaaggaagtt agggatactc ttatattgtg 2280tgaggccttc
tggcccaagt cggaggaccc aatttgatac aacctatcat aggagaaaag
2340aagtggagac taaattaaag taacaaaatt ttaaagcaca ctttctagta
tatatacttc 2400ttttttttat agtatagaaa agaagagatt ttgtgcttta
gtgtatagat agagtctact 2460tagtataggt tatacttcta gttccttgag
aagattgata caactagtag tatttttttt 2520cttttgggtt ggcttggagt
actattttaa gttattggaa actagctata gtaaatgttg 2580taaagttgtg
atattgttcc tctcaatttg catataattt gaaatatttt gtacctacta
2640gctagtctct aaattatgtt tccattgctt gtaattgcaa ttttatttga
attttgtgct 2700atcattatta gattagcaaa aaaaaaaaaa aaaaa
27352688PRTSolanum tuberosum 2Met Tyr Tyr Gln Gly Thr Ser Asp Asn
Thr Asn Ile Gln Ala Asp His1 5 10 15Gln Gln Arg His Asn His Gly Asn
Ser Asn Asn Asn Asn Ile Gln Thr 20 25 30Leu Tyr Leu Met Asn Pro Asn
Asn Tyr Met Gln Gly Tyr Thr Thr Ser35 40 45Asp Thr Gln Gln Gln Gln
Gln Leu Leu Phe Leu Asn Ser Ser Pro Ala50 55 60Ala Ser Asn Ala Leu
Cys His Ala Asn Ile Gln His Ala Pro Leu Gln65 70 75 80Gln Gln His
Phe Val Gly Val Pro Leu Pro Ala Val Ser Leu His Asp 85 90 95Gln Ile
Asn His His Gly Leu Leu Gln Arg Met Trp Asn Asn Gln Asp 100 105
110Gln Ser Gln Gln Val Ile Val Pro Ser Ser Thr Gly Val Ser Ala
Thr115 120 125Ser Cys Gly Gly Ile Thr Thr Asp Leu Ala Ser Gln Leu
Ala Phe Gln130 135 140Arg Pro Ile Pro Thr Pro Gln His Arg Gln Gln
Gln Gln Gln Gln Gly145 150 155 160Gly Leu Ser Leu Ser Leu Ser Pro
Gln Leu Gln Gln Gln Ile Ser Phe 165 170 175Asn Asn Asn Ile Ser Ser
Ser Ser Pro Arg Thr Asn Asn Val Thr Ile 180 185 190Arg Gly Thr Leu
Asp Gly Ser Ser Ser Asn Met Val Leu Gly Ser Lys195 200 205Tyr Leu
Lys Ala Ala Gln Glu Leu Leu Asp Glu Val Val Asn Ile Val210 215
220Gly Lys Ser Ile Lys Gly Asp Asp Gln Lys Lys Asp Asn Ser Met
Asn225 230 235 240Lys Glu Ser Met Pro Leu Ala Ser Asp Val Asn Thr
Asn Ser Ser Gly 245 250 255Gly Gly Glu Ser Ser Ser Arg Gln Lys Asn
Glu Val Ala Val Glu Leu 260 265 270Thr Thr Ala Gln Arg Gln Glu Leu
Gln Met Lys Lys Ala Lys Leu Leu275 280 285Ala Met Leu Glu Glu Val
Glu Gln Arg Tyr Arg Gln Tyr His His Gln290 295 300Met Gln Ile Ile
Val Leu Ser Phe Glu Gln Val Ala Gly Ile Gly Ser305 310 315 320Ala
Lys Ser Tyr Thr Gln Leu Ala Leu His Ala Ile Ser Lys Gln Phe 325 330
335Arg Cys Leu Lys Asp Ala Ile Ala Glu Gln Val Lys Ala Thr Ser Lys
340 345 350Ser Leu Gly Glu Glu Glu Gly Leu Gly Gly Lys Ile Glu Gly
Ser Arg355 360 365Leu Lys Phe Val Asp His His Leu Arg Gln Gln Arg
Ala Leu Gln Gln370 375 380Ile Gly Met Met Gln Pro Asn Ala Trp Arg
Pro Gln Arg Gly Leu Pro385 390 395 400Glu Arg Ala Val Ser Val Leu
Arg Ala Trp Leu Phe Glu His Phe Leu 405 410 415His Pro Tyr Pro Lys
Asp Ser Asp Lys Ile Met Leu Ala Lys Gln Thr 420 425 430Gly Leu Thr
Arg Ser Gln Val Ser Asn Trp Phe Ile Asn Ala Arg Val435 440 445Arg
Leu Trp Lys Pro Met Val Glu Glu Met Tyr Leu Glu Glu Val Lys450 455
460Asn Gln Glu Gln Asn Ser Thr Asn Thr Ser Gly Asp Asn Lys Asn
Lys465 470 475 480Glu Thr Asn Ile Ser Ala Pro Asn Glu Glu Lys His
Pro Ile Ile Thr 485 490 495Ser Ser Leu Leu Gln Asp Gly Ile Thr Thr
Thr Gln Ala Glu Ile Ser 500 505 510Thr Ser Thr Ile Ser Thr Ser Pro
Thr Ala Gly Ala Ser Leu His His515 520 525Ala His Asn Phe Ser Phe
Leu Gly Ser Phe Asn Met Asp Asn Thr Thr530 535 540Thr Thr Val Asp
His Ile Glu Asn Asn Ala Lys Lys Gln Arg Asn Asp545 550 555 560Met
His Lys Phe Ser Pro Ser Ser Ile Leu Ser Ser Val Asp Met Glu 565 570
575Ala Lys Ala Arg Glu Ser Ser Asn Lys Gly Phe Thr Asn Pro Leu Met
580 585 590Ala Ala Tyr Ala Met Gly Asp Phe Gly Arg Phe Asp Pro His
Asp Gln595 600 605Gln Met Thr Ala Asn Phe His Gly Asn Asn Gly Val
Ser Leu Thr Leu610 615 620Gly Leu Pro Pro Ser Glu Asn Leu Ala Met
Pro Val Ser Gln Gln Asn625 630 635 640Tyr Leu Ser Asn Asp Leu Gly
Ser Arg Ser Glu Met Gly Ser His Tyr 645 650 655Asn Arg Met Gly Tyr
Glu Asn Ile Asp Phe Gln Ser Gly Asn Lys Arg 660 665 670Phe Pro Thr
Gln Leu Leu Pro Asp Phe Val Thr Gly Asn Leu Gly Thr675 680
68531898DNASolanum tuberosum 3atgactttca ggtctagtct tccactagac
ctccgtgaaa tttcaacaac aaatcatcaa 60gttggaatac tatcatcatc accattacca
tcaccaggaa caaataccaa taatatcaat 120catactcgag gattaggggc
atcatcatct ttttcgattt ctaatgggat gatattgggt 180tctaagtacc
taaaagttgc acaagatctt cttgatgaag ttgttaatgt tggaaaaaac
240atcaaattat cagatggctt agagagtggt gcaaaggaga aacacaaatt
ggacaatgaa 300ttaatatctt tggctagtga tgatgttgaa agcagcagcc
aaaaaaatag tggtgttgaa 360cttacaacag ctcaaagaca agaacttcaa
atgaagaaag ccaagcttgt tagcatgctt 420gatgaggtgg atcaaaggta
tagacaatac catcaccaaa tgcaaatgat tgcaacatca 480tttgagcaaa
caacaggaat tggatcatca aaatcataca cacaacttgc tttgcacaca
540atttcaaagc aatttagatg tttaaaagat gcaatttctg ggcaaataaa
ggacactagc 600aaaactttag gggaagaaga aaacattgga ggcaaaattg
aaggatcaaa gttgaaattt 660gtggatcatc atttacgcca acaacgtgca
ctacaacaat tagggatgat gcaaaccaat 720gcatggaagc ctcaaagagg
tttgccagaa agagcggttt cagttctccg cgcttggctt 780ttcgagcatt
ttcttcatcc gtatcccaaa gattcagata aaatcatcct tgctaagcaa
