Sheet B
GRANT AGENCY OF THE ACADEMY OF SCIENCES
Justification of the proposal
(minimum 3 pages, maximum 10 pages)
Introduction - Secretory pathway and the regulation of plant development
The secretory apparatus of plant cells, being for many years neglected as a "boring" executor of cell wall excretion,
became now rapidly very popular among plant physiologists as soon as its functioning was intimately linked to the
morphogenetic role of auxin. There is at present strong evidence that polar transport and distribution of IAA within a plant
are driven by polar distribution of IAA influx and efflux carriers within the cell plasma membrane (recently reviewed in
Friml 2003). Both exocytosis and endocytosis participate in carriers recycling (Geldner et al. 2003). Likewise, cloning of
mutant loci of knolle (syntaxin) and gnom (ARF GEF) embryonic mutants of Arabidopsis in the Tübingen laboratory of
Gerd Jürgens brought the focus of plant developmental biology to the importance of vectorial vesicle transport and cell wall
deposition for correct plant morphogenesis, and demonstrated how secretory pathways and IAA gradients are intimately
linked together (Geldner et al. 2003). However, at present we have very scarce knowledge about the molecular mechanisms
guiding the dynamics of plant endomembrane system and even less is known about the process of polarized and localized
secretion in plants. Genomic analyses suggest that the basic mechanisms of vesicle trafficking machinery are conserved
between plants and other eukaryotes.
Many proteins have been functionally connected with vesicular transport, owing mainly to genetic studies in budding
yeast and biochemical investigations in mammalian cells. Two major classes of proteins have been found to ensure
specificity of vesicle destination to target membranes, proteins from the SNARE superfamily and small GTPases from the
RAB family. Experimental studies and genomic searches indicate that SNAREs and RABs are ubiquitous components of all
eukaryotes that are probably functionally conserved. Distinct members of RAB and SNARE families seem to regulate
distinct steps of the secretory and endocytic pathways.
We and others have shown that RAB GTPases are active in plants along with their regulators (Moore et al. 1997,
Zarsky et al. 1997, Rutherford and Moore 2002). The whole set of SNAREs and associated proteins are also present in
plants and their role e.g. in vacuole biogenesis, gravitropism or cytokinesis has been studied in some detail (Sanderfoot and
Raikhel 2003, Muller et al. 2003).
The exocyst – the tethering complex for localized exocytosis
Another class of proteins involved in the spatial specificity of vesicle targeting – the so-called tethering factors – is
emerging from recent studies. They are believed to tether transport vesicles to their target membranes, acting there as
effectors of a RAB GTPase specific for the given transport step. In contrast to SNAREs and RABs, different tethering
factors bear a little resemblance to one another.
The only tethering/docking complex described so far in plant cells resides on the vacuole and seems to be homologous
to the C type VPS system (or the HOPS complex) from yeast and mammals (Rojo et al. 2003).
The exocyst, sometimes referred to as the sec6/8 complex, is a conserved protein complex comprising eight distinct
subunits and experimentally characterized from yeast and mammalian cells (reviewed e.g. in Hsu et al. 1999). Six of many
identified SEC loci (Novick et al. 1980), SEC3, SEC5, SEC6, SEC8, SEC10, and SEC15, turned out to encode subunits of a
protein complex containing two additional subunits Exo70p and Exo84p (TerBush et al., 1996, Guo et al. 1999a). Based on
sequence similarity to the yeast proteins, the mammalian complex was purified and proved to contain eight proteins
orthologous to the yeast subunits (Kee et al. 1997, Matern et al. 2001). The exocyst localizes to specific domains of the
plasma membrane characterized by the local maxima of secretion, e.g. the very tip of the emerging bud or the neck region
during cytokinesis in yeast, and the region of tight junctions in mammalian epithelial cells. At least in yeast the exocyst
functions as an effector of an exocytosis-specific RAB GTPase (Sec4) and probably tethers secretory vesicles to the plasma
membrane prior to the formation of the SNARE complex involved in the actual membrane fusion (Guo et al. 1999b).
Therefore, the exocyst in concert with the actin cytoskeleton governed by RHO and RAB GTPases is believed to be
responsible for localized secretory vesicle fusion (e.g. Novick and Guo 2002).
The only published attempt to identify the potential plant components of the exocyst using a complementation screen of
the Arabidopsis cDNA library was unsuccessful. Only a non-related suppressor complementing the sec15-1 yeast mutation
and representing the RING finger-containing E3 ubiquitin ligase was found (Matsuda and Nakano, 1998; Matsuda et al.
2001).
