U.S. patent application number 13/060911 was filed with the patent office on 2011-06-30 for the an3 protein complex and its use for plant growth promotion.
This patent application is currently assigned to BASF Plant Science Company GmbH. Invention is credited to Geert De Jaeger, Dirk Inze, Aurine Verkest.
Application Number | 20110162110 13/060911 |
Document ID | / |
Family ID | 41258864 |
Filed Date | 2011-06-30 |
United States Patent
Application |
20110162110 |
Kind Code |
A1 |
De Jaeger; Geert ; et
al. |
June 30, 2011 |
THE AN3 Protein Complex and its Use for Plant Growth Promotion
Abstract
The present invention relates to an AN3-based protein complex.
It relates further to the use of the complex to promote plant
growth, and to a method for stimulating the complex formation, by
overexpressing at least two member of the complex.
Inventors: |
De Jaeger; Geert; (Evergem,
BE) ; Verkest; Aurine; (Gent, BE) ; Inze;
Dirk; (Moorsel-Aalst, BE) |
Assignee: |
BASF Plant Science Company
GmbH
Ludwigshafen
DE
|
Family ID: |
41258864 |
Appl. No.: |
13/060911 |
Filed: |
August 31, 2009 |
PCT Filed: |
August 31, 2009 |
PCT NO: |
PCT/EP2009/061206 |
371 Date: |
February 25, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61190543 |
Aug 29, 2008 |
|
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|
Current U.S.
Class: |
800/290 ;
435/188; 530/370 |
Current CPC
Class: |
C12N 15/8216 20130101;
C12N 15/8261 20130101; Y02A 40/146 20180101 |
Class at
Publication: |
800/290 ;
530/370; 435/188 |
International
Class: |
A01H 1/06 20060101
A01H001/06; C07K 14/415 20060101 C07K014/415; C12N 9/96 20060101
C12N009/96 |
Claims
1. An isolated AN3-based protein complex, comprising at least the
proteins AN3p and one or more proteins encoded by AT4G16143,
AT1G09270, AT3G06720, AT5G53480, AT3G60830, AT1G18450, AT2G46020,
AT2G28290, AT1G21700, AT5G14170, AT4G17330, AT4G27550, AT1G65980,
AT5G55210, AT3G15000, AT4G35550, AT1G20670, AT1G08730, AT5G13030,
AT2G18876, AT5G17510, AT1G05370, AT4G21540, AT1G23900 or AT5
G23690.
2. An isolated AN3-based protein complex comprising at least the
proteins AN3p and one or more proteins selected from the group
consisting of ARP4 (AT1G18450), ARP7 (AT3G60830), SNF2 (AT2G46020),
SYD (AT2G28290), SWI3C (AT1G21700) and SWP73B (AT5G14170).
3. The isolated AN3-based protein complex of claim 2, wherein said
protein complex comprises at least AN3p, an actin related protein
selected from the group consisting of ARP4 and ARP7, an ATPase
selected from the group consisting of SNF2 (BRM) and SYD, and a
SWIRM domain containing protein.
4. The isolated AN3-based protein complex of claim 3, wherein said
SWIRM domain containing protein is SWI3C.
5. A method of promoting plant growth comprising simultaneously
overexpressing at least two proteins of the complex of claim 1.
6. A method to promote AN3-based protein complex formation
comprising simultaneously ovexpressing at least two proteins of the
complex.
Description
[0001] The present invention relates to an AN3-based protein
complex. It relates further to the use of the complex to promote
plant growth, and to a method for stimulating the complex
formation, by overexpressing at least two members of the
complex.
[0002] The demand for more plant derived products has spectacularly
increased. In the near future the challenge for agriculture will be
to fulfill the growing demands for feed and food in a sustainable
manner. Moreover plants start to play an important role as energy
sources. To cope with these major challenges, a profound increase
in plant yield will have to be achieved. Biomass production is a
multi-factorial system in which a plethora of processes are fed
into the activity of meristems that give rise to new cells,
tissues, and organs. Although a considerable amount of research on
yield performance is being performed little is known about the
molecular networks underpinning yield (Van Camp, 2005). Many genes
have been described in Arabidopsis thaliana that, when mutated or
ectopically expressed, result in the formation of larger
structures, such as leaves or roots. These so-called "intrinsic
yield genes" are involved in many different processes whose
interrelationship is mostly unknown.
[0003] One of these "intrinsic yield genes", AN3 (also known as
GIF1), was identified in search of GRF (growth regulating factor)
interactors (Kim and Kende, 2004) and by analysis of narrow-leaf
Arabidopsis mutants (Horiguchi et al., 2005). AN3 is a homolog of
the human SYT (synovial sarcoma translocation) protein and is
encoded by a small gene family in the Arabidopsis genome. SYT is a
transcription co-activator whose biological function, despite the
implication of its chromosomal translocation in tumorigenesis, is
still unclear (Clark et al., 1994; de Bruijn et al., 1996). Using
the yeast GAL4 system, AN3 was shown to possess transactivation
activity (Kim and Kende, 2004). This together with yeast two-hybrid
and in vitro binding assays demonstrating interaction of AN3 with
several GRFs (Kim and Kende, 2004; Horiguchi et al., 2005),
suggests a role of AN3 as transcription co-activator of GRFs. GRF
(growth regulating factor) genes occur in the genomes of all seed
plants thus far examined and encode putative transcription factors
that play a regulatory role in growth and development of leaves
(Kim et al., 2003). In support of a GRF and AN3 transcription
activator and co-activator complex, grf and an3 mutants display
similar phenotypes, and combinations of grf and an3 mutations
showed a cooperative effect (Kim and Kende, 2004). The an3 mutant
narrow-leaf phenotype is shown to result of a reduction in cell
numbers. Moreover, ectopic expression of AN3 resulted in transgenic
plants with larger leaves consisting of more cells, indicating that
AN3 controls both cell number and organ size (Horiguchi et al.,
2005). Although the function of AN3 in plant growth regulation is
not known, these results show that AN3 fulfills the requirements of
an "intrinsic yield gene".