840acagggctaa caaggagcca ggtatcaaat tggtttataa atgctagagt
tagactatgg 900aagccaatgg tagaagaaat gtacatggaa gaagtgaaga
aaaacaatca agaacaaaat 960attgagccta ataacaatga aattgttggc
tcaaaatcaa gtgttccaca agagaaatta 1020ccaattagta gcaatattat
tcataatgct tctccaaatg atatttctac ttccaccatt 1080tcaacatctc
cgacgggtgg cggcggttcg attccgactc agacggttgc aggtttctcc
1140ttcattaggt cattaaacat ggagaacatt gatgatcaaa ggaacaacaa
aaaggcaaga 1200aatgagatgc aaaattgttc aactagtact attctctcaa
tggaaagaga aatcataaat 1260aaagttgtgc aagatgagac aatcaaaagt
gaaaagttca acaacacaca aacaagagaa 1320tgttactctc taatgactcc
aaattacaca atggatgatc aatttggaac aaggttcaat 1380aatcaaaatc
atgaacaatt ggcaacaaca acaacttttc atcaaggaaa tggtcatgtt
1440tctcttactt tagggcttcc accaaattct gaaaaccaac acaattacat
tggattggaa 1500aatcattaca atcaacctac acatcatcca aatattagct
atgaaaacat tgattttcag 1560agtggaaagc gatacgccac tcaactatta
caagattttg tttcttgatg atatatataa 1620tttgcaggta aatcagcttg
aaattacatc atgacaggtc ttgaataaaa gaaggggagt 1680tgagatttag
tgatcatata aatatgtata ggtagaaatt ttagttagta tatataggtt
1740atacttctag tttcttaatg aagatacaag ttttgttgtt atttttgtat
tgaggtaact 1800agctagcttg gattatttaa agttggtgca tgcaactaaa
gaagaagaaa aaataatcta 1860tatatgcaaa ctacagtata ttgtaaattt tgtgcttc
18984535PRTSolanum tuberosum 4Met Thr Phe Arg Ser Ser Leu Pro Leu
Asp Leu Arg Glu Ile Ser Thr1 5 10 15Thr Asn His Gln Val Gly Ile Leu
Ser Ser Ser Pro Leu Pro Ser Pro 20 25 30Gly Thr Asn Thr Asn Asn Ile
Asn His Thr Arg Gly Leu Gly Ala Ser35 40 45Ser Ser Phe Ser Ile Ser
Asn Gly Met Ile Leu Gly Ser Lys Tyr Leu50 55 60Lys Val Ala Gln Asp
Leu Leu Asp Glu Val Val Asn Val Gly Lys Asn65 70 75 80Ile Lys Leu
Ser Asp Gly Leu Glu Ser Gly Ala Lys Glu Lys His Lys 85 90 95Leu Asp
Asn Glu Leu Ile Ser Leu Ala Ser Asp Asp Val Glu Ser Ser 100 105
110Ser Gln Lys Asn Ser Gly Val Glu Leu Thr Thr Ala Gln Arg Gln
Glu115 120 125Leu Gln Met Lys Lys Ala Lys Leu Val Ser Met Leu Asp
Glu Val Asp130 135 140Gln Arg Tyr Arg Gln Tyr His His Gln Met Gln
Met Ile Ala Thr Ser145 150 155 160Phe Glu Gln Thr Thr Gly Ile Gly
Ser Ser Lys Ser Tyr Thr Gln Leu 165 170 175Ala Leu His Thr Ile Ser
Lys Gln Phe Arg Cys Leu Lys Asp Ala Ile 180 185 190Ser Gly Gln Ile
Lys Asp Thr Ser Lys Thr Leu Gly Glu Glu Glu Asn195 200 205Ile Gly
Gly Lys Ile Glu Gly Ser Lys Leu Lys Phe Val Asp His His210 215
220Leu Arg Gln Gln Arg Ala Leu Gln Gln Leu Gly Met Met Gln Thr
Asn225 230 235 240Ala Trp Lys Pro Gln Arg Gly Leu Pro Glu Arg Ala
Val Ser Val Leu 245 250 255Arg Ala Trp Leu Phe Glu His Phe Leu His
Pro Tyr Pro Lys Asp Ser 260 265 270Asp Lys Ile Ile Leu Ala Lys Gln
Thr Gly Leu Thr Arg Ser Gln Val275 280 285Ser Asn Trp Phe Ile Asn
Ala Arg Val Arg Leu Trp Lys Pro Met Val290 295 300Glu Glu Met Tyr
Met Glu Glu Val Lys Lys Asn Asn Gln Glu Gln Asn305 310 315 320Ile
Glu Pro Asn Asn Asn Glu Ile Val Gly Ser Lys Ser Ser Val Pro 325 330
335Gln Glu Lys Leu Pro Ile Ser Ser Asn Ile Ile His Asn Ala Ser Pro
340 345 350Asn Asp Ile Ser Thr Ser Thr Ile Ser Thr Ser Pro Thr Gly
Gly Gly355 360 365Gly Ser Ile Pro Thr Gln Thr Val Ala Gly Phe Ser
Phe Ile Arg Ser370 375 380Leu Asn Met Glu Asn Ile Asp Asp Gln Arg
Asn Asn Lys Lys Ala Arg385 390 395 400Asn Glu Met Gln Asn Cys Ser
Thr Ser Thr Ile Leu Ser Met Glu Arg 405 410 415Glu Ile Ile Asn Lys
Val Val Gln Asp Glu Thr Ile Lys Ser Glu Lys 420 425 430Phe Asn Asn
Thr Gln Thr Arg Glu Cys Tyr Ser Leu Met Thr Pro Asn435 440 445Tyr
Thr Met Asp Asp Gln Phe Gly Thr Arg Phe Asn Asn Gln Asn His450 455
460Glu Gln Leu Ala Thr Thr Thr Thr Phe His Gln Gly Asn Gly His
Val465 470 475 480Ser Leu Thr Leu Gly Leu Pro Pro Asn Ser Glu Asn
Gln His Asn Tyr 485 490 495Ile Gly Leu Glu Asn His Tyr Asn Gln Pro
Thr His His Pro Asn Ile 500 505 510Ser Tyr Glu Asn Ile Asp Phe Gln
Ser Gly Lys Arg Tyr Ala Thr Gln515 520 525Leu Leu Gln Asp Phe Val
Ser530 53551920DNASolanum tuberosum 5ggggagcgag tggttccgac
aaggtatggt aatgggtgga ggtgcaagta gtcaacaatt 60gggatatgca aaaaatcata
ctcctaatgt ggcggagtcc atgcaacttt ttctaatgaa 120tccacaacca
aggtcacctt ctccatctcc tcctaattca acttcttcta cgcttcacat
180gttgttacca aacccatcat ctacttcaac acttcaaggg tttcctaatc
cggccgaagg 240atctttcggt caattcatta catgggggaa tggaggagca
agtgctgcca cagccaccca 300tcatctcaat gcccagaatg aaatcggagg
agtaaacgtt gtagaaagtc aaggcctatc 360tctatccttg tcttcttcgt
tacagcacaa ggcggaggaa ttacaaatga gcggagaagc 420tggaggaatg
atgttcttca atcaaggagg gtctagtact tccgggcagt atcgatacaa
480gaatttgaat atgggtggat caggagtaag cccaaacatt catcaagtcc
atgttgggta 540tgggtcatca ttaggagtgg tcaatgtgtt gaggaattcc
aaatacgcga aagctgccca 600agaactactg gaagaattct gcagtgttgg
aagaggtaaa ttgaagaaga ctaacaacaa 660agcagcagcc aataacccta
atacgaaccc tagtggcgct aacaatgaag cttcttcaaa 720agatgttcct
actttgtccg ctgctgatag aattgagcat cagagaagga aggtcaaact
780tttatctatg gttgatgagg tagataggag gtacaatcat tactgtgaac
aaatgcagat 840ggttgtaaat tcgtttgatt tagtgatggg tttcggcaca
gcagttccct acacagcact 900tgcacagaag gcaatgtcaa gacatttcag