Contemporary knowledge of the Exo70 subunit is of the utmost importance for our project. Apart from the interactions
within the exocyst complex, Exo70 was shown to communicate in yeast with two RHO GTPases, Rho3 and Rho4,
preferentially in their GTP-bound form (Robinson et al 1999, Adamo et al. 1999). Very recently, an exciting observation
led to the discovery of a RHO GTPase interacting with the mammalian Exo70. Inoue et al. (2003) showed that the Rhorelated TC10 GTPase activated by an insulin-triggered signaling cascade recruits Exo70 to the plasma membrane, where a
multiprotein complex is assembled that includes also other exocyst subunits. This leads to targeting of the glucose
transporter Glut4-containing vesicles to the plasma membrane (Inoue et al. 2003).
There are preliminary reports pointing to other Exo70-interacting proteins in yeast and mammals, opening also the
possibility that Exo70 may have a role in the nucleus as a chromatin component (e.g. BIND database at http://bind.ca complex 11988).
Our preliminary data
Sequencing of the Arabidopsis and rice genomes offered a great opportunity to search for possible plant exocyst
subunits taking advantage of the bioinformatic approach. Indeed, our BLAST searches of the plant sequence data confirmed
that plant genomes do code for proteins obviously homologous to all eight known exocyst subunits (Cvrckova et al 2001,
Elias 2002, Elias et al. 2003). These homologues are discernible in sequences from angiosperms, gymnosperms as well as
the moss Physcomitrella patens. On the other hand, analysis of the recently completed draft genome sequence of the green
alga Chlamydomonas reinhardtii revealed that the exocyst complex is probably absent from this flagellate (Elias,
unpublished). Work in our laboratory is in progress, aimed at purification and functional characterization of the exocyst in
Arabidopsis thaliana.
Published data and our own searches of genome sequences from various eukaryotes indicate that individual exocyst
subunits are encoded by single-copy genes in most organisms.
In angiosperms, the situation is strikingly different. For instance, in Arabidopsis, only the Sec6, Sec8 and Sec10 subunits
are represented as single-copy genes. On the other side, there are two paralogues of each SEC3, SEC5 and SEC15, and three
paralogues of EXO84 in the Arabidopsis genome. Plant genomes are known to contain multiple paralogues, often as a result
of specific mode of plant genome evolution comprising rounds of polyploidisation followed by a massive gene loss. It
seems, however, that this explanation is not relevant with respect to the multiplicity of the exocyst genes in plants. For
instance, phylogenetic analysis indicates that the multiplication of the SEC15 and EXO84 genes is ancient and occurred
prior to the divergence of lineages leading to current dicots (Arabidopsis) and monocots (rice). It is tempting to speculate
that the paralogous genes have been kept to fulfil different or at least partially non-overlapping functions. In contrast, the
two SEC3 paralogues in Arabidopsis are very recent, as they are almost identical in sequence and the two genes form a
tandem duplication on the chromosome I that may have arisen by a recent event of non-reciprocal crossing-over. We infer
that the two Arabidopsis SEC3 genes are probably to a large extent functionally redundant.
We started to clone exocyst subunits from Arabidopsis and characterize them in the framework of our project within the
research center “Signaling pathways in plants”, which is due to be terminated next year. We have concentrated first mostly
upon the Sec6 subunits, prepared antibodies and found that it is expressed and membrane-bound during pollen tube growth
(Elias et al. 2003).
The Exo70 subunit
Surprisingly, we found that the plant EXO70 family, in pronounced contrast to other eukaryotes, comprises more than 20
paralogues in one angiosperm plant species, as exemplified by both Arabidopsis and rice. An overview of the Arabidopsis
EXO70 family is provided in Table 1. The large EXO70 family indeed seems to be idiomatic to plants (Cvrckova et al.
2001, Elias et al. 2003). In Arabidopsis it is possible to define 8, and in rice even 9, different EXO70 subfamilies (fig. 1.).
During the aforementioned research center project we were able just to open the topic. We cloned one member of the
Arabidopsis Exo70 family, AtExo70-G1, and studied its intracellular localization using transient expression assays of a
GFP-fusion in tobacco leaves and protoplasts. It localizes to distinct patches mostly at the plasmalemma, but the signal (in
contrast to GFP-AtSec10 as analyzed by confocal microscopy) is present also in the cytoplasm and the nucleus (Elias et al.
2003; Drdova, unpublished). We used the same construct in our first attempt to transiently express the exocyst subunits by
micro-projectile bombardment (Bio-Rad DNA gun in the laboratories of the Institute of Experimental Botany in Olomouc).