[0004] In our ambition to decipher the molecular network
underpinning yield enhancement mechanism a genome-wide protein
centered approach was undertaken to study AN3 interacting proteins
in Arabidopsis thaliana cell suspension cultures. The tandem
affinity purification (TAP) technology combined with mass
spectrometry (MS) based protein identification resulted in the
isolation and identification of 25 AN3 interacting proteins that
may function in the regulation of plant growth (Table 2).
Surprisingly, we isolated several proteins belonging to
multiprotein complexes. Moreover, many interactors are completely
uncharacterized. Reports on few of the AN3 interactors show that
they are implicated in several developmental processes (Wagner
& Meyerowitz, 2002; Meagher et al., 2005; Sarnowski et al.,
2005; Hurtado et al., 2006; Kwon et al., 2006) but so far none of
the identified genes have been associated with stimulation of plant
growth.
[0005] A first aspect of the invention is an isolated AN3-based
protein complex, comprising at least the proteins AN3p and one or
more of the proteins selected from the group encoded by AT4G16143,
AT1G09270, AT3G06720, AT5G53480, AT3G60830, AT1G18450, AT2G46020,
AT2G28290, AT1G21700, AT5G14170, AT4G17330, AT4G27550, AT1G65980,
AT5G55210, AT3G15000, AT4G35550, AT1G20670, AT1G08730, AT5G13030,
AT2G 18876, AT5G17510, AT1G05370, AT4G21540, AT1G23900 and
AT5G23690 (genes listed in Table II). Preferably, said AN3-based
protein complex comprises at least the proteins AN3p and one or
more proteins selected from the group consisting of ARP4
(AT1G18450), ARP7 (AT3G60830), SNF2 (AT2G46020), SYD (AT2G28290),
SWI3C (AT1G21700) and SWP73B (AT5G14170). Even more preferably,
said AN3-based protein complex comprises at least AN3p, an actin
related protein selected from the group consisting of ARP4 and
ARP7, an ATPase selected from the group consisting of SNF2 (BRM)
and SYD and a SWIRM domain containing protein. Preferably, said
SWIRM domain containing protein is SWI3C. An AN3-based protein
complex as used here means that AN3p is interacting, directly or
indirectly, with the other proteins of the complex. A direct
interaction is an interaction where at least one domain of AN3p
interacts with one or more domains or the interaction partner. An
indirect interaction is an interaction where AN3p itself is not
interacting with the interacting protein by one of its domains, but
where said interacting protein is interacting with a protein that
is directly or indirectly interacting with AN3p.
[0006] A further aspect of the invention is the use of a protein
complex according to the invention to promote plant growth.
Preferably, said use is an overexpression of the protein complex,
by overexpressing at least two members of the protein complex.
Promotion of plant growth, as used here, is an increase in plant
biomass in plants where the protein complex is used, compared with
the same plant where the complex is not used, grown under the same
conditions, except for the conditions needed for the use of the
complex, if any. Such conditions may be, as a non limited example,
the addition of one or more compounds to induce one or more
promoters of one or more genes encoding a protein of the complex.
Alternatively, the same plant is an untransformed parental plant,
grown under the same conditions as the transformed plant, wherein
the complex is used. Preferably, promotion of plant growth results
in an increased yield. This yield can be a total increase in plant
biomass, or a partial increase of yield, such as, but not limited
to seed yield, leave yield or root yield.
[0007] Still another aspect of the invention is a method to promote
AN3-based protein complex formation, by simultaneous overexpression
of at least two proteins of the complex. Proteins of the complex,
beside AN3p itself, are listed in table II. Preferably, said
overexpression is an overexpression of AN3p and one or more
proteins selected from the group consisting of ARP4 (AT1G18450),
ARP7 (AT3G60830), SNF2 (AT2G46020), SYD (AT2G28290), SWI3C
(AT1G21700) and SWP73B (AT5G14170). Even more preferably, said
overexpression is an overexpression of at least AN3p, an actin
related protein selected from the group consisting of ARP4 and
ARP7, an ATPase selected from the group consisting of SNF2 (BRM)
and SYD and a SWIRM domain containing protein. Preferably, said
SWIRM domain containing protein is SWI3C.
[0008] Methods for obtaining overexpression are known to the person
skilled in the art, and comprise, but are not limited to placing
the gene encoding the protein to be overexpressed after a strong
promoter such as the Cauliflower Mosaic Virus 35S promoter.
Simultaneous overexpression as used here means that there is an
overlap in timeframe for all the proteins to be overexpressed,
whereby the level of said proteins is increased when compared to a
non-overexpressed control. It does not necessarily mean that all
genes should be induced at the same moment. Depending upon the
turnover of the messenger RNA and/or the protein, one gene may be
induced before or after another, as long as there is an overlap in
time where both proteins are present in a concentration that is
higher than the normal (non-overexpressed) concentration.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1. Expression analysis of GS-tagged GFP and AN3 in
transgenic cell suspension cultures.
[0010] The total protein extract of 2-day-old wild-type and N- and
C-terminal GS-tagged GFP and AN3 overexpressing cultures (60 .mu.g)
was separated by 12% SDS-PAGE and immunoblotted. For detection of
GS-tagged proteins, blots were incubated with human blood plasma
followed by incubation with anti-human IgG coupled to horseradish
peroxidase. Protein gel blots were developed by Chemiluminiscent
detection. The expected recombinant molecular masses for GS-tagged
GFP and AN3 are 52.8 kDa and 43.5 kDa, respectively (indicated with
a black dot).
[0011] FIG. 2. Analysis of the TAP protein eluates.
[0012] GS-tagged protein complexes were purified from transgenic
plant cell suspension cultures, precipitated with TCA (25%, v/v),
separated on 4-12% NuPAGE gels, and visualized with colloidal
Coomassie G-250 staining. Bait proteins are indicated with a
dot.