gtgtttaaag gatgcaatag gagcacaatt 960gaagcagagt tgtgagttat
taggagagaa agatgcagga aattcgggat tgactaaagg 1020agaaactccg
aggcttaaga tgcttgaaca aagtttgagg caacaaaggg cgtttcacca
1080aatgggaatg atggaacaag aagcttggag accacaaaga ggcttacctg
aacgttctgt 1140caacatttta agagcttggc tttttgagca ttttctacac
ccgtatccaa gtgatgctga 1200taaacatctg ttggcaagac agactggtct
ctccagaaat caggtatcaa attggttcat 1260taatgctagg gttcggttgt
ggaaacccat ggtagaagat atgtatcaac aagaagccaa 1320agatgaagat
ggagatggag atgagaagag ccaaagccaa aacagtggca ataacataat
1380tgcacaaaca ccaacgccta atagcctgac taacacttca tctactaata
tgacgacgac 1440aacagcccct acaactacga cagctctagc tgctgcagag
acaggaacag ctgccactcc 1500cataactgtt acctcaagca aaagatccca
aatcaatgcc acggatagtg acccttcact 1560tgtagcaatc aattccttct
ctgaaaacca agctactttt ccgaccaaca ttcatgatcc 1620cgacgattgc
cgtcgcggca acttatccgg tgacgacggg accaccacac atgatcatat
1680ggggtccacc atgataaggt ttgggaccac tgctggtgac gtgtcactca
ccttagggtt 1740acgacatgca ggaaatttac cagagaatac tcatttcttt
ggttaattaa tacgtatttt 1800ccccatagta attaattaaa actgaatttg
cttgagctca tcataattta tgcattgctt 1860tttgttataa gaaattccat
aaattagctt tgtgttaaaa aaaaaaaaaa aaaaaaaaaa 19206586PRTSolanum
tuberosum 6Met Val Met Gly Gly Gly Ala Ser Ser Gln Gln Leu Gly Tyr
Ala Lys1 5 10 15Asn His Thr Pro Asn Val Ala Glu Ser Met Gln Leu Phe
Leu Met Asn 20 25 30Pro Gln Pro Arg Ser Pro Ser Pro Ser Pro Pro Asn
Ser Thr Ser Ser35 40 45Thr Leu His Met Leu Leu Pro Asn Pro Ser Ser
Thr Ser Thr Leu Gln50 55 60Gly Phe Pro Asn Pro Ala Glu Gly Ser Phe
Gly Gln Phe Ile Thr Trp65 70 75 80Gly Asn Gly Gly Ala Ser Ala Ala
Thr Ala Thr His His Leu Asn Ala 85 90 95Gln Asn Glu Ile Gly Gly Val
Asn Val Val Glu Ser Gln Gly Leu Ser 100 105 110Leu Ser Leu Ser Ser
Ser Leu Gln His Lys Ala Glu Glu Leu Gln Met115 120 125Ser Gly Glu
Ala Gly Gly Met Met Phe Phe Asn Gln Gly Gly Ser Ser130 135 140Thr
Ser Gly Gln Tyr Arg Tyr Lys Asn Leu Asn Met Gly Gly Ser Gly145 150
155 160Val Ser Pro Asn Ile His Gln Val His Val Gly Tyr Gly Ser Ser
Leu 165 170 175Gly Val Val Asn Val Leu Arg Asn Ser Lys Tyr Ala Lys
Ala Ala Gln 180 185 190Glu Leu Leu Glu Glu Phe Cys Ser Val Gly Arg
Gly Lys Leu Lys Lys195 200 205Thr Asn Asn Lys Ala Ala Ala Asn Asn
Pro Asn
Thr Asn Pro Ser Gly210 215 220Ala Asn Asn Glu Ala Ser Ser Lys Asp
Val Pro Thr Leu Ser Ala Ala225 230 235 240Asp Arg Ile Glu His Gln
Arg Arg Lys Val Lys Leu Leu Ser Met Val 245 250 255Asp Glu Val Asp
Arg Arg Tyr Asn His Tyr Cys Glu Gln Met Gln Met 260 265 270Val Val
Asn Ser Phe Asp Leu Val Met Gly Phe Gly Thr Ala Val Pro275 280
285Tyr Thr Ala Leu Ala Gln Lys Ala Met Ser Arg His Phe Arg Cys
Leu290 295 300Lys Asp Ala Ile Gly Ala Gln Leu Lys Gln Ser Cys Glu
Leu Leu Gly305 310 315 320Glu Lys Asp Ala Gly Asn Ser Gly Leu Thr
Lys Gly Glu Thr Pro Arg 325 330 335Leu Lys Met Leu Glu Gln Ser Leu
Arg Gln Gln Arg Ala Phe His Gln 340 345 350Met Gly Met Met Glu Gln
Glu Ala Trp Arg Pro Gln Arg Gly Leu Pro355 360 365Glu Arg Ser Val
Asn Ile Leu Arg Ala Trp Leu Phe Glu His Phe Leu370 375 380His Pro
Tyr Pro Ser Asp Ala Asp Lys His Leu Leu Ala Arg Gln Thr385 390 395
400Gly Leu Ser Arg Asn Gln Val Ser Asn Trp Phe Ile Asn Ala Arg Val
405 410 415Arg Leu Trp Lys Pro Met Val Glu Asp Met Tyr Gln Gln Glu
Ala Lys 420 425 430Asp Glu Asp Gly Asp Gly Asp Glu Lys Ser Gln Ser
Gln Asn Ser Gly435 440 445Asn Asn Ile Ile Ala Gln Thr Pro Thr Pro
Asn Ser Leu Thr Asn Thr450 455 460Ser Ser Thr Asn Met Thr Thr Thr
Thr Ala Pro Thr Thr Thr Thr Ala465 470 475 480Leu Ala Ala Ala Glu
Thr Gly Thr Ala Ala Thr Pro Ile Thr Val Thr 485 490 495Ser Ser Lys
Arg Ser Gln Ile Asn Ala Thr Asp Ser Asp Pro Ser Leu 500 505 510Val
Ala Ile Asn Ser Phe Ser Glu Asn Gln Ala Thr Phe Pro Thr Asn515 520
525Ile His Asp Pro Asp Asp Cys Arg Arg Gly Asn Leu Ser Gly Asp
Asp530 535 540Gly Thr Thr Thr His Asp His Met Gly Ser Thr Met Ile
Arg Phe Gly545 550 555 560Thr Thr Ala Gly Asp Val Ser Leu Thr Leu
Gly Leu Arg His Ala Gly 565 570 575Asn Leu Pro Glu Asn Thr His Phe
Phe Gly 580 58572103DNASolanum tuberosummisc_feature(5)..(5)n is a,
c, g, or t 7aaccnaaaaa agagatcgaa ttcggcacga gtgatcatgg tccttcgtct
tctaagaaca 60ttattagtga acaattttac caacatggta gtcatgaaaa tatgttgaca
acaacaacta 120ctcatcatga tgatcatcaa ggctcgtggc atcacgataa
taacagaaca ttacttgttg 180atgatccatc tatgagatgt gttttccctt
gtgaaggaaa tgaaaggcca agtcatggac 240tttcattatc tctttgttcc
tcaaatccat caagtattgg tttacaatct tttgaactta 300gacatcaaga
tttgcaacaa ggattaatac atgatggatt tttgggtaaa tctacaaata
360tacaacaagg gtattttcat catcatcatc aagttaggga ctcgaaatat
ttaggtccgg 420ctcaagagtt gctcagtgag ttctgtagtc tcggaataaa
gaagaataat gatcattctt 480cttcaaaagt acttctaaag caacatgaga
gtactgctag tacttcaaaa aagcaacttt 540tacagtctct tgaccttttg
gaacttcaaa aaagaaagac aaaattgctt caaatgcttg 600aagaggtgga
tagaaggtac aagcattatt gtgatcaaat gaaggctgtt gtatcatcat
660ttgaagcagt ggctggaaat ggagcagcaa cagtttactc agccttagca