Preliminary data show a very restricted cortical localization of AtExo70-G1 in onion skin cells. Preparation of a
recombinant AtExo70-G1 protein, which will be used for immunization of mice or rabbits in order to obtain polyclonal
antibodies, is in progress.
Fig. 1. Phylogenetic analysis of the plant Exo70 protein family.
The tree was constructed using the neighbour-joining method based on the well-conserved regions of
a multiple alignment of deduced protein sequences of Arabidopsis and rice Exo70 homologues.
Bootstrap values (in percentages, computed from 500 replicates) are given only for nodes with >50%
support. The main nine Exo70 subgroups are indicated by letters A-J.
gene
locus
chromosome
gene (accession
number)
cDNA (accession
number)
Insertional mutant
AtEXO70-A1
At5g03540
V
AL162751 .1
AY072155.1
SALK_014826
F12E4.320
AY133751.1
AtEXO70-A2
At5g52340
V
AB019226.1
FLAG_264F01
K24M7.7
Working hypothesis and aims of
the project
AtEXO70-A3
At5g52350
V
AB019226 .1
SALK_046855
K24M7.8
Our working
hypothesis isAt5g58430
that, like in otherV eukaryotes, AB025632
exocyst .1complex participates
localization of
AtEXO70-B1
AY094480.1 in theGABI_156G02
MCK7.32
BT000827.1
secretion/exocytosis
to specific At1g07000
membrane domains inI plant cells. Since
all eight plant AY075660.1
exocyst subunits are
homologous to
AtEXO70-B2
AC067971 .5
Garlic_752_F02
AY101526.1
Garlic_621_A03
yeast and mammalian ones, weF10K1.28
suspect that the plant exocyst is also structurally similar
to its mammalian
and yeast
AtEXO70-C1
At5g13150
V
AL391711 .1
GABI_334D05
T19L5.110
GABI_336E08
counterpart. However, the presence
of so many paralogues of the EXO70-like genes raises several questions,
which are the
AtEXO70-C2
At5g13990
V
AB005230. 2
BT003863.1
Garlic_147_D04
main subject of this project.
MAC12.17
AtEXO70-D1
At1g72470
I
AC016529 .7
SALK_074641
T10D10.6
SALK_074650
1)
It is unknown whether all plant Exo70 homologues act as subunits of the exocyst complex orSALK_074660
if they may be
AtEXO70-D2(see the possible
At1g54090function of theI yeast Exo70 AC006577
.2
AY093196.1
SALK_003651
bifunctional
in chromatin)
or even
specialized for different
function.
F15I1.17
AtEXO70-D3
At3g14090
III
AP000600 .1
AY056159.1
cDNA)for the authentic exocyst
2)
We anticipate that theMAG2.5
A subfamily of Exo70 isoforms is the most likely (partial
candidate
AtEXO70-E1
At3g29400
III
AP001309 .1
SALK_084145
subunit, as Exo70-A MUO10.14
is most similar at the sequences from other eukaryotes. Therefore, we will start with the
AtEXO70-E2
At5g61010
V
AB006696 .1
AY050411.1
Exo70-A subfamily. MSL3.1
AY059656.1
AtEXO70-F1
At5g50380
V
AB012248 .1
SALK_036927
3)
However, if most or MXI22.10
all Exo70 isoforms represent genuine exocyst subunits, we expect that they will have a
AtEXO70-G1
At4g31540
IV
AL080283 .1
AY139762.1
SALK_056871
F3L17.110 of subcellular localization AL161579.2
SALK_019409
crucial role in a specification
of the plant exocyst. In order to get insight
into these
SALK_048154
questions,
we will select
one isoform as anI example from
each Arabidopsis
Exo70 subfamily and
attempt their
AtEXO70-G2
At1g51640
AC025294
.14
SALK_067521
F19C24.13
basic
characterizationAt3g55150
with respect to subcellular
localization,
AtEXO70-H1
III
AL132954tissue/developmental
.1
AY074262.1specificity of expression,
T26I12.30
AY096453.1
interacting proteins and
mutant phenotype.
