Báo cáo khoa học: Expression of MsPG3-GFP fusions in Medicago truncatula Ôhairy rootsÕ reveals preferential tip localization of the protein in root hairs pot

Expression of MsPG3-GFP fusions in Medicago truncatula Ôhairy rootsÕ reveals preferential tip localization of the protein in root hairs Ignacio D. Rodrı ´ guez-Llorente 1 , Javier Pe ´ rez-Hormaeche 1 , Mohammed Dary 1 , Miguel A. Caviedes 1 , Adam Kondorosi 2,3 , Pascal Ratet 2 and Antonio J. Palomares 1 1 Departamento de Microbiologı ´ a y Parasitologı ´ a, Facultad de Farmacia, Universidad de Sevilla, Spain; 2 Institut des Sciences Ve ´ ge ´ tales, Centre National de la Recherche Scientifique, Gif sur Yvette, France; 3 Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary Tip growth is a specialized type of polar growth where new cell wall is deposited in a localized region of the cell, the growing tip. These cells show a characteristic zonation, with a high accumulation of secretory vesicles containing cell wall components at the tip, followed by an organelle-enriched zone. MsPG3 is a Medicago sativa polygalacturonase gene isolated in our laboratory, specifically expressed during the interaction of this plant with its symbiotic partner Sinorhiz- obium meliloti and which might participate in tip growth processes during symbiosis. We have used MsPG3-GFP fusions to study in vivo protein transport processes and localization during root hair growth. Different MsPG3-GFP fusions were expressed in Medicago truncatula Ôhairy rootsÕ following a protocol developed for this study and also tested by transient expression in onion epidermal cells. Preferential accumulation of an MsPG3-GFP fusion protein in the tip of the growing root hair at different developmental stages was found, confirming the delivery of MsPG3 to the newly synthesized cell wall. This indicates that this protein may participate in tip growth processes during symbiosis and, in addition, that this fusion could be a useful tool to study this process in plants. Keywords:GFP;hairyroot;Medicago truncatula; polygal- acturonase; tip growth. Plant cells grow either by diffuse growth, over a wide region, or by tip growth, limited to the apex. Tip growth is a specialized type of polar growth where new cell wall is deposited in a localized region of the cell, the growing tip. These cells show a characteristic zonation, with a high accumulation of secretory vesicles containing cell wall components at the tip, followed by an organelle-enriched zone [1]. Cell wall produced and deposited by tip growth is a mechanism used in various cellular systems including pollen tubes, fungal hyphae, developing root hairs and Rhizobium- induced infection threads. Pollen tubes have been used as a model system to investigate the tip growth process in plants [2]. More recently, root hairs in Arabidopsis have become a model system for tip growth [3]. The isolation and phenotypic characterization of mutants with defects in specific aspects of root hair growth has led to the definition of four stages in Arabidopsis root hair morphogenesis: the selection of a growing site, bulge formation, tip growth and polarized extension [3,4]. In the same way, four stages have been described in Vicia sativa spp. nigra L. (vetch) root hair development: bulging, growing, growth terminating and full growth hair [5]. While recent studies with Arabidopsis mutants have provided new insights into how the tip growth is governed [1], the mechanisms directing the growth specifically to the tip are still unknown. Only the importance of microtubules in this process has been described [6–8]. Polygalacturonases (PGs, EC 3.2.1.15) are cell-wall- degrading enzymes involved in the degradation of pectins that are complex polysaccharides found in the middle lamella and primary cell wall of higher plants. In a previous study [9], we characterized a Medicago sativa PG gene (MsPG3) specifically expressed during symbiosis with Sinorhizobium meliloti. Our results suggested that MsPG3 may participate in several steps of the infection process, including infection thread formation and reinitiation of the root hair tip growth induced by the Nod factor during the early steps of the plant–bacterial interaction [10,11]. The primary aim of this research was to study the cellular localization of the MsPG3 protein in vivo, using green fluorescent protein (GFP), in order to understand better its role during the early steps