EXAMPLES
Materials and Methods to the Examples
Vector Construction
[0013] Construction of N- and C-terminal GS-tagged GFP and AN3
under the control of the 35S (CaMV) promoter was obtained by
Multisite Gateway LR reactions. The coding regions, without (-) and
with (+) stopcodon, were amplified by polymerase chain reaction
(PCR) and cloned into the Gateway pDONR221 vector (Invitrogen)
resulting in pEntryL1L2-GFP(-) pEntryL1L2-GFP(+), pEntryL1L2-AN3(-)
and pEntryL1L2-AN3(+). The Pro.sub.35S:GFP-GS- and
Pro.sub.35S:AN3-GS-containing plant transformation vectors were
obtained by Multisite Gateway LR reaction between
pEntryL4R1-Pro.sub.35S, pEntryL1L2-GFP(-) or pEntryL1L2-AN3(-), and
pEntryR2L3-GS and the destination vector pKCTAP, respectively (Van
Leene et al., 2007). To obtain the Pro35S:GS-GFP and Pro35S:GS-AN3
vectors Multisite LR recombination between pEntryL4L3-Pro.sub.35S
and pEntryL1L2-GFP(+) or pEntryL1L2-AN3(+) with pKNGSTAP
occurred.
[0014] All entry and destination vectors were checked by sequence
analysis. Expression vectors were transformed to Agrobacterium
tumefaciens strain C58C1 Rif.sup.R (pMP90) by electroporation.
Transformed bacteria were selected on yeast extract broth plates
containing 100 .mu.g/mL rifampicin, 40 .mu.g/mL gentamicin, and 100
.mu.g/mL spectinomycin.
Cell Suspension Cultivation
[0015] Wild-type and transgenic Arabidopsis thaliana cell
suspension PSB-D cultures were maintained in 50 mL MSMO medium
(4.43 g/L MSMO, Sigma-Aldrich), 30 g/L sucrose, 0.5 mg/L NAA, 0.05
mg/L kinetin, pH 5.7 adjusted with 1M KOH) at 25.degree. C. in the
dark, by gentle agitation (130 rpm). Every 7 days the cells were
subcultured in fresh medium at a 1/10 dilution.
Cell Culture Transformation
[0016] The Arabidopsis culture was transformed by Agrobacterium
co-cultivation as described previously (Van Leene et al., 2007).
The Agrobacterium culture exponentially growing in YEB (OD.sub.600
between 1.0 and 1.5) was washed three times by centrifugation (10
min at 5000 rpm) with an equal volume MSMO medium and resuspended
in cell suspension growing medium until an OD.sub.600 of 1.0. Two
days after subcultivation, 3 mL suspension culture was incubated
with 200 .mu.L washed Agrobacteria and 200 .mu.M acetoseringone,
for 48 h in the dark at 25.degree. C. with gentle agitation (130
rpm). Two days after co-cultivation, 7 mL MSMO containing a mix of
three antibiotics (25 .mu.g/mL kanamycin, 500 .mu.g/mL
carbenicellin, and 500 .mu.g/mL vancomycin) was added to the cell
cultures and grown further in suspension under standard conditions
(25.degree. C., 130 rpm and continuous darkness). The stable
transgenic cultures were selected by sequentional dilution in a 1:5
and 1:10 ratio in 50 mL fresh MSMO medium containing the
antibiotics mix, respectively at 11, and 18 days post
co-cultivation. After counter selecting the bacteria, the
transgenic plant cells were further subcultured weekly in a 1:5
ratio in 50 mL MSMO medium containing 25 .mu.g/mL kanamycin for two
more weeks. Thereafter the cells were weekly subcultured in fresh
medium at a 1/10 dilution.
Expression Analysis of Cell Suspension Cultures
[0017] Transgene expression was analyzed in a total protein extract
derived from exponentially growing cells, harvested two days after
subculturing. Equal amounts of total protein were separated on 12%
SDS-PAGE gels and blotted onto Immobilon-P membranes (Millipore,
Bedford, Mass.). Protein gel blots were blocked in 3% skim milk in
20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Triton X-100. For
detection of GS-tagged proteins, blots were incubated with human
blood plasma followed by incubation with anti-human IgG coupled to
horseradish peroxidase (HRP; GE-Healthcare). Protein gel blots were
developed by Chemiluminiscent detection (Perkin Elmer, Norwalk,
Conn.).
Protein Extract Preparation
[0018] Cell material (15 g) was grinded to homogeneity in liquid
nitrogen. Crude protein extract were prepared in an equal volume
(w/v) of extraction buffer (25 mM Tris-HCl, pH 7.6, 15 mM
MgCl.sub.2, 5 mM EGTA, 150 mM NaCl, 15 mM p-nitrophenylphosphate,
60 mM .beta.-glycerophosphate, 0.1% (v/v) Nonidet P-40 (NP-40), 0.1
mM sodium vanadate, 1 mM NaF, 1 mM DTT, 1 mM PMSF, 10 .mu.g/mL
leupeptin, 10 .mu.g/mL aprotinin, 5 .mu.g/mL antipain, 5 .mu.g/mL
chymostatin, 5 .mu.g/mL pepstatin, 10 .mu.g/mL soybean trypsin
inhibitor, 0.1 mM benzamidine, 1 .mu.M
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), 5%
(v/v) ethylene glycol) using an Ultra-Turrax T25 mixer (IKA Works,
Wilmington, N.C.) at 4.degree. C. The soluble protein fraction was
obtained by a two-step centrifugation at 36900 g for 20 min and at
178000 g for 45 min, at 4.degree. C. The extract was passed through
a 0.45 .mu.m filter (Alltech, Deerfield, Ill.) and the protein
content was determined with the Protein Assay kit (Bio-Rad,
Hercules, Calif.).