AtEXO70-H2
At2g39380
II
AC004218 .3
F12L6.4
AtEXO70-H3
At3g09530
III
AC016661 .7
SALK_034560
F11F8.11
SALK_034709
AtEXO70-H4
At3g09520
III
AC016661 .7
SALK_003200
F11F8.10
SALK_052208
AtEXO70-H5
At2g28640
II
AC007171 .5
SALK_007810
T8O18.7
AtEXO70-H6
At1g07725
I
AC007583 .2
SALK_016535
F24B9.17
SALK_016542
AtEXO70-H7
At5g59730
V
AB006705 .2
AY045671.1
SALK_009200
MTH12.6
AY060527.1
SALK_009208
AY034910.1
AY063096.1
AF360179.1
AtEXO70-H8
At2g28650
II
AC007171 .5
AK118418.1
SALK_014867
T8O18.6
SALK_018915
Table 1
Genes of the EXO70 family in Arabidopsis thaliana. All 23 paralogues are included and listed according to the proposed
nomenclature reflecting phylogenetic relationships (fig. 1.). Accession numbers refer to the international GenBank/EMBL/DDBJ
sequence database. cDNA clones are available from the Arabidopsis Biological Resource Center (http://arabidopsis.org/abrc/) or
the RIKEN Bio Resource Center (http://www.brc.riken.go.jp/lab/epd/Eng/index.html). The sixth column indicate identification
numbers of Arabidopsis lines potentially bearing a T-DNA insertion in the region of the respective gene. Seeds from these lines
are available from collections of the Nottingham Arabidopsis Stock Centre (http://nasc.nott.ac.uk/) (SALK lines), the Torrey Mesa
Research Institute (http://www.nadii.com/pages/collaborations/garlic_files/) (Garlic lines), the Versailles Collection (http://flagdbgenoplante-info.infobiogen.fr/projects/fst/) (FLAG lines) or the MPI for Plant Breeding Research, Collone (GABI lines).
Experimental plan:
We will combine molecular biological, biochemical, microscopical and genetic approaches. The experimental procedure
will be divided into several steps:
I. Preparation of DNA construct and antibodies.
To maximize efficiency of preparation of DNA constructs we will extensively take advantage of the GATEWAY
cloning technology. We will clone expression constructs for selected Exo70 isoforms and prepare recombinant proteins in
bacteria. Yeast and mammalian Exo70 subunits were shown to be well soluble proteins easy to isolate by affinity
purification. Polyclonal antibodies will be raised in mice and rabbits and after thorough characterization used for
immunolocalization and immunoprecipitation experiments. In parallel we will prepare constructs for Agrobacteriummediated stable and transient transformation of Arabidopsis and tobacco plants. The respective binary vectors compatible
with the GATEWAY technology are mostly available (e.g. http://www.psb.rug.ac.be/gateway/list-of-constructs.html).
Constructs for the 2-hybrid system will be made by the conventional cloning strategy.
II. Systematic study of plant Exo70-interacting proteins
a – Using the 2-hybrid system. The two-hybrid system in yeast will be used to study expected interactions with plant
members of the RHO family, Rop GTPases, and other exocyst subunits. New putative interactors will be detected by
screening publicly available Arabidopsis two-hybrid cDNA libraries.
b – Purification of Exo70-containing complexes from plant cells. Study of Exo70-containing complexes will be
attempted first using anti-GFP antibodies for immunoprecipitation from the experiments and plants planned in III-b.
At the same time we will prepare transgenic Arabidopsis plants or cell lines expressing selected Exo70 isoforms as
translational fusions with the TAP tag (Rigaut et al. 1999); the transgenic plant material will be used for tandem
affinity purification of Exo70-containing complexes as described by e.g. Gavin et al. (2002). If successful, we will
try to characterize subunit composition of the purified complexes using the MALDI-TOF approach (at the Dept. of
Biochemistry, Faculty of Sciences, Charles University - dr. Bezouška). We will also use previously prepared antiSec6 polyclonal mouse antibodies to study Exo70 containing complexes (see Preliminary data).
III. Study of the isoform-specific features of expression and subcellular localization
a – Promoter::GUS fusions. We will prepare promoter::GUS fusion transgenic Arabidopsis plants for selected
members from all 8 subgroups of plant EXO70s, starting with two AtEXO70-G1 and AtExo70-A1. Using
histochemical detection of GUS activity we will study tissue- and/or developmental-specific expression of EXO70s.
We will also collect publicly available data from DNA chips and microarrays (Affymetrix and co.)
b – Intracellular localization of Exo70. In a similar way we will prepare Exo70-GFP fusions in order to study specific
cellular localization of selected Exo70s using transient expression assays – especially upon infiltration of tobacco
leaves – and stably transformed Arabidopsis plants. In specific instances – especially with respect to the study of
Exo70s expression in pollen tubes – we will use DNA-biolistics (Biorad DNA gun available in Olomouc – see also
preliminary data). Using quantitative image analysis (Lucia) we will look particularly for possible differences in the
fluorescence distribution between the cytoplasm and nuclei. We will combine here fluorescence and confocal
microscopy. In parallel, we will prepare polyclonal antibodies against selected plant Exo70 recombinat proteins
expressed in bacteria, which could be used for indirect immunofluorescence localization of Exo70s;
immunolocalization will be attempted also with tagged proteins.