tcaagggcta 720tgtcaaggca ttttagatgt ttaagagatg gaattgtggc
acaaattaag gccacaaaaa 780tggctatggg agaaaaagac agtactagta
ctcttattcc tggttcaaca agaggtgaaa 840caccaagact cagacttctt
gatcaaactt taaggcaaca aaaggctttc caacagatga 900atatgatgga
gactcatcca tggagaccgc aacgtggtct cccagaaaga tcagtctccg
960ttctccgcgc ttggctcttt gaacactttc ttcacccgta cccaagtgat
gttgataaac 1020acattttagc tcgccaaact ggtctttcaa gaagccaggt
gtctaattgg ttcattaatg 1080caagggtaag gctatggaag ccaatggtgg
aagaaatgta cttagaagaa acaaaagaag 1140aagaaaatgt tggatctcca
gatggatcaa aagccctaat tgatgacatg acaattcatc 1200aatcacacat
tgatcatcat caagctgatc aaaagccaaa tcttgtaaga attgactctg
1260aatgcatatc ttccatcata aatcatcaac ctcatgagaa aaatgatcaa
aactatggag 1320taattagagg tggagatcaa tcgtttggcg cgattgagct
agatttttca acaaatattg 1380cttatggtac tagtggtggt gaccatcatc
atcatggagg gggtgtttct ttaacattgg 1440gattacaaca acatggtgga
agtggtggat catcaatggg gttaactaca ttttcatcac 1500aaccatctca
taatcaaagt tcactttttt atccaagaga tgatgatcaa gttcaatatt
1560catcactttt ggatagtgaa aatcagaatt tgccatatag aaaccttgat
gggggcacaa 1620cttcttcatg atttggctgg ttaaaaaatg acagagattc
ttcattttgg accttattat 1680atactctaat tttaatatat attggtgatg
aatgatgata aaaaaaaaaa aaaaaaaaaa 1740aaaaaaaaaa aaaaaaaaaa
acctcgancc cggtcgactn tanancccta tagngagtcg 1800tnttnctgca
nanatctntg aatcgtaaat nctgaaaaac cccgcaagtt cacttcaact
1860gngcatcgng cnccatctca atttctttca tttatncatc gttttgcctt
nttttatgta 1920actatnctcc tntaagtttc aatcttggcc atgtaacctn
tgatctntaa aattttttaa 1980atgactanaa ttaatgccca tntttttttt
ggacctaaat tnttcatgaa aatntnttnc 2040nagggcttnt tcaaaanctt
tggacttntt cnccanaggt ttggtcaagt ntccaatcaa 2100ggt
21038589PRTSolanum tuberosum 8Met Val Asn His Gln Leu Gln Asn Phe
Glu Thr Asn Pro Glu Met Tyr1 5 10 15Asn Leu Ser Ser Thr Thr Ser Ser
Met Asp Gln Met Ile Gly Phe Pro 20 25 30Pro Asn Asn Asn Asn Pro His
His Val Leu Trp Lys Gly Asn Phe Pro35 40 45Asn Lys Ile Asn Gly Val
Asp Asp Asp Asp His Gly Pro Ser Ser Ser50 55 60Lys Asn Ile Ile Ser
Glu Gln Phe Tyr Gln His Gly Ser His Glu Asn65 70 75 80Met Leu Thr
Thr Thr Thr Thr His His Asp Asp His Gln Gly Ser Trp 85 90 95His His
Asp Asn Asn Arg Thr Leu Leu Val Asp Asp Pro Ser Met Arg 100 105
110Cys Val Phe Pro Cys Glu Gly Asn Glu Arg Pro Ser His Gly Leu
Ser115 120 125Leu Ser Leu Cys Ser Ser Asn Pro Ser Ser Ile Gly Leu
Gln Ser Phe130 135 140Glu Leu Arg His Gln Asp Leu Gln Gln Gly Leu
Ile His Asp Gly Phe145 150 155 160Leu Gly Lys Ser Thr Asn Ile Gln
Gln Gly Tyr Phe His His His His 165 170 175Gln Val Arg Asp Ser Lys
Tyr Leu Gly Pro Ala Gln Glu Leu Leu Ser 180 185 190Glu Phe Cys Ser
Leu Gly Ile Lys Lys Asn Asn Asp His Ser Ser Ser195 200 205Lys Val
Leu Leu Lys Gln His Glu Ser Thr Ala Ser Thr Ser Lys Lys210 215
220Gln Leu Leu Gln Ser Leu Asp Leu Leu Glu Leu Gln Lys Arg Lys
Thr225 230 235 240Lys Leu Leu Gln Met Leu Glu Glu Val Asp Arg Arg
Tyr Lys His Tyr 245 250 255Cys Asp Gln Met Lys Ala Val Val Ser Ser
Phe Glu Ala Val Ala Gly 260 265 270Asn Gly Ala Ala Thr Val Tyr Ser
Ala Leu Ala Ser Arg Ala Met Ser275 280 285Arg His Phe Arg Cys Leu
Arg Asp Gly Ile Val Ala Gln Ile Lys Ala290 295 300Thr Lys Met Ala
Met Gly Glu Lys Asp Ser Thr Ser Thr Leu Ile Pro305 310 315 320Gly
Ser Thr Arg Gly Glu Thr Pro Arg Leu Arg Leu Leu Asp Gln Thr 325 330
335Leu Arg Gln Gln Lys Ala Phe Gln Gln Met Asn Met Met Glu Thr His
340 345 350Pro Trp Arg Pro Gln Arg Gly Leu Pro Glu Arg Ser Val Ser
Val Leu355 360 365Arg Ala Trp Leu Phe Glu His Phe Leu His Pro Tyr
Pro Ser Asp Val370 375 380Asp Lys His Ile Leu Ala Arg Gln Thr Gly
Leu Ser Arg Ser Gln Val385 390 395 400Ser Asn Trp Phe Ile Asn Ala
Arg Val Arg Leu Trp Lys Pro Met Val 405 410 415Glu Glu Met Tyr Leu
Glu Glu Thr Lys Glu Glu Glu Asn Val Gly Ser 420 425 430Pro Asp Gly
Ser Lys Ala Leu Ile Asp Asp Met Thr Ile His Gln Ser435 440 445His
Ile Asp His His Gln Ala Asp Gln Lys Pro Asn Leu Val Arg Ile450 455
460Asp Ser Glu Cys Ile Ser Ser Ile Ile Asn His Gln Pro His Glu
Lys465 470 475 480Asn Asp Gln Asn Tyr Gly Val Ile Arg Gly Gly Asp
Gln Ser Phe Gly 485 490 495Ala Ile Glu Leu Asp Phe Ser Thr Asn Ile
Ala Tyr Gly Thr Ser Gly 500 505 510Gly Asp His His His His Gly Gly
Gly Val Ser Leu Thr Leu Gly Leu515 520 525Gln Gln His Gly Gly Ser
Gly Gly Ser Ser Met Gly Leu Thr Thr Phe530 535 540Ser Ser Gln Pro
Ser His Asn Gln Ser Ser Leu Phe Tyr Pro Arg Asp545 550 555 560Asp
Asp Gln Val Gln Tyr Ser Ser Leu Leu Asp Ser Glu Asn Gln Asn 565 570
575Leu Pro Tyr Arg Asn Leu Asp Gly Gly Thr Thr Ser Ser 580
58591939DNASolanum tuberosum 9acgagcgttt atgagacagc cgggttgttg
tctgaaatgt tcaattttca gacaacatcc 60acggctgcaa ctgaattgtt gcagaatcaa
ttgtcaaata actatagaca cccgaatcaa 120cagccacatc atcaacctcc
gaccagggag tggtttggta acagacaaga gatcgtagtt 180ggtggaagtt
tgcaggtaac atttggggat acaaaagatg atgtgaatgc gaaggtatta
240ttgagtaacc gtgatagtgt aactgattat tatcagcgtc aacacaatca
agtaccaagt 300ataaataccg cggagtccat gcaacttttt cttatgaatc