Tandem Affinity Purification
[0019] Purifications were performed as described by Burckstummer et
al. (2006), with some modifications. Briefly, 200 mg total protein
extract was incubated for 1 h at 4.degree. C. under gentle rotation
with 100 .mu.L IgG Sepharose 6 Fast Flow Flow beads (GE-Healthcare,
Little Chalfont, UK), pre-equilibrated with 3 mL extraction buffer.
The IgG Sepharose beads were transferred to a 1 mL Mobicol column
(MoBiTec, Goettingen, Germany) and washed with 10 mL IgG wash
buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% NP-40, 5%
ethylene glycol) and 5 mL Tobacco (Nicotiana tabacum L.) Etch Virus
(TEV) buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% (v/v)
NP-40, 0.5 mM EDTA, 1 mM PMSF, 1 .mu.M E64, 5% (v/v) ethylene
glycol). Bound complexes were eluted via AcTEV digest
(2.times.100U, Invitrogen) for 1 h at 16.degree. C. The IgG eluted
fraction was incubated for 1 h at 4.degree. C. under gentle
rotation with 100 .mu.L Streptavidin resin (Stratagene, La Jolla,
Calif.), pre-equilibrated with 3 mL TEV buffer. The Streptavidin
beads were packed in a Mobicol column, and washed with 10 mL TEV
buffer. Bound complexes were eluted with 1 mL streptavidin elution
buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% (v/v) NP-40, 0.5
mM EDTA, 1 mM PMSF, 1 .mu.M E64, 5% (v/v) ethylene glycol, 20 mM
Desthiobiotin), and precipitated using TCA (25% v/v). The protein
pellet was washed twice with ice-cold aceton containing 50 mM HCl,
redissolved in sample buffer and separated on 4-12% gradient NuPAGE
gels (Invitrogen). Proteins were visualized with colloidal
Coomassie brilliant blue staining.
Proteolysis and Peptide Isolation
[0020] After destaining, gel slabs were washed for 1 hour in
H.sub.2O, polypeptide disulfide bridges were reduced for 40 min in
25 mL of 6.66 mM DTT in 50 mM NH.sub.4HCO.sub.3 and sequentially
the thiol groups were alkylated for 30 min in 25 mL 55 mM IAM in 50
mM NH.sub.4HCO.sub.3. After washing the gel slabs 3 times with
water, complete lanes from the protein gels were cut into slices,
collected in microtiter plates and treated essentially as described
before with minor modifications (Van Leene et al., 2007). Per
microtiterplate well, dehydrated gel particles were rehydrated in
20 .mu.L digest buffer containing 250 ng trypsin (MS Gold; Promega,
Madison, Wis.), 50 mM NH.sub.4HCO.sub.3 and 10% CH.sub.3CN (v/v)
for 30 min at 4.degree. C. After adding 10 .mu.L of a buffer
containing 50 mM NH.sub.4HCO.sub.3 and 10% CH.sub.3CN (v/v),
proteins were digested at 37.degree. C. for 3 hours. The resulting
peptides were concentrated and desalted with microcolumn solid
phase tips (PerfectPure.TM. C18 tip, 200 nL bed volume; Eppendorf,
Hamburg, Germany) and eluted directly onto a MALDI target plate
(Opti-TOF.TM. 384 Well Insert; Applied Biosystems, Foster City,
Calif.) using 1.2 .mu.L of 50% CH.sub.3CN: 0.1% CF.sub.3COOH
solution saturated with .alpha.-cyano-4-hydroxycinnamic acid and
spiked with 20 fmole/.mu.L Glu1-Fibrinopeptide B (Sigma-Aldrich),
20 fmole/.mu.L des-Pro2-Bradykinin (Sigma-Aldrich), and 20
fmole/.mu.L Adrenocorticotropic Hormone Fragment 18-39 human
(Sigma-Aldrich).
Acquisition of Mass Spectra
[0021] A MALDI-tandem MS instrument (4800 Proteomics Analyzer;
Applied Biosystems) was used to acquire peptide mass fingerprints
and subsequent 1 kV CID fragmentation spectra of selected peptides.
Peptide mass spectra and peptide sequence spectra were obtained
using the settings essentially as presented in Van Leene et al.
(2007). Each MALDI plate was calibrated according to the
manufacturers' specifications. All peptide mass fingerprinting
(PMF) spectra were internally calibrated with three internal
standards at m/z 963.516 (des-Pro2-Bradykinin), m/z 1570.677
(Glu1-Fibrinopeptide B), and m/z 2465,198 (Adrenocorticotropic
Hormone Fragment 18-39) resulting in an average mass accuracy of 5
ppm.+-.10 ppm for each analyzed peptide spot on the analyzed MALDI
targets. Using the individual PMF spectra, up to sixteen peptides,
exceeding a signal-to-noise ratio of 20 that passed through a mass
exclusion filter were submitted to fragmentation analysis.
MS-Based Protein Homology Identification
[0022] PMF spectra and the peptide sequence spectra of each sample
were processed using the accompanied software suite (GPS Explorer
3.6, Applied Biosystems) with parameter settings essentially as
described in Van Leene et al. (2007). Data search files were
generated and submitted for protein homology identification by
using a local database search engine (Mascot 2.1, Matrix Science).
An in-house nonredundant Arabidopsis protein database called SNAPS
Arabidopsis thaliana version 0.4 (SNAPS=Simple Nonredundant
Assembly of Protein Sequences, 77488 sequence entries, 30468560
residues; available at http://www.ptools.ua.ac.be/snaps) was
compiled from nine public databases. Protein homology
identifications of the top hit (first rank) with a relative score
exceeding 95% probability were retained. Additional positive
identifications (second rank and more) were retained when the score
exceeded the 98% probability threshold.