of symbiosis. GFP is commonly used for in vivo protein localization, as the mechanism of fluorophore formation, involving intramolecular autoxida- tion, does not require exogenous cofactors [12]. A major advantage of GFP is the maintenance of its fluorescence when fused to other proteins making it a very useful reporter protein. To test their functionality, MsPG3-GFP fusions were transiently expressed in cells from epidermal peels of onion (Allium cepa) [13]. In addition we have modified the protocol for the generation of transgenic Ôhairy rootsÕ for Correspondence to A. J. Palomares, Departamento de Microbiologı ´ a y Parasitologı ´ a, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain. Fax: + 34 954556924, Tel.: + 34 954556924; E-mail: palomares@us.es Abbreviations: BFA, brefeldin A; ER, endoplasmic reticulum; GFP, green fluorescent protein; MS, Murashige and Skoog media; PG, polygalacturonase; t-nos, nopaline synthase terminator. Enzymes: Polygalacturonases (PGs, EC 3.2.1.15). (Received 10 September 2002, revised 13 November 2002, accepted 21 November 2002) Eur. J. Biochem. 270, 261–269 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03384.x Vicia hirsuta plants described by Quandt et al.[14]toobtain rapidly transgenic roots of M. truncatula expressing the GFP fusions in root hairs. By using this system we showed that MsPG3 is specif- ically exported to the growing part of the root hair cell during its development, indicating that the protein may participate in tip growth processes during symbiosis. In addition, the protocol described in this work for M. trun- catula hairy root production may be useful to study in vivo those proteins that are involved in root hair growth and elongation, and the fusions could also be used as tools to examine the secretory system activity in different physiolo- gical, biochemical and developmental contexts. Materials and methods Construction of GFP fusion proteins Oligonucleotide primers used in this work were: 5038 ()12): 5¢-CTAA GAATTCACATGGATAGGA AA-3¢; PG3B (1984): 5¢-GG GGATCCGCTTCTGCTGC AGTTGTGC-3¢;EPS-1(72):5¢-CC CCATGGCTAAT ATCTTTGATATAAA-3¢;EPS-2(49):5¢-CCACCAG GATTGGGACCACGCC-3¢;SPS-1()33): 5¢-C CCCGGG AGTGAAAAAAGCAAAGTTCAAC-3¢; SPS-2 (105): 5¢-CCC CCATGGCTCCTCCAAATGATTTTATATC-3¢. The underlined sequences indicate the EcoRI (5038), BamHI (PG3B), NcoI (EPS-1 and SPS-2) and SmaI (SPS-1) restriction enzyme cleavage sites used for cloning. Distances from the oligonucleotide 5¢ ends to the ATG of the MsPG3 genomic sequence (EMBL data bank, accession no. Y11118) are given, except for EPS-2, designed from the gfp sequence [15]. MsPG3-gfp translational fusions were made as described below. pgc-gfp-t-nos. The pgc fragment containing the complete MsPG3 coding sequence including introns was obtained by PCR amplification from plasmid DNA using oligonucleo- tides 5038 and PG3B. This fragment was cloned in the pGEM-T easy vector (Promega) and transferred as an EcoRI-BamHI fragment to a Bluescript-derived plasmid (pKSgfp, Stratagene) containing a BamHI, NcoI-gfp-t-nos- NotI cassette from pmon30049 [15] (t-nos is the nopaline synthase terminator). pgte-gfp-t-nos. The pgte fragment containing the two first exons of MsPG3 and corresponding to an EcoRI-NcoI fragment of pgc, was cloned as an NcoI fragment in-frame in the NcoIsiteofthegfp-nos cassette of pKSgfp. pgwsp-gfp-t-nos. Using oligonucleotides EPS-1 and EPS-2, the pgwsp fragment lacking the signal peptide was amplified from fragment pgte-gfp, NcoI restricted and cloned as a NcoI fragment in-frame with gfp in pKSgfp plasmid. pgsp-gfp-t-nos. Oligonucleotides SPS-1 and SPS-2 were used to amplify a 105 bp DNA fragment containing the sequence of the predicted MsPG3 signal peptide. The NcoI and SmaI restriction sites generated by PCR were used to clone the pgsp fragment in-frame with the gfp coding sequence in pKSgfp plasmid. All of these MsPG3-gfp-t-nos fragments were transferred from pKSgfp plasmid to a pK18-derived plasmid [16] containing the CaMV35S promoter (pK35). In the same way, a gfp-t-nos fragment from pmon30049 was placed in this plasmid under the control of CaMV35S promoter. These plasmids, named pPGC-GFP, pPGTE-GFP, pPGWSP-GFP, pPGSP-GFP and p35S-cyt-GFP (Fig. 1), were used for particle bombardment experiments. For hairy root experiments 35S-pgte-gfp-t-nos and 35S- pgwsp-gfp-t-nos cassettes were cloned as HindIII-SacI fragments into pLP100 binary vector [17] to obtain pPGTE-GFPb and pPGWSP-GFPb plasmids. Plasmid pLP35GFP [18] was used as a cytoplasmic GFP control. Finally, these binary vectors were transformed into Agro- bacterium rhizogenes Arqua1 [14] by electroporation. A plasmid containing the modified gfp cassette mgfp4-ER (with a peptide targeting system) [19] has been used as a control of GFP fluorescence localization in the endoplasmic reticulum (ER), both in onion and M. truncatula roots. The construct containing this cassette was called GFP-ER (Fig. 1). All the constructs used in this study are listed in Table 1, indicating their functional domains and their expected targeting. Fig. 1. Schematic representation of GFP fusions used in this work. 35S, constitutive promoter from cauliflower mosaic virus; gfp, S65T- intron green fluorescent protein [15]; t-nos, nopaline synthase termi- nator; pgc, construction including the MsPG3 complete coding sequence; pgte, construction including the MsPG3 first two exons and the first intron; pgwsp, construction including the pgte part of MsPG3 without the signal peptide; pgsp, construction including only the MsPG3 signal peptide; mgfp4-ER,modifiedgfp cassette with a peptide for ER targeting [19]. The restriction sites relevant for the construc- tions are: B, BamHI; H, HindIII; E, EcoRI; Nc, NcoI; N, NotI; S, SacI; Sm, SmaI. 262 I. D. Rodrı ´ guez-Llorente et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Transformation of onion cells by particle bombardment Onion plant material and the gold particle bombardment protocol are described in Scott et al. [13]. This study used the same equipment and protocol, apart from the prepar- ation of cells. Particles were bombarded directly onto onion pieces that were peeled just before observation, instead of bombarding onion peels placed on agar plates. To visualize GFP in the cell wall, onion pieces were bathed in 20 m M sodium phosphate buffer (pH 7.0) following the 22 h incubation period needed for gfp expression as described in Scott et al. [13]. Generation of M. truncatula Ôhairy rootsÕ Seeds of M. truncatula (Gaertn.) R-108–1 (3c) were surface sterilized in 8 gÆL )1 Bayrochlor mini (Bayrol GMBH) in a shaking Erlenmeyer flask for 45 min. Thereafter, they were rinsed six times in sterile water. The seeds were allowed to swell overnight by incubation in sterile water. Pregermi- nated seeds were dried briefly by removing all the water and transferred to agar plates containing 0.5· Murashige and Skoog (MS) salts and vitamins (Sigma, M 0404), 10 gÆL )1 sucrose and 9 gÆL )1 kalys 575 agar (Mayoly Spindler, France). Ten seedlings were placed on 12 cm square plates which were then incubated vertically in a 25 °Cgrowth cabinet in the dark. After 24 h the plantlets were transferred to a growth chamber with a 16 h photoperiod (120– 130 mEÆm )2 Æs )1 ), at 25 °C with a relative humidity of 60%. When the first leaves appeared, around three days later, the plants were placed again in the dark for two days to elongate the hypocotyls to facilitate bacterial infection. When the hypocotyl length reached 3 cm, the plates were placed again in the growth chamber with the photoperiod described above. M. truncatula plantlets having hypocotyls of 3–3.5 cm in length, 24–48 h after the last transfer into light, were infected with A. rhizogenes by stab inoculation. One side of the hypocotyl was stabbed three to five times (approxi- mately 1/3 distance from the hook of the primary root to the cotyledons) with an Agrobacterium-containing needle. Thereafter, A. rhizogenes was taken again from the plate with the needle and placed carefully on the wounded area of the hypocotyl. Plates containing infected plantlets were returned to the growth chamber in the conditions described previously. Between 2 and 3 weeks after this procedure, hairy roots were obtained in 25–30% of the plants. When these roots were at least 2 cm long, the main root was excised and the resulting composite plants were transferred to fresh 0.5· MS with 200 mgÆL )1 cefotaxime (Sigma C7912) to avoid Agrobacterium proliferation. The composite plants were grown in the conditions described above, keeping the roots in the dark. One week later, Ôhairy rootsÕ with a length of 4 cm were checked for GFP expression by epifluores- cence microscopy as described below. Positive roots were then placed in the dark individually on fresh medium with antibiotic, as every root is a single transformation