IV. Reverse-genetic analysis
We will study macroscopic and microscopic phenotypes of EXO70 insertional mutants available form public mutant
collections with already sequenced flanking regions (SALK, GARLIC etc., see also Table 1). If needed, we would also use
the opportunity to screen by PCR mutant collections of dr. C. Koncz in MPI (Köln, Germany) and the John Innes Center
(UK) collection (TIPNET collaboration with dr. Liam Dolan laboratory). We will prepare double or triple mutants by
crossing to overcome expected functional redundancy. The set of Exo70s used for GUS and GFP fusions will be also used
to prepare anti-sense Arabidopsis transgenic plants.
Time plan and distribution of work:
(IEB = Inst. of Exp. Bot; DPF = Dept. of Pl. Physiol.)
During the first two years the proposed project will be in parallel partially funded by the EU-RTN project TIPNET
devoted to the study of polarized growth of plant cells. This research and training project is providing a post-doc salary
(Antonio Torres) and mobility. That’s why we are not asking for travel money at the beginning of the project.
1.year - During the first year we will use the methodological (but not thematic!) overlap with the ending research center
program on the exocyst; therefore, we are asking less money for the first year. This will help us to keep the momentum in
this highly competitive field of plant cell inquiry. We will work (in both laboratories) on the cloning of expression,
transformation, GFP, 2-hybrid, RNAi and TAP-tagged Exo70 constructs. At the same time we will continuously work on
the completion of our collection of Arabidopsis EXO70 insertional mutants (at present we have 2 candidates in the lab). We
will start to transform plants as soon as the constructs are prepared – most of the stable transformation work will be done at
the IEB. Transient expressions of GFP fusions will be performed at both laboratories. We will use the confocal microscope
of our consorcium located in the Academy of Sciences campus in Krc, Prague.
2.year – Constructs and recombinant proteins from the first year will be used for custom preparation of polyclonal
antibodies. We will continue in transformation of Arabidopsis by appropriate constructs. Effort will be made especially to
use extensively GFP constructs in transient assays as the fastest way to address biological function of different Exo70
isoforms.
DPF laboratory will start to analyze tissue specific expression of EXO70 promoter::GUS fusions of first
transgenics. At the IEB 2-hybrid screen of Exo70 interactions within the exocyst as well as with new partners (using
Arabidopsis 2-hybrid libraries) will be initiated. We will continue in characterization of insertional Arabidopsis mutants
(IEB).
3.year – It is expected that this research area will be developing very fast, so we will not only continue in our experiments as
initiated during the first two years of work but we will most probably modify and specify our targets according to our own
data as well as those published by others. We should be able to select most promising Exo70s according to tissue specificity
discovered especially during the 2nd year. We will concentrate on a few selected contrasting cases in order to address the
question if differential expression is linked with tissue specificity or cellular localization or both. As a main focus we will
continue in using the TAP-tagged transgenic Arabidopsis plants to study Exo70-containing complexes by affinity
chromatography. If successful, we will attempt at the DPF the first MALDI-TOF analysis of purified proteins in
collaboration with dr. Bezouška from the Dept. of Biochemistry.
4.year – Based on the experience collected, we will continue with the emphasis on: 1. efforts to immunoprecipitate Exo70
complexes by well characterized and immuno-purified antibodies form the previous years (IEB mostly); 2. efforts to
localize Exo70 not only by GFP-fusions but also by immunofluorescence; 3. efforts to study co-localization of Exo70 and
other exocyst subunits by co-expression of spectral variants of GFP and differently labelled antibodies; 4. finalization of
characterization of mutant and RNAi plants.
Conditions for the implementation of the project
Both partner investigators are established scientists with teams of experienced young researchers. Both participating
laboratories are well equipped to run plant molecular biology research with the whole set of important techniques. The only
new techniques related to the project are routine use of GATEWAY system and TAP-tag affinity purification of protein
complexes. Extensive cloning related to the project will be distributed between both participating groups. IEB laboratory
will then be devoted more to the whole plant work and GFP localizations, while DPF laboratory will concentrate on protein
and biochemical techniques as affinity purification attempts etc.
Expected outcome
Apart from the contribution of this project to the basic plant cell science in the form of publications, four PhD students and
at least one undergraduate will participate on this project in the context of intensive interactions of EU laboratories.
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