Example 1
Expression Analysis of GS-Tagged GFP and AN3 Overexpressing Cell
Lines
[0023] Before performing TAP purifications stably transformed cell
suspension cultures were screened on the protein expression level
of the transgenes. Protein gel blotting of equal amounts of total
protein extract derived from wild-type (PSB-D) cultures and GS-GFP,
GFP-GS, GS-AN3, and AN3-GS overexpressing cell lines showed clear
expression of the GS-tagged proteins (FIG. 1).
Example 2
TAP Purification of Wild-Type and GS-Tagged GFP Overexpressing
Cultures
[0024] Despite the two successive purification steps performed
within TAP purifications, background proteins co-purified by
non-specific binding are an issue. Contaminating proteins due to
experimental background were determined by purifications on
wild-type and transgenic cultures overexpressing N- and C-terminal
GS-tagged nuclear localized green fluorescent protein (GFP).
Non-specific co-purified proteins were precipitated, separated on
gel, stained (FIG. 2), trypsin digested and identified
unambiguously by MALDI-TOF/TOF. Most contaminants are high abundant
proteins, such as chaperones, cytoskeleton proteins, ribosomal
proteins, metabolic enzymes, or protein translation factors (Table
1). Identical or similar proteins were found as common contaminants
in other plant protein-protein interaction studies (Rohila et al.,
2006; Van Leene et al., 2007).
Example 3
TAP Isolation and MS Identification of AN3 Interacting Proteins
[0025] In order to identify the interaction partners of AN3 in
vivo, we performed tandem affinity (TAP) purifications on N- and
C-terminal GS-fusions of AN3 ectopically expressed under control of
the constitutive 35SCaMV promoter in transgenic Arabidopsis
suspension cultures. Two independent TAP purifications were
performed on extracts from AN3-GS and GS-AN3 lines, harvested two
days after sub-culturing into fresh medium. The affinity purified
proteins were separated on a 4-12% NuPAGE gel and stained with
Coomassie Brilliant Blue. The purification profiles from transgenic
cultures overexpressing AN3 is shown in FIG. 2. Protein bands were
cut, in-gel digested with trypsin and subjected to MALDI-TOF/TOF
mass spectrometry for protein identification. After substracting
background proteins, identified by the control purifications
described in example 2 and in other analyses (GUS and cytosolic
GFP, Van Leene et al., 2007), from the obtained hit list we
identified 25 AN3 interacting proteins (Table 2). These can be
divided into two groups: 14 proteins were confirmed experimentally
and 11 proteins were identified only in one out of four TAP
experiments.
Example 4
Isolation and Subunit Identification of AN3 Interacting SWI/SNF
Chromatin Remodeling Complexes in Plants
[0026] Among the experimentally confirmed AN3 interactors six
proteins act as subunits of macromolecular machines that remodel
chromatin structure. A database survey (ChromDB, Gendler et al.,
2008) illustrates that all of them belong to the SWI/SNF ATPase
family. SWI/SNF chromatin remodeling ATPases are conserved in the
animal and the plant kingdom and regulate transcriptional programs
in response to endogenous and exogenous cues. This suggests that
the transcriptional activity of AN3 is regulated through chromatin
remodeling. In agreement, the human AN3 homolog SYT was also shown
to interact with the SWI/SNF complex components BRM and Brg1
(Thaete et al., 1999; Perani et al., 2003; Ishida et al.,
2004).
[0027] Although the functional role of several putative SWI/SNF
complex components has been studied in Arabidopsis, so far no
complete plant chromatin remodeling complex has been isolated and
characterized. The co-purification with AN3 gives for the first
time prove of the in vivo physical composition of plant SWI/SNF
complexes which before was based solely on homology analyses and
the interpretation of genetic and in vitro interactions. A
literature survey illustrates that SWI/SNF ATPase subunits control
multiple developmental pathways in Arabidopsis. Null mutants of the
two isolated ATPases SYD (At2g28290) and BRM (SNF2) (At2g46020)
display pleiotropic developmental defects. Both mutants are slow
growing and dwarfed, have defects in cotyledon separation, and
exhibit reduced apical dominance (Wagner & Meyerowitz, 2002;
Farrona et al., 2004; Hurtado et al., 2006; Kwon et al., 2006; Su
et al., 2006). Null mutants in BRM (SNF2) also have unique root
growth defects and are male sterile (Wagner & Meyerowitz, 2002;
Hurtado et al., 2006; Kwon et al., 2006). Core complex Swi3c
(At1g21700) mutants closely resemble brm mutants (Sarnowski et al.,
2005). Mutants of the accessory components ARP4 and ARP7 display
pleiotropic defects with less resemblance to the syd, brm and swi3c
phenotypes (Meagher et al., 2005). Down-regulation of ARP4 resulted
in phenotypes including altered organization of plant organs, early
flowering, delayed flower senescence and partial sterility
(Kandasamy et al., 2005a). ARP7 knockdown results in dwarfed plants
with small rosette leaves, highly retarded root growth, altered
flower development and reduced fertility (Kandasamy et al., 2005b).
Finally, RNAi-mediated silencing of the accessory SWI/SNF complex
component SWP73B (At5g14170) resulted in dwarfed plants with
shorter roots (Crane & Gelvin, 2007).
Example 5
Isolation and Identification of AN3 Interactors
[0028] With the exception of the SWI/SNF chromatin remodeling
complex subunits all other 19 identified AN3 interactors are not or
poorly characterized. Table 3 gives an overview of there GO
biological process and molecular function.
[0029] Among them four interactors (At4g16143, At1g09270, At3g06720
and At5g53480) are involved in nucleocytoplasmic trafficking which
identifies AN3 as one of the targets of plant nuclear transporters.
Indeed a precise cellular localization is essential for protein
function and nuclear localization is a key to the function of
transcription factors. In plants, nucleocytoplasmic trafficking
plays a critical role in various biological processes (Meier, 2007;
Xu & Meier, 2008) and nuclear transporters have been shown to
be involved in regulating different signal transduction pathways
during plant development (Bollman et al., 2003) and in plant
responses to biotic (Palma et al., 2005) and abiotic stresses
(Verslues et al., 2006).