event. At this stage, fast lateral root development took place until enough material for further detailed analysis was obtained. These transgenic roots can be maintained for months in plates if transferred on fresh medium every three weeks. GFP detection Roots and onion cells were examined using a Polyvar microscope with two types of filters giving an excitation spectrum between 450 and 495 nm (B1) or between 475 and 495 nm (B4) and a stop filter at 520 nm (B1) or between 520 and 560 nm (B4). Images were recorded using a Leica DC200 camera. Confocal images were obtained in a Sarastro 2000 Confocal Microscope (Molecular Dynamics). Results Testing the expression capacity of GFP fusions in onion epidermal cells Scott et al. [13] have developed a rapid transient expression system using onion skin cells to express GFP fusion proteins. The onion epidermis has large, living, transparent cells, ideal for visualizing GFP. We thus used this system to test the capacity of various MsPG3-gfp fusions to be expressed and easily detected before root transformation. Onion cells expressing the cytoplasmic GFP fusion (cyt- GFP) showed a cytoplasmic and nuclear GFP localization (Fig. 2A). This previously reported nuclear localization of GFP [20] is due to its small molecular weight (27 kDa). As a second control, we expressed the GFP-ER construct in onion cells, which targets GFP to the ER (Fig. 1B). Fluorescence in GFP-ER expressing cells could be observed in the perinuclear region and in the cortical zone of the cell as a reticulate pattern of fluorescence (Fig. 2B). When the PGC-GFP construct, corresponding to the fusion of the complete MsPG3 coding sequence to GFP, was delivered into onion epidermal cells only a weak expression sur- rounding the nucleus was found (data not shown), probably corresponding to a weak gfp expression localized in the ER surrounding this nucleus. Scott et al. [13] demonstrated that GFP fusions targeted to the cell wall could not be detected due to the low pH of this compartment, but could be visualized by incubating the tissue in a medium buffered at pH 7.0. As the MsPG3 protein might be exported to the cell wall compartment, these cells were bathed overnight in a medium buffered at pH 7.0. Under these conditions, fluorescence appeared in the border of the cell (Fig. 2C), suggesting that the fusion protein containing the full Table 1. Polygalacturonase-gfp andcontrolfusionsusedinthisstudy. The main characteristics of the constructs and the expected subcellular localization of the codified proteins are indicated. GFP fusion Functional domains Expected targeting cyt-GFP None Cytoplasm and nucleus PGC-GFP Complete MsPG3 gene Cell wall PGTE-GFP First two exons and first intron of MsPG3 Endoplasmic reticulum?/cell wall? PGWSP-GFP PGTE without the signal peptide Cytoplasm and nucleus PGSP-GFP MsPG3 signal peptide Endoplasmic reticulum GFP-ER Peptide targeting system Endoplasmic reticulum Ó FEBS 2003 Tip localization of MsPG3-GFP fusions (Eur. J. Biochem. 270) 263 MsPG3 coding sequence was exported to the plasma membrane, or more probably to the cell wall. Interestingly, onion cells transformed with the PGTE- GFP construct (containing only the two first exons of MsPG3) showed a spotted staining pattern with fluorescent bodies (Fig. 2D). In addition, it was possible to detect fluorescence in the transvacuolar strands as well as around the nucleus (Fig. 2D) suggesting ER targeting. To verify that the spotted structures represented Golgi stacks, their sensitivity to brefeldin A (BFA) was tested. When onion peels expressing PGTE-GFP were incubated with BFA the pattern of fluorescence appeared different (Fig. 2E). Bigger structures rather than the small spots were detected, suggesting the localization of GFP within a BFA-sensitive Fig. 2. Transient expression of the GFP fusion proteins in onion epidermal cells. A-G: epifluorescence microscopy (B1 filter). (A) Cell expressing cytoplasmic GFP (cyt-GFP). Fluorescence appears inside the nucleus and in transvacuolar strands. (B) Cell expressing the GFP-ER fusion. Fluorescence is observed in the perinuclear region and in the cortical ER. (C) Cell expressing the PGC-GFP construct and bathed at pH 7.0. Fluorescence is detected in the cell wall. (D) Cell expressing the PGTE-GFP construct. A spotted pattern of fluorescence is detected, in addition to fluorescence associated to the transvacuolar strands and to the