cacaaccaag ttcaccatca 360caatctactc cttcaactct tcatcaaggg
ttttctagcc cggtcggagg gcattttagt 420caattcatgt gtggaggagc
aagtacttct tcaaatccaa ttggaggagt aaatgtgatt 480gatcaagggc
aaggtctttc attgtccttg tcatctactt tacaacattt ggaagcatcc
540aaagtggaag atttgaggat gaatagtgga ggagaaatgt tgtttttcaa
tcaagaaagt 600caaaatcatc ataatattgg ttttgggtca tcactaggac
tagtcaatgt gttgaggaat 660tcaaagtatg tcaaagcaac acaagagttg
ttggaagagt tttgttgtgt tgggaagggt 720caattgttca agaaaatcaa
caaagtttct aggaataaca acacaagtac atcacccatt 780attaacccta
gtggaagtaa taacaataat tcatcttctt caaaggctat tatccctcct
840aatttgtcaa ctgcagagag acttgatcat caaagaagga aggtcaaact
tttatccatg 900cttgatgagg tagagaaaag atacaaccac tattgtgaac
aaatgcagat ggtagtaaac 960tcattcgatc tagtgatggg ttttggagct
gcagttcctt acacagcact agcacagaaa 1020gccatgtcta ggcatttcaa
gtgtttaaaa gatggcgtgg cggcgcaatt gaagaagaca 1080tgtgaggcac
taggtgaaaa agatgcaagc agtagttcag gactgactaa aggagaaaca
1140ccaaggctta aggtgcttga acaaagcttg aggcaacaaa gagcttttca
acaaatggga 1200atgatggaac aagaagcttg gaggccacaa agaggattgc
ctgaacgatc tgtcaatatt 1260ttaagagctt ggcttttcga acattttcta
catccgtatc caagtgatgc agataagcat 1320cttttggcac gacagactgg
tctctccaga aaccaggtag caaactggtt cataaatgcg 1380agggtgagat
tgtggaaacc catggtagaa gaaatgtatc aaagagaggt taatgaagat
1440gatgttgatg acatgcaaga aaaccaaaac agtacaaata cacaaatacc
aacgcctaat 1500attattatta caaccaattc taacattaca gaaacaaaat
cagctgccac tgccacaatt 1560gcttcagaca aaaaacccca aatcaatgtc
tctgaaattg acccttcaat tgtcgcaatg 1620aatacacatt attcttcctc
tatgccaact caattaacca atttccccac tattcaagat 1680gagtccgacc
acatcttata tcgccgcagt ggagcggaat atgggaccac aaatatggct
1740agtaattctg aaattggatc caacatgata acatttggga ccactacggc
tagtgatgtt 1800tcacttacct taggactgcg ccatgcgggt aatttacctg
agaatactca tttttccggt 1860taattaagat agtgtattca aacactgcta
cataaattat gattttatat atatatatat 1920tgtcatccga ttagtttat
193910620PRTSolanum tuberosum 10Thr Ser Val Tyr Glu Thr Ala Gly Leu
Leu Ser Glu Met Phe Asn Phe1 5 10 15Gln Thr Thr Ser Thr Ala Ala Thr
Glu Leu Leu Gln Asn Gln Leu Ser 20 25 30Asn Asn Tyr Arg His Pro Asn
Gln Gln Pro His His Gln Pro Pro Thr35 40 45Arg Glu Trp Phe Gly Asn
Arg Gln Glu Ile Val Val Gly Gly Ser Leu50 55 60Gln Val Thr Phe Gly
Asp Thr Lys Asp Asp Val Asn Ala Lys Val Leu65 70 75 80Leu Ser Asn
Arg Asp Ser Val Thr Asp Tyr Tyr Gln Arg Gln His Asn 85 90 95Gln Val
Pro Ser Ile Asn Thr Ala Glu Ser Met Gln Leu Phe Leu Met 100 105
110Asn Pro Gln Pro Ser Ser Pro Ser Gln Ser Thr Pro Ser Thr Leu
His115 120 125Gln Gly Phe Ser Ser Pro Val Gly Gly His Phe Ser Gln
Phe Met Cys130 135 140Gly Gly Ala Ser Thr Ser Ser Asn Pro Ile Gly
Gly Val Asn Val Ile145 150 155 160Asp Gln Gly Gln Gly Leu Ser Leu
Ser Leu Ser Ser Thr Leu Gln His 165 170 175Leu Glu Ala Ser Lys Val
Glu Asp Leu Arg Met Asn Ser Gly Gly Glu 180 185 190Met Leu Phe Phe
Asn Gln Glu Ser Gln Asn His His Asn Ile Gly Phe195 200 205Gly Ser
Ser Leu Gly Leu Val Asn Val Leu Arg Asn Ser Lys Tyr Val210 215
220Lys Ala Thr Gln Glu Leu Leu Glu Glu Phe Cys Cys Val Gly Lys
Gly225 230 235 240Gln Leu Phe Lys Lys Ile Asn Lys Val Ser Arg Asn
Asn Asn Thr Ser 245 250 255Thr Ser Pro Ile Ile Asn Pro Ser Gly Ser
Asn Asn Asn Asn Ser Ser 260 265 270Ser Ser Lys Ala Ile Ile Pro Pro
Asn Leu Ser Thr Ala Glu Arg Leu275 280 285Asp His Gln Arg Arg Lys
Val Lys Leu Leu Ser Met Leu Asp Glu Val290 295 300Glu Lys Arg Tyr
Asn His Tyr Cys Glu Gln Met Gln Met Val Val Asn305 310 315 320Ser
Phe Asp Leu Val Met Gly Phe Gly Ala Ala Val Pro Tyr Thr Ala 325 330
335Leu Ala Gln Lys Ala Met Ser Arg His Phe Lys Cys Leu Lys Asp Gly
340 345 350Val Ala Ala Gln Leu Lys Lys Thr Cys Glu Ala Leu Gly Glu
Lys Asp355 360 365Ala Ser Ser Ser Ser Gly Leu Thr Lys Gly Glu Thr
Pro Arg Leu Lys370 375 380Val Leu Glu Gln Ser Leu Arg Gln Gln Arg
Ala Phe Gln Gln Met Gly385 390 395 400Met Met Glu Gln Glu Ala Trp
Arg Pro Gln Arg Gly Leu Pro Glu Arg 405 410 415Ser Val Asn Ile Leu
Arg Ala Trp Leu Phe Glu His Phe Leu His Pro 420 425 430Tyr Pro Ser
Asp Ala Asp Lys His Leu Leu Ala Arg Gln Thr Gly Leu435 440 445Ser
Arg Asn Gln Val Ala Asn Trp Phe Ile Asn Ala Arg Val Arg Leu450 455
460Trp Lys Pro Met Val Glu Glu Met Tyr Gln Arg Glu Val Asn Glu
Asp465 470 475 480Asp Val Asp Asp Met Gln Glu Asn Gln Asn Ser Thr
Asn Thr Gln Ile 485 490 495Pro Thr Pro Asn Ile Ile Ile Thr Thr Asn
Ser Asn Ile Thr Glu Thr 500 505 510Lys Ser Ala Ala Thr Ala Thr Ile
Ala Ser Asp Lys Lys Pro Gln Ile515 520 525Asn Val Ser Glu Ile Asp
Pro Ser Ile Val Ala Met Asn Thr His Tyr530 535 540Ser Ser Ser Met
Pro Thr Gln Leu Thr Asn Phe Pro Thr Ile Gln Asp545 550 555 560Glu
Ser Asp His Ile Leu Tyr Arg Arg Ser Gly Ala Glu Tyr Gly Thr 565 570
575Thr Asn Met Ala Ser Asn Ser Glu Ile Gly Ser Asn Met Ile Thr Phe
580 585 590Gly Thr Thr Thr Ala Ser Asp Val Ser Leu Thr Leu Gly Leu
Arg His595 600 605Ala Gly Asn Leu Pro Glu Asn Thr His Phe Ser
Gly610 615 620112128DNASolanum tuberosummisc_feature(2078)..(2078)n