[0030] Another AN3 interactor, that is yet not characterized, is
the trehalose phosphatase/synthase 4 (TPS4). Several studies in
plants imply an important role of trehalose biosynthesis for plant
growth, development and stress tolerance (Grennan, 2007). In the
case of Arabidopsis TPS1, knockout mutants display an embryo lethal
phenotype, suggesting a role of this gene in plant development
(Eastmond et al., 2002). In addition, overexpression of TPS1 shed
light on its role as a regulator of glucose, abscisic acid, and
stress signalling (Avonce et al., 2004). The latter study, together
with a recent analysis of a rice TPS triggering abiotic stress
response gene induction when overexpressed (Ge et al., 2008),
suggests a possible role for TPS genes in regulating
transcriptional signaling pathways.
[0031] The other identified interactors indicate links of AN3
function in multiple processes. Several studies demonstrate the
involvement of sphingosine kinases in plant cell signaling (Coursol
et al., 2003; Coursol et al., 2005; Worral et al., 2008), whereas
reports on myosin homologues (Peremyslov et al., 2008; Jiang et
al., 2007) implicate roles of protein and organelle trafficking in
plant development. The connections between these genes, the other
identified interactors and AN3 will be interesting to study in the
future.
TABLE-US-00001 TABLE 1 List of co-purifying proteins during TAP
experiments of untransformed cell cultures, and of cultures
ectopically expressing nuclear localized GFP Accession number
Protein name Mock GFP At1g06780 glycosyl transferase family 8
protein + At1g07930 elongation factor 1-alpha + At1g09080 luminal
binding protein 3 (BiP-3) (BP3) + At1g13440 glyceraldehyde
3-phosphate dehydrogenase, cytosolic, + At1g31230 bifunctional
aspartate kinase/homoserine dehydrogenase + At1g34610 Ulp1 protease
family protein + At1g50010 tubulin alpha chain + At1g61210 WD-40
repeat family protein/katanin p80 subunit, putative + At1g75010
MORN repeat-containing protein + At1g79920 heat shock protein 70,
putative + At1g79930 heat shock protein, putative + At2g07620
putative helicase + At2g21410 vacuolar proton ATPase, putative +
At2g26570 expressed protein + At3g07160 glycosyl transferase family
48 protein + At3g09170 Ulp1 protease family protein + At3g09440
heat shock cognate 70 kDa protein 3 + At3g11950 ATHST;
prenyltransferase + At3g12580 heat shock protein 70, putative +
At3g17390 S-adenosylmethionine synthetase, putative + At3g18530
expressed protein + At3g26020 serine/threonine protein phosphatase
2A regulatory subunit B' + At3g42100 AT hook motif-containing
protein-related + At3g48870 ATP-dependent Clp protease ATP-binding
subunit (ClpC) + At3g49640 nitrogen regulation family protein +
At3g54940 cysteine proteinase, putative + At4g00020 BRCA2A (breast
cancer 2 like 2A) + At4g09800 40S ribosomal protein S18 + At4g14960
tubulin alpha chain + At4g18080 hypothetical protein + At4g20160
expressed protein + At4g20890 tubulin beta chain + At4g31820
phototropic-responsive NPH3 family protein + At4g33200 myosin,
putative + At5g02490 heat shock cognate 70 kDa protein 2 +
At5g02500 heat shock cognate 70 kDa protein 1 + At5g08670 ATP
synthase beta chain, mitochondrial + At5g08680 ATP synthase beta
chain, mitochondrial + At5g08690 ATP synthase beta chain,
mitochondrial + At5g09810 actin 7 (ACT7)/actin 2 + + At5g18110
Novel cap-binding protein (nCBP) + At5g28540 luminal binding
protein 1 (BiP-1) (BP1) + + At5g35360 acetyl-CoA carboxylase,
biotin carboxylase subunit (CAC2) + At5g40060 disease resistance
protein (TIR-NBS-LRR class), putative + At5g42020 luminal binding
protein 2 (BiP-2) (BP2) + At5g44340 tubulin beta chain + At5g60390
elongation factor 1-alpha + At5g62700 tubulin beta chain +
TABLE-US-00002 TABLE 2 List of AN3-copurified proteins identified
by MS. The last column tells in how many of the four independent
experiments an interactor was identified. Protein Best ion MW
Peptide score/ score/ AGI code Description (kDa) count threshold
threshold AT4G16143 importin alpha-2, putative (IMPA2) 49.5 13
388/61 84/28 2 AT1G09270 importin alpha-1 subunit, putative (IMPA4)
59.4 6 74/61 37/31 1 AT3G06720 importin alpha-1 subunit, putative
(IMPA1) 58.6 8 160/61 62/28 2 AT5G53480 importin beta-2, putative
96.2 16 295/61 50/32 2 AT3G60830 actin-related protein 7 (ARP7)
39.9 12 285/61 53/28 3 AT1G18450 actin-related protein 4 (ARP4)
48.9 12 230/61 44/28 2 AT2G46020 transcription regulatory protein
SNF2 (ATPase) 245.4 31 351/61 57/31 2 AT2G28290 chromatin
remodeling protein, SYD ATPase 389.8 22 118/61 53/31 4 AT1G21700
SWIRM domain-containing protein/DNA-binding family protein 88.2 5
32/32 2 AT5G14170 SWIB complex BAF60b domain-containing protein
59.2 18 302/61 43/31 2 AT4G17330 G2484-1, agenet (tudor-like)
domain-containing protein 113.3 25 317/61 61/32 3 AT4G27550
trehalose phosphatase/synthase 4 89.4 15 68/61 2 AT1G65980