perinuclear region. (E) Reorganization of PGTE-GFP-labelled structures after BFA treatment (50 lg/mL). (F) Cell expressing the PGTE-GFP construct and bathed at pH 7.0. Fluorescence is observed in the cell wall. (G) Expression of PGWSP-GFP construct in onion cells. Fluorescence can be observed in the nucleus and in the cytoplasm (border of the cell and transvacuolar strands). (H) Expression of PGSP-GFP detected by confocal scanning microscopy. Fluorescence can be observed associated to the cortical ER and in the ER located around the nucleus and in the transvacuolar strands. TVS, transvascular strands; N, nucleus; C, cortical ER. Bars ¼ 50 lm. Confocal image has been coloured. 264 I. D. Rodrı ´ guez-Llorente et al.(Eur. J. Biochem. 270) Ó FEBS 2003 compartment probably representing the Golgi apparatus. When these peels were bathed at pH 7.0 we observed the same expression pattern as that detected with the complete MsPG3 (Fig. 2F). These experiments indicate that the protein fusion containing the first two exons (PGTE-GFP) is sufficient for exporting the GFP to the cell wall. In addition, this construct allows the detection of the different compartments used by the protein during the exportation process, and was thus later chosen for root transformation (see below). The first step in MsPG3 export pathway is probably peptide penetration in the ER mediated by the presence of a signal peptide, described in all plant PGs cloned to date. The activity of the predicted MsPG3 signal peptide was deter- mined using two new GFP fusions. Using the PCR technique, the sequence of the predicted signal peptide was amplified from the pgte fragment to obtain fragment pgwsp. The PGWSP-GFP construct expressed in onion cells showed fluorescence in the cytoplasm and in the nucleus (Fig. 2G), similar to the one observed using the cytoplasmic GFP (Fig. 2A). Finally, an MsPG3 fragment coding only for the first 35 amino acids of MsPG3 and including the 22 amino acids of the predicted signal peptide was fused to gfp (PGSP-GFP) and used to transform onion cells. The fluorescence pattern observed using the PGSP-GFP con- struct indicated an ER localization of the GFP (Fig. 2H), as we obtained the same pattern of expression in cells transformed with the GFP-ER construct. Using confocal scanning microscopy, fluorescence was detected in tubules and lamellar regions of the cortical ER, in the ER present in transvacuolar strands and in the ER surrounding the nucleus (Fig. 2H). MsPG3 export during M. truncatula root hair development To study MsPG3 localization and export during root hair development, M. truncatula Ôhairy rootsÕ expressing the cyt- GFP, GFP-ER, PGWSP-GFP and PGTE-GFP constructs were obtained. Ten out of 40 of the plants infected with A. rhizogenes containing T-DNA expressing cyt-GFP pro- duced between two and four hairy roots. Eight of them were identified as transgenic roots. The same number of Ôhairy rootsÕ was generated in approximately 30% (12/40) of the plants transformed with PGTE-GFP. In this case, 10 roots showed GFP activity. Similar results were obtained using the PGWSP-GFP and GFP-ER constructs. Despite the relatively low efficiency of Ôhairy rootÕ generation of our protocol, they were easily identified as GFP fluorescent roots. We used a binary vector carrying a CaMV35S promoter-gus fusion as a control with similar results. Here, Ôhairy rootsÕ were produced in 30% of the infected plants, indicating that the transformation efficiency in these experiments is independent of the construct used. None of the control roots showed fluorescence when the roots were young. The emission of weak yellow autofluorescence was detected in old roots (data not shown). Roots expressing GFP were placed individually on plates and lateral roots emerged very quickly. Young lateral roots with root hairs were cut and tested for GFP emission under the epifluorescence microscope. Roots transformed with cyt-GFP showed cytoplasmic (around the cell, due to the presence of a large vacuole) and nuclear fluorescence localization (Fig. 3A). The expression pattern of the cyto- plasmic GFP was the same in all the cells of the root, including root hairs (Fig. 3B). This result was also observed with confocal scanning microscopy (Fig. 4A). Cells from the root border expressing the cyt-GFP construct showed fluorescence in the nucleus and the border of the cell. The same pattern of expression was found in roots