is a, c, g, or t 11caagggcttt cacttagcct gtcctcgtcc cagcagccgg
ggtttgggaa cttcacggcg 60gcgcgtgagc ttgtttcttc gccttcgggt tcggcttcag
cttcagggat acaacaacaa 120caacagcaac aacagagtat tagtagtgtg
cctttgagtt ctaagtacat gaaggctgca 180caagagctac ttgatgaagt
tgtaaatgtt ggaaaatcaa tgaaaagtac taatagtact 240gatgttgttg
ttaataatga tgtcaagaaa tcgaagaata tgggcgatat ggacggacag
300ttagacggag ttggagcaga caaagacgga gctccaacaa ctgagctaag
tacaggggag 360agacaagaaa ttcaaatgaa gaaagcaaaa cttgttaaca
tgcttgacga ggtggagcag 420aggtatagac attatcatca ccaaatgcag
tcagtgatac attggttaga gcaagctgct 480ggcattggat cagcaaaaac
atatacagca ttggctttgc agacgatttc gaagcaattt 540aggtgtctta
aggacgcgat aattggtcaa atacgatcag caagccagac gttaggcgaa
600gaagatagtt tgggagggaa gattgaaggt tcaaggctta aatttgttga
taatcagcta 660agacagcaaa gggctttgca acaattggga atgatccagc
ataatgcttg gagacctcag 720agaggattgc ccgaacgagc tgtttctgtt
cttcgcgctt ggctttttga acatttcctc 780catccttatc ccaaggattc
agacaaaatg atgctagcaa aacaaacagg actaactagg 840agtcaggtgt
cgaattggtt catcaatgct cgagttcgtc tttggaagcc aatggtggaa
900gagatgtact tggaagagat aaaagaacac gaacagaatg ggttgggtca
agaaaagacg 960agcaaattag gtgaacagaa cgaagattca acaacatcaa
gatccattgc tacacaagac 1020aaaagccctg gttcagatag ccaaaacaag
agttttgtct caaaacagga caatcatttg 1080cctcaacaca accctgcttc
accaatgccc gatgtccaac gccacttcca tacccctatc 1140ggtatgacca
tccgtaatca gtctgctggt ttcaacctca ttggatcacc agagatcgaa
1200agcatcaaca ttactcaagg gagtccaaag aaaccgagga acaacgagat
gttgcattca 1260ccaaacagca ttccatccat caacatggat gtaaagccta
acgaggaaca aatgtcgatg 1320aagtttggtg atgataggca ggacagagat
ggattctcac taatgggagg accgatgaac 1380ttcatgggag gattcggagc
ctatcccatt ggagaaattg ctcggtttag caccgagcaa 1440ttctcagcac
catactcaac cagtggcaca gtttcactca ctcttggcct accacataac
1500gaaaacctct caatgtctgc aacacaccac agtttccttc caattccaac
acaaaacatc 1560caaattggaa
gtgaaccaaa tcatgagttt ggtagcttaa acacaccaac atcagctcac
1620tcaacatcaa gcgtctatga aaccttcaac attcagaaca gaaagaggtt
cgccgcaccc 1680ttgttaccag attttgttgc ctgatcacaa aaacaaaaac
aggttttggc aacagacaaa 1740cttctgtcgc taaacaagga catgatttag
cgacagataa cttcagtcgc taacttagcg 1800actgaaaact tctgtcgcta
agcatgaaca tgtattagcg acatacagta tgcaactgta 1860tgtcactaaa
caagaacatg atgaattagt gacggacaac ttctgtcgct aaacaacaaa
1920aaaaaatcca tgttttagta tattgtttct cattctatca tatcatggta
gtgtaaagaa 1980tcaagaaaca agttttacat agtaacagtc tttatacatt
ggagatgaag aaccatttaa 2040gttcttcaaa atagatagat tttctaggtt
acttctanaa gatatatata tggttgaggg 2100tttgtatatt aaaaaaaaaa aaaaaaaa
212812567PRTSolanum tuberosum 12Gln Gly Leu Ser Leu Ser Leu Ser Ser
Ser Gln Gln Pro Gly Phe Gly1 5 10 15Asn Phe Thr Ala Ala Arg Glu Leu
Val Ser Ser Pro Ser Gly Ser Ala 20 25 30Ser Ala Ser Gly Ile Gln Gln
Gln Gln Gln Gln Gln Gln Ser Ile Ser35 40 45Ser Val Pro Leu Ser Ser
Lys Tyr Met Lys Ala Ala Gln Glu Leu Leu50 55 60Asp Glu Val Val Asn
Val Gly Lys Ser Met Lys Ser Thr Asn Ser Thr65 70 75 80Asp Val Val
Val Asn Asn Asp Val Lys Lys Ser Lys Asn Met Gly Asp 85 90 95Met Asp
Gly Gln Leu Asp Gly Val Gly Ala Asp Lys Asp Gly Ala Pro 100 105
110Thr Thr Glu Leu Ser Thr Gly Glu Arg Gln Glu Ile Gln Met Lys
Lys115 120 125Ala Lys Leu Val Asn Met Leu Asp Glu Val Glu Gln Arg
Tyr Arg His130 135 140Tyr His His Gln Met Gln Ser Val Ile His Trp
Leu Glu Gln Ala Ala145 150 155 160Gly Ile Gly Ser Ala Lys Thr Tyr
Thr Ala Leu Ala Leu Gln Thr Ile 165 170 175Ser Lys Gln Phe Arg Cys
Leu Lys Asp Ala Ile Ile Gly Gln Ile Arg 180 185 190Ser Ala Ser Gln
Thr Leu Gly Glu Glu Asp Ser Leu Gly Gly Lys Ile195 200 205Glu Gly
Ser Arg Leu Lys Phe Val Asp Asn Gln Leu Arg Gln Gln Arg210 215
220Ala Leu Gln Gln Leu Gly Met Ile Gln His Asn Ala Trp Arg Pro
Gln225 230 235 240Arg Gly Leu Pro Glu Arg Ala Val Ser Val Leu Arg
Ala Trp Leu Phe 245 250 255Glu His Phe Leu His Pro Tyr Pro Lys Asp
Ser Asp Lys Met Met Leu 260 265 270Ala Lys Gln Thr Gly Leu Thr Arg
Ser Gln Val Ser Asn Trp Phe Ile275 280 285Asn Ala Arg Val Arg Leu
Trp Lys Pro Met Val Glu Glu Met Tyr Leu290 295 300Glu Glu Ile Lys
Glu His Glu Gln Asn Gly Leu Gly Gln Glu Lys Thr305 310 315 320Ser
Lys Leu Gly Glu Gln Asn Glu Asp Ser Thr Thr Ser Arg Ser Ile 325 330
335Ala Thr Gln Asp Lys Ser Pro Gly Ser Asp Ser Gln Asn Lys Ser Phe
340 345 350Val Ser Lys Gln Asp Asn His Leu Pro Gln His Asn Pro Ala
Ser Pro355 360 365Met Pro Asp Val Gln Arg His Phe His Thr Pro Ile
Gly Met Thr Ile370 375 380Arg Asn Gln Ser Ala Gly Phe Asn Leu Ile
Gly Ser Pro Glu Ile Glu385 390 395 400Ser Ile Asn Ile Thr Gln Gly
Ser Pro Lys Lys Pro Arg Asn Asn Glu 405 410 415Met Leu His Ser Pro
Asn Ser Ile Pro Ser Ile Asn Met Asp Val Lys 420 425 430Pro Asn Glu
Glu Gln Met Ser Met Lys Phe Gly Asp Asp Arg Gln Asp435 440 445Arg
Asp Gly Phe Ser Leu Met Gly Gly Pro Met Asn Phe Met Gly Gly450 455
460Phe Gly Ala Tyr Pro Ile Gly Glu Ile Ala Arg Phe Ser Thr Glu
Gln465 470 475 480Phe Ser Ala Pro Tyr Ser Thr Ser Gly Thr Val Ser