thioredoxin-dependent peroxidase 17.4 8 80/61 2 AT5G55210 expressed
protein 18.5 4 105/61 49/31 2 AT3G15000 expressed protein similar
to DAG protein 42.8 3 38/30 2 AT4G35550 homeobox-leucine zipper
protein (HB-2)/HD-ZIP protein 29.6 3 33/28 1 AT1G20670 DNA-binding
bromodomain-containing protein 72.9 16 75/61 1 AT1G08730 myosin
heavy chain (PCR43) (Fragment) 174.6 18 70/61 1 AT5G13030 expressed
protein 71.1 3 31/29 1 AT2G18876 expressed protein 43.5 11 67/61 1
AT5G17510 expressed protein 42.5 3 37/28 1 AT1G05370 expressed
protein 49.9 12 66/61 1 AT4G21540 putative sphingosine kinase
(SphK) 141.7 9 69/61 1 AT1G23900 gamma-adaptin 96.4 19 78/61 1
AT5G23690 polynucleotide adenylyltransferase family protein 59.6 11
66/61 1
TABLE-US-00003 TABLE 3 AGI Code Name/Description GO Biological
Process GO Molecular Function At4g16143 Importin alpha-2 (IMP2)
Protein import into nucleus Protein transporter activity At1g09270
Importin alpha-1 (IMPA4) Intracellular protein transport Protein
transporter activity At3g06720 Importin alpha-1 (IMPA1)
Intracellular protein transport Protein transporter activity
At5g53480 Importin beta-2 Protein import into nucleus Protein
transporter activity At4g17330 G2484-1 protein unknown RNA binding
At4g27550 Trehalose phosphatase/synthase 4 Trehalose biosynthesis
Trehalose phosphate (TPS4) synthase activity At1g65980
Thioredoxin-dependent peroxidase 1 unknown Antioxidant activity
(TPX1) At5g55210 Expressed protein unknown unknown At3g15000
Expressed protein similar to DAG unknown unknown protein At4g35550
Wuschel-related homeobox 13 Regulation of transcription DNA binding
(WOX13) At1g20670 Bromodomain-containing protein unknown DNA
binding At1g08730 Myosin-like protein XIC Actin filament-based
movement Protein binding At5g13030 Expressed protein unknown
unknown At2g18876 Expressed protein unknown unknown At5g17510
Expressed protein unknown unknown At1g05370 Expressed protein
unknown unknown At4g21540 Putative sphingosine kinase Activation of
protein kinase C Kinase activity activity At1g23900 Gamma-adaptin
Vesicle-mediated transport Clathrin binding At5g23690
Polynucleotide adenylyltransferase RNA processing RNA binding
protein
REFERENCES
[0032] Avonce N, Leyman B, Mascorro-Gallardo J O, Van Dijck P,
Thevelein J M, Iturriaga G (2004) The Arabidopsis trehalose-6-P
synthase AtTPS1 gene is a regulator of glucose, abscisic acid, and
stress signaling. Plant Physiol 136: 3649-3659 [0033] Bollman K M,
Aukerman M J, Park M-Y, Hunter C, Berardini T Z, Poethig R S (2003)
HASTY, the Arabidopsis ortholog of expotin 5/MSN5, regulates phase
change and morphogenesis. Development 130: 1439-1504 [0034]
Burckstummer T, Bennett K L, Preradovic A, Schutze G, Hantschel O,
Superti-Firga G, Bauch A (2006) An efficient tandem affinity
purification procedure for interaction proteomics in mammalian
cells. Nat Methods 3: 1013-1019. [0035] Clark J, Rocques P J, Crew
A J, Gill S, Shipley J, Chan A M, Guterson B A, Cooper C S (1994)
Identification of novel genes, SYT and SSX, in the
t(X;19)(p11.2;q11.2) translocation found in human synovial sarcoma.
Nat Genet. 7: 502-508 [0036] Coursol S, Fan L M, Le Stunff H,
Spiegel S, Gilroy S, Assmann S M (2003) Sphingolipid signalling in
Arabidopsis guard cells involves heterotrimeric G proteins. Nature
423: 651-654 [0037] Coursol S, Le Stunff H, Lynch D V, Gilroy S,
Assmann S M, Spiegel S (2005) Arabidopsis sphingosine kinase and
the effects of phytosphingosine-1-phosphate on stomatal aperture.
Plant Physiol 137: 724-737 [0038] Crane Y M, Gelvin S B (2007)
RNAi-mediated gene silencing reveals involvement of Arabidopsis
chromatin-related genes in Agrobacterium-mediated root
transformation. Proc Natl Acad Sci USA 104: 15156-15161 [0039] de
Bruijn D R, Baats E, Zechner U, de Leeuw B, Balemans M, Olde
Weghuis D, Hirning-Folz U, Geurts van Kessel A G (1996) Isolation
and characterization of the mouse homolog of SYT, a gene implicated
in the development of human synovial sarcomas. Oncogene 13: 643-648
[0040] Eastmond P J, van Dijken A J H, Spielman M, Kerr A, Tissier
A F, Dickinson H G, Jones J D G, Smeekens S C, Graham I A (2002)
Trehalose-6-phosphate synthase 1, which catalyses the first step in
trehalose synthesis, is essential for Arabidopsis embryo
maturation. Plant J 29: 225-235 [0041] Farrona S, Hurtado L, Bowman
J L, Reyes J C (2004) The Arabidopsis thaliana SNF2 homolog AtBRM
controls shoot development and flowering. Development 131:
4964-4795 [0042] Ge L F, Chao D Y, Shi M, Zhu M Z, Gao J P, Lin H X
(2008) Overexpression of the trehalose-6-phosphate phosphatase gene
OsTPP1 confers stress tolerance in rice and results in the
activation of stress responsive genes. Planta 228: 191-201 [0043]
Gendler K, Paulsen T, Napoli C (2008) ChromDB: the chromatin
database. Nucleic Acids Res 36: D298-302 [0044] Grennan A K (2007)
