Grant Agency of the Academy of Sciences

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 Rho- related 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.

Working hypothesis and aims of the project

Our working hypothesis is that, like in other eukaryotes, exocyst complex participates in the localization of secretion/exocytosis to specific membrane domains in plant cells. Since all eight plant exocyst subunits are homologous to yeast and mammalian ones, we suspect that the plant exocyst is also structurally similar to its mammalian and yeast counterpart. However, the presence of so many paralogues of the EXO70-like genes raises several questions, which are the main subject of this project.

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 cDNA (accession Insertional mutant number) number) AtEXO70-A1 At5g03540 V AL162751 .1 AY072155.1 SALK_014826 F12E4.320 AY133751.1 AtEXO70-A2 At5g52340 V AB019226.1 FLAG_264F01 K24M7.7 AtEXO70-A3 At5g52350 V AB019226 .1 SALK_046855 K24M7.8 AtEXO70-B1 At5g58430 V AB025632 .1 AY094480.1 GABI_156G02 MCK7.32 BT000827.1 AtEXO70-B2 At1g07000 I AC067971 .5 AY075660.1 Garlic_752_F02 F10K1.28 AY101526.1 Garlic_621_A03 AtEXO70-C1 At5g13150 V AL391711 .1 GABI_334D05 T19L5.110 GABI_336E08 AtEXO70-C2 At5g13990 V AB005230. 2 BT003863.1 Garlic_147_D04 MAC12.17 AtEXO70-D1 At1g72470 I AC016529 .7 SALK_074641 T10D10.6 SALK_074650 SALK_074660 AtEXO70-D2 At1g54090 I AC006577 .2 AY093196.1 SALK_003651 F15I1.17 AtEXO70-D3 At3g14090 III AP000600 .1 AY056159.1 MAG2.5 (partial cDNA) AtEXO70-E1 At3g29400 III AP001309 .1 SALK_084145 MUO10.14 AtEXO70-E2 At5g61010 V AB006696 .1 AY050411.1 MSL3.1 AY059656.1 AtEXO70-F1 At5g50380 V AB012248 .1 SALK_036927 MXI22.10 AtEXO70-G1 At4g31540 IV AL080283 .1 AY139762.1 SALK_056871 F3L17.110 AL161579.2 SALK_019409 SALK_048154 AtEXO70-G2 At1g51640 I AC025294 .14 SALK_067521 F19C24.13 AtEXO70-H1 At3g55150 III AL132954 .1 AY074262.1 T26I12.30 AY096453.1 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://flagdb-genoplante- info.infobiogen.fr/projects/fst/) (FLAG lines) or the MPI for Plant Breeding Research, Collone (GABI lines). 1. It is unknown whether all plant Exo70 homologues act as subunits of the exocyst complex or if they may be bifunctional (see the possible function of the yeast Exo70 in chromatin) or even specialized for different function.

2. We anticipate that the A subfamily of Exo70 isoforms is the most likely candidate for the authentic exocyst subunit, as Exo70-A is most similar at the sequences from other eukaryotes. Therefore, we will start with the Exo70-A subfamily.

3. However, if most or all Exo70 isoforms represent genuine exocyst subunits, we expect that they will have a crucial role in the specification of subcellular localization of the plant exocyst.

In order to get insight into these questions, we will select one isoform as an example from each Arabidopsis Exo70 subfamily and attempt their basic characterization with respect to subcellular localization, tissue/developmental specificity of expression, interacting proteins and mutant phenotype.

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 constructs 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 Agrobacterium- mediated 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. 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.

III. 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 anti- Sec6 polyclonal mouse antibodies to study Exo70 containing complexes (see Preliminary data).

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 (2nd year results). 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.

References

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