Leu Thr Leu Gly 485 490 495Leu Pro His Asn Glu Asn Leu Ser Met Ser
Ala Thr His His Ser Phe 500 505 510Leu Pro Ile Pro Thr Gln Asn Ile
Gln Ile Gly Ser Glu Pro Asn His515 520 525Glu Phe Gly Ser Leu Asn
Thr Pro Thr Ser Ala His Ser Thr Ser Ser530 535 540Val Tyr Glu Thr
Phe Asn Ile Gln Asn Arg Lys Arg Phe Ala Ala Pro545 550 555 560Leu
Leu Pro Asp Phe Val Ala 565132065DNASolanum tuberosum 13atctccaagt
aaaaaggtta ttgagaaaag taacacagat ggcgacttat tttcctagtc 60caaacaatca
aagagatgct gatcagacat ttcaatattt taggcaatct ttgcctgagt
120cttattcaga agcttcaaat gctccagaaa acatgatggt attcatgaac
tattcttctt 180ctggggcata ttcagatatg ttgacgggta cttcccaaca
acaacacaac tgcatcgata 240tcccatctat aggagccacg cctttcaaca
catcccaaca agaaatattg tcaaatcttg 300gaggatcgca gatggggatt
caggattttt cttcatggag agatagcaga aatgagatgc 360tagctgataa
tgtctttcaa gttgcacaaa atgtgcaggg tcaaggatta tccctcagtc
420ttggctccaa tataccatct ggaattggaa tttcacatgt ccaatctcag
aatcctaacc 480aaggtggcgg ttttaacatg tcctttggag atggtgataa
ttcccaacca aaagaacaaa 540gaaatgcaga ttattttcct ccggataatc
ctggaaggga cttggatgct atgaaagggt 600ataattctcc atatggtacg
tcgagtattg caaggaccat tcccagctcg aagtatttga 660aagcagctca
atatttgctt gatgaggttg ttagtgtcag aaaggccatc aaggagcaaa
720attctaagaa agagttgaca aaggattcca gagagtctga tgtggactcg
aaaaatatat 780catcagatac tcctgcaaat gggggttcaa atcctcatga
gtccaaaaac aaccaaagtg 840aactttcacc taccgagaag caagaagtgc
agaacaaact ggccaaactt ctgtcaatgc 900tggatgagat tgatagaagg
tacagacaat attatcatca gatgcaaata gtggtttcat 960catttgatgt
ggtagctgga gaaggagcag ctaaaccata cacagctctt gctctccaga
1020caatttcccg acacttccgt tgcttgcgtg atgcaatctg cgatcagatt
cgagcatcac 1080gaagaagtct tggagagcaa gatgcttcag aaaacagcaa
agcgattgga atatcacgcc 1140tgcgttttgt ggatcatcat attagacagc
agagagccct gcagcagctt ggtatgatgc 1200aacaacatgc ctggaggcct
cagaggggat tgcctgaaag ctctgtttca gttttgcgtg 1260cttggctctt
tgagcacttt cttcatccct acccgaaaga ttctgacaaa attatgctag
1320caaggcaaac tggcttaacg agaagtcagg tatcaaattg gttcataaat
gcacgggtgc 1380gtctttggaa acccatggtt gaggaaatgt acaaagaaga
ggctggtgat gctaaaatag 1440actcaaattc ttcatcggat gttgccccca
gacttgcaac aaaagactca aaagttgaag 1500aaagaggaga attgcaccag
aatgcagctt cagaatttga gcagtacaat agtggccaaa 1560tcctggagtc
aaaatctaac catgaagctg atgtagaaat ggagggagca agtaatgcag
1620aaactcaaag tcaatctgga atggaaaacc aaacaggcga acccctgcct
gctatggata 1680attgcaccct ttttcaggac gcatttgttc aaagcaacga
tagattctca gaatttggta 1740gttttggaag tggaaatgta ctacccaatg
gagtttcact tacattgggg ctgcagcaag 1800gtgaaggaag caacctacct
atgtccatcg aaactcacgt tagttatgta ccattaaggg 1860cagatgacat
gtatagtaca gcacctacta ctatggtccc tgaaacagca gaattcaact
1920gcttggattc tgggaatagg cagcaaccat tttggctcct accatctgct
acatgatttt 1980gtatgtgttg tagaattaaa ctgcaagttt tgagtacatc
aacattcatc ttcaaaaaaa 2040aaaaaaaaaa aaaaaaaaaa aaaaa
206514645PRTSolanum tuberosum 14Met Ala Thr Tyr Phe Pro Ser Pro Asn
Asn Gln Arg Asp Ala Asp Gln1 5 10 15Thr Phe Gln Tyr Phe Arg Gln Ser
Leu Pro Glu Ser Tyr Ser Glu Ala 20 25 30Ser Asn Ala Pro Glu Asn Met
Met Val Phe Met Asn Tyr Ser Ser Ser35 40 45Gly Ala Tyr Ser Asp Met
Leu Thr Gly Thr Ser Gln Gln Gln His Asn50 55 60Cys Ile Asp Ile Pro
Ser Ile Gly Ala Thr Pro Phe Asn Thr Ser Gln65 70 75 80Gln Glu Ile
Leu Ser Asn Leu Gly Gly Ser Gln Met Gly Ile Gln Asp 85 90 95Phe Ser
Ser Trp Arg Asp Ser Arg Asn Glu Met Leu Ala Asp Asn Val 100 105
110Phe Gln Val Ala Gln Asn Val Gln Gly Gln Gly Leu Ser Leu Ser
Leu115 120 125Gly Ser Asn Ile Pro Ser Gly Ile Gly Ile Ser His Val
Gln Ser Gln130 135 140Asn Pro Asn Gln Gly Gly Gly Phe Asn Met Ser
Phe Gly Asp Gly Asp145 150 155 160Asn Ser Gln Pro Lys Glu Gln Arg
Asn Ala Asp Tyr Phe Pro Pro Asp 165 170 175Asn Pro Gly Arg Asp Leu
Asp Ala Met Lys Gly Tyr Asn Ser Pro Tyr 180 185 190Gly Thr Ser Ser
Ile Ala Arg Thr Ile Pro Ser Ser Lys Tyr Leu Lys195 200 205Ala Ala
Gln Tyr Leu Leu Asp Glu Val Val Ser Val Arg Lys Ala Ile210 215
220Lys Glu Gln Asn Ser Lys Lys Glu Leu Thr Lys Asp Ser Arg Glu
Ser225 230 235 240Asp Val Asp Ser Lys Asn Ile Ser Ser Asp Thr Pro
Ala Asn Gly Gly 245 250 255Ser Asn Pro His Glu Ser Lys Asn Asn Gln
Ser Glu Leu Ser Pro Thr 260 265 270Glu Lys Gln Glu Val Gln Asn Lys
Leu Ala Lys Leu Leu Ser Met Leu275 280 285Asp Glu Ile Asp Arg Arg
Tyr Arg Gln Tyr Tyr His Gln Met Gln Ile290 295 300Val Val Ser Ser
Phe Asp Val Val Ala Gly Glu Gly Ala Ala Lys Pro305 310 315 320Tyr
Thr Ala Leu Ala Leu Gln Thr Ile Ser Arg His Phe Arg Cys Leu 325 330
335Arg Asp Ala Ile Cys Asp Gln Ile Arg Ala Ser Arg Arg Ser Leu Gly
340 345 350Glu Gln Asp Ala Ser Glu Asn Ser Lys Ala Ile Gly Ile Ser
Arg Leu355 360 365Arg Phe Val Asp His His Ile Arg Gln Gln Arg Ala
Leu Gln Gln Leu370 375 380Gly Met Met Gln Gln His Ala Trp Arg Pro
Gln Arg Gly Leu Pro Glu385 390 395 400Ser Ser Val Ser Val Leu Arg
Ala Trp Leu Phe Glu His Phe Leu His 405 410 415Pro Tyr Pro Lys Asp
Ser Asp Lys Ile Met Leu Ala Arg Gln Thr Gly 420 425 430Leu Thr Arg