The role of trehalose biosynthesis in plants 136: 3649-3659. [0045]
Horiguchi G, Kim G-T, Tsukaya H (2005) The transcription factor
AtGRF5 and the transcription coactivator AN3 regulate cell
proliferation in leaf primordial of Arabidopsis thaliana. Plant J
43: 68-78 [0046] Hurtado L, Farrona S, Reyes J C (2006) The
putative SWI/SNF complex subunit BRAHMA activates flower homeotic
genes in Arabidopsis thaliana. Plant Mol Biol 62: 291-304 [0047]
Ishida M, Tanaka S, Ohki M, Ohta T (2004) Transcriptional
co-activator activity of SYT is negatively regulated by BRM and
Brg1. Genes Cells 9: 419-428 [0048] Jiang S-Y, Cai M, Ramachandran
S (2007) ORYZA SATIVA MYOSIN XI B controls pollen development by
photoperiod-sensitive protein localizations. Developmental Biology
304: 579-592 [0049] Kandasamy M K, Deal R B, McKinney E C, Meagher
R B (2005a) Silencing the nuclear actin-related AtARP4 in
Arabidopsis has multiple effects on plant development, including
early flowering and delayed floral senescence. Plant J 41: 845-858
[0050] Kandasamy M K, McKinney E C, Deal R B, Meagher R B (2005b)
Arabidopsis ARP7 is an essential actin-related protein required for
normal embryogenesis, plant architecture, and floral organ
abscission. Plant Physiol 138: 2019-2032 [0051] Kim J H, Choi D,
Kende H (2003) The AtGRF family of putative transcription factors
is involved in leaf and cotyledon growth in Arabidopsis. Plant J
36: 94-104 [0052] Kim J H, Kende H (2004) A transcriptional
coactivator, AtGIF1, is involved in regulating leaf growth and
morphology in Arabidopsis. Proc Natl Acad Sci USA 1001: 13374-13379
[0053] Kwon C S, Hibara K-I, Pfluger J, Bezhani S, Metha H, Aida M,
Tasaka M, Wagner D (2006) A role for chromatin remodeling in
regulation of CUC gene expression in the Arabidopsis cotyledon
boundary. Development 133: 3223-3230 [0054] Meagher R B, Deal R B,
Kandasamy M K, McKinney E C (2005) Nuclear actin-related proteins
as epigenetic regulators of development. Plant Physiol 139:
1579-1585 [0055] Meier I (2007) Composition of the plant nuclear
envelope: theme and variations. J Exp Bot 58: 27-34 [0056] Palma K,
Zhang Y, Li X (2005) An importin alpha homolog, MOS6, plays an
important role in plant innate immunity. Curr Biol 15: 1129-1135
[0057] Perani M, Ingram C J, Cooper C S, Garrett M D, Goodwin G H
(2003) Conserved SNH domain of the proto-oncoprotein SYT interacts
with components of the human chromatin remodeling complexes, while
the QPGY repeat domain forms homo-oligomers. Oncogene 22: 8156-8167
[0058] Peremyslov V V, Prokhnevsky A I, Avisar D, Dolja V V (2008)
Two class XI myosins function in organelle trafficking and root
hair development in Arabidopsis. Plant Phys 146:1109-1116 [0059]
Rohila J S, Chen M, Chen S, Chen J, Cerny R, Dardick C, Canlas P,
Xu X, Gribskov M, Kanrar S, Zhu J-K, Ronald P, Fromm M E (2006)
Protein-protein interactions of tandem affinity purification tagged
protein kinases in rice. Plant J 46: 1-13 [0060] Sarnowski T J,
Rios G, Jasik J, Swiezewski S, Kaczanowski S, Li Y, Kwiatkowska A,
Pawlikowska K, Kozbial M, Koncz C, Jerzmanowski A (2005) SWI3
subunits of putative SWI/SNF chromatin-remodeling complexes play
distinct roles during Arabidopsis development. Plant Cell 17:
2454-2472 [0061] Su Y, Kwon C S, Bezhani S, Huvermann B, Chen C,
Peragine A, Kennedy J F, Wagner D (2006) The N-terminal ATPase
AT-hook-containing region of the Arabidopsis chromatin-remodeling
protein SPLAYED is sufficient for biological activity. Plant J 46:
685-699 [0062] Thaete C, Brett D, Monaghan P, Whitehouse S, Rennie
G, Rayner E, Cooper C S, Goodwin G (1999) Functional domains of the
SYT and SYT-SSX synovial sarcoma translocation proteins and
co-localization with the SNF protein BRM in the nucleus. Hum Mol
Genet. 8: 585-591 [0063] Van Camp W (2005) Yield enhancement genes:
seeds for growth. Curr Opin Biotech 16: 147-153 [0064] Van Leene J,
Stals H, Eeckhout D, Persiau G, Van De Slijke E, Van Isterdael G,
De Clercq A, Bonnet E, Laukens K, Remmerie N, Henderickx K, De
Vijlder T, Abdelkrim A, Pharazyn A, Van Onckelen H, Inze D, Witters
E, De Jaeger G (2007) A tandem affinity purification-based
technology platform to study the cell cycle interactome in
Arabidopsis thaliana. Mol Cell Proteomics 6: 1226-1238 [0065]
Verslues P E, Gou Y, Dong C-H, Ma W, Zhu J-K (2006) Mutation of
SAD2, an importin beta-domain protein in Arabidopsis, alters
abscisic acid sensitivity. Plant J 47: 776-787 [0066] Wagner D,
Meyerowitz E M (2002) SPLAYED, a novel SWI/SNF ATPase homolog,
controls reproductive development in Arabidopsis. Current Biol 12:
85-94 [0067] Worrall D, Liang Y K, Alvarez S, Holroyd G H, Spiegel
S, Panagopulos M, Gray J E, Hetherington A M (2008) Involvement of
sphingosine kinase in plant cell signalling. Plant J (Epub ahead of
print) [0068] Xu X M, Meier I (2008) The nuclear pore comes to the
fore. Trends Plant Sci 13: 20-27
* * * * *
References