Ser Gln Val Ser Asn Trp Phe Ile Asn Ala Arg Val Arg435 440 445Leu
Trp Lys Pro Met Val Glu Glu Met Tyr Lys Glu Glu Ala Gly Asp450 455
460Ala Lys Ile Asp Ser Asn Ser Ser Ser Asp Val Ala Pro Arg Leu
Ala465 470 475 480Thr Lys Asp Ser Lys Val Glu Glu Arg Gly Glu Leu
His Gln Asn Ala 485 490 495Ala Ser Glu Phe Glu Gln Tyr Asn Ser Gly
Gln Ile Leu Glu Ser Lys 500 505 510Ser Asn His Glu Ala Asp Val Glu
Met Glu Gly Ala Ser Asn Ala Glu515 520 525Thr Gln Ser Gln Ser Gly
Met Glu Asn Gln Thr Gly Glu Pro Leu Pro530 535 540Ala Met Asp Asn
Cys Thr Leu Phe Gln Asp Ala Phe Val Gln Ser Asn545 550 555 560Asp
Arg Phe Ser Glu Phe Gly Ser Phe Gly Ser Gly Asn Val Leu Pro 565 570
575Asn Gly Val Ser Leu Thr Leu Gly Leu Gln Gln Gly Glu Gly Ser Asn
580 585 590Leu Pro Met Ser Ile Glu Thr His Val Ser Tyr Val Pro Leu
Arg Ala595 600 605Asp Asp Met Tyr Ser Thr Ala Pro Thr Thr Met Val
Pro Glu Thr Ala610 615 620Glu Phe Asn Cys Leu Asp Ser Gly Asn Arg
Gln Gln Pro Phe Trp Leu625 630 635 640Leu Pro Ser Ala Thr
645157PRTSolanum tuberosum 15Val Ser Leu Thr Leu Gly Leu1
5161383DNASolanum tuberosum 16gagtttctct cccttttaaa aaagaaaaaa
aaaacacaac acccacttca aatatcaaac 60aaatttctca tttgattatt tctaagtgat
ttacactact ttgtattttt gtttgttttt 120ttttagatat atatatggat
gatgaaatgt atggttttca ttcaacaaga gacgattacg 180cggataaagc
tttgatgtca ccggagaatt tgatgatgca aactgagtac aacaatttcc
240acaactatac caactcgtcc atcttgactt ctaatccgat gatgtttgga
tccgatgata 300ttcaattatc atcggaacaa actaattctt tcagtactat
gactcttcaa aataatgata 360atatttatca aattagaagt ggaaattgtg
gcggaggcag tggcagtggt ggtagcagta 420aggatcataa tgataataac
aataataatg aagattatga tgaagatggt tcaaatgtta 480tcaaggctaa
aatcgtctca catccttatt atcctaaatt actcaacgct tatattgatt
540gccaaaaggt tggagcacca gcgggtatag taaatctgct ggaagaaata
aggcaacaaa 600ctgattttcg taaaccaaac gctacttcta tatgtatagg
agctgatcct gaacttgatg 660agtttatgga aacgtattgt gatatattgt
tgaagtataa gtccgatctg tctaggcctt 720ttgatgaagc aacaacgttc
ctcaacaaga ttgaaatgca actaggtaat ctttgcaaag 780atgatggtgg
tgtatcatca gatgaggagt taagttgtgg tgaggcagat gcatcaatga
840gaagtgagga taatgaactc aaagatagac tcctacgtaa gtttggaagt
catttaagta 900gtctaaagtt ggaattttca aagaaaaaga agaaagggaa
gctaccaaaa gaggcaaggc 960aaatgttact tgcatggtgg gatgatcact
ttagatggcc ttaccctacg gaggctgata 1020agaattcact agcagaatca
acaggacttg atccaaagca gatcaacaat tggtttataa 1080atcaaaggaa
gagacattgg aaaccatcag agaatatgca gttagctgtt atggataatc
1140taagctctca gttcttctca tcagatgatt gagtttgaat ggaaattgtg
aaaatactgc 1200tcttcatttc tctttttatt atatataata tataaatagt
atatttttgg gaaagaaaga 1260agttatttta ttaatcaaaa tctctataaa
taatggtaga gattaattaa tgttgaattc 1320ttcttgatca tgtaaatatt
caatctagct aattgtcaaa attaatgctt acctaaaaaa 1380aaa
138317345PRTSolanum tuberosum 17Met Asp Asp Glu Met Tyr Gly Phe His
Ser Thr Arg Asp Asp Tyr Ala1 5 10 15Asp Lys Ala Leu Met Ser Pro Glu
Asn Leu Met Met Gln Thr Glu Tyr 20 25 30Asn Asn Phe His Asn Tyr Thr
Asn Ser Ser Ile Leu Thr Ser Asn Pro35 40 45Met Met Phe Gly Ser Asp
Asp Ile Gln Leu Ser Ser Glu Gln Thr Asn50 55 60Ser Phe Ser Thr Met
Thr Leu Gln Asn Asn Asp Asn Ile Tyr Gln Ile65 70 75 80Arg Ser Gly
Asn Cys Gly Gly Gly Ser Gly Ser Gly Gly Ser Ser Lys 85 90 95Asp His
Asn Asp Asn Asn Asn Asn Asn Glu Asp Tyr Asp Glu Asp Gly 100 105
110Ser Asn Val Ile Lys Ala Lys Ile Val Ser His Pro Tyr Tyr Pro
Lys115 120 125Leu Leu Asn Ala Tyr Ile Asp Cys Gln Lys Val Gly Ala
Pro Ala Gly130 135 140Ile Val Asn Leu Leu Glu Glu Ile Arg Gln Gln
Thr Asp Phe Arg Lys145 150 155 160Pro Asn Ala Thr Ser Ile Cys Ile
Gly Ala Asp Pro Glu Leu Asp Glu 165 170 175Phe Met Glu Thr Tyr Cys
Asp Ile Leu Leu Lys Tyr Lys Ser Asp Leu 180 185 190Ser Arg Pro Phe
Asp Glu Ala Thr Thr Phe Leu Asn Lys Ile Glu Met195 200 205Gln Leu
Gly Asn Leu Cys Lys Asp Asp Gly Gly Val Ser Ser Asp Glu210 215
220Glu Leu Ser Cys Gly Glu Ala Asp Ala Ser Met Arg Ser Glu Asp
Asn225 230 235 240Glu Leu Lys Asp Arg Leu Leu Arg Lys Phe Gly Ser
His Leu Ser Ser 245 250 255Leu Lys Leu Glu Phe Ser Lys Lys Lys Lys
Lys Gly Lys Leu Pro Lys 260 265 270Glu Ala Arg Gln Met Leu Leu Ala
Trp Trp Asp Asp His Phe Arg Trp275 280 285Pro Tyr Pro Thr Glu Ala
Asp Lys Asn Ser Leu Ala Glu Ser Thr Gly290 295 300Leu Asp Pro Lys
Gln Ile Asn Asn Trp Phe Ile Asn Gln Arg Lys Arg305 310 315 320His
Trp Lys Pro Ser Glu Asn Met Gln Leu Ala Val Met Asp Asn Leu 325 330
335Ser Ser Gln Phe Phe Ser Ser Asp Asp 340
3451820DNAartificialPrimer 18aagaagaaga agaaagggaa
201917DNAartificialPrimer 19atgaaccagt tgttgat 172010DNASolanum
tuberosum 20ttgacttgac 102124DNAartificialPrimer 21ggatccttga
agtggctctt ctct 242225DNAartificialPrimer 22aatctagaga cactctcttt
ttcgt 252324DNAartificialPrimer 23ctatttgact tcacacggtt attt
242424DNAartificialPrimer 24aaataaccgt gtgaagtcaa atag
24258DNASolanum tuberosum 25tgacagst 8269DNASolanum tuberosum
26tgacttgac 9279DNASolanum tuberosum 27tgaswtgac 92810DNASolanum
tuberosum 28tgattgacag 10
* * * * *
References