transformed with the PGWSP-GFP construct (fusion without the signal peptide), both in meristematic root cells (Fig. 3C) and root hairs (Fig. 3D). When roots were transformed with the PGTE-GFP construct, different localization of the fluorescence was observed. Differentiated cells exhibited stronger GFP fluor- escence in the periphery of the cell and around the nucleus, suggesting cell wall localization and possibly perinuclear ER localization (Fig. 3E), in agreement with the results obtained with onion cells. Meanwhile, the cells that were developing to form root hairs at the bulge stage exhibited fluorescent vesicles accumulating at the place where the root hair was emerging (Fig. 3E). This specific targeting of the fluorescence to the growing part of the root hair was also clearly observed using confocal scanning microscopy, both in a cell in the bulge stage (Fig. 4B) and in a growing root hair (Fig. 4C). This pattern of fluorescence probably represents Golgi stacks or transport vesicles, accumulating at the growing part of the cell. At a later stage of development, the growth terminating stage, root hairs showed GFP fluorescence close to the root hair tip, in the elongating root hair region, in addition to the perinuclear localization (Fig. 3F). In roots transformed with the GFP- ER construct the fluorescence appeared mainly around the nucleus, where the density of ER is high (Fig. 3G,H). This fluorescence was also observed in the border of the cells with weak intensity (Fig. 3G). This pattern of fluorescence had common localizations with the one described for roots transformed with the PGTE-GFP construct (everything related to the ER), but it also showed certain differences. One difference was clearly observed in the growth termin- ating stage of the root hairs, as in that case fluorescence was associated only with the perinuclear region, without the accumulation in the root hair tip (Fig. 3H). Discussion We have previously cloned, sequenced and partially char- acterized an M. sativa polygalacturonase gene (MsPG3) specifically expressed during the interaction of this plant with S. meliloti [9]. Our previous results suggested that MsPG3 might be involved in the early stages of the interaction, including infection thread formation and rein- itiation of the root hair tip growth induced by the Nod factor during the early steps of the plant–bacterial interac- tion [10,11]. These two processes are related to tip growth, defined as a specialized type of growth where organelles are arranged in zones and where new cell wall is deposited in a localized region of the cell, the growing tip. This growing mechanism is used in several important cellular systems including pollen tubes, fungal hyphae, root hairs and Rhizobium-induced infection threads. We have used the reporter protein GFP fused to various part of the MsPG3 coding sequence [12] to study the Ó FEBS 2003 Tip localization of MsPG3-GFP fusions (Eur. J. Biochem. 270) 265 Fig. 3. Expression of the GFP fusion proteins in M. truncatula hairy roots detected by epifluorescence microscopy. Nuclear and cytoplasmic fluorescence localization in root cells (A) and root hair cells (B) transformed with the cyt-GFP. Nuclear and cytoplasmic fluorescence localization in root cells (C) and root hair cells (D) expressing the PGWSP-GFP fusion. (E,F) GFP expression in roots transformed with PGTE-GFP. (E) Strong fluorescence can be observed around the nuclear region and in the periphery of the root cell. Fluorescent bodies, with a preferential accumulation in the region of the emerging root hair, in a cell in the bulge stage (ERH) can be observed. (F) Root hair in growth terminating stage with fluorescence localized in the root hair tip. Fluorescence around the nucleus is also observed. (G,H) Root cells expressing the GFP-ER fusion. (G) Fluorescence observed in the cell border and the perinuclear region, containing higher density ER. The intensity of the latter fluorescence is weak. (H) Root hair in growth terminating stage. Fluorescence is mainly observed around the nucleus, without accumulation at the root hair tip. ERH, emerging root hair; N, nucleus; T, root hair tip. Bars ¼ 20 lm in panels A, C, E and G and 10 lminpanelsB,D,FandH. 266 I. D. Rodrı ´ guez-Llorente et al.(Eur. J. Biochem. 270) Ó FEBS 2003 localization of our protein. GFP coding sequences have been fused at either the 5¢ or 3¢ end of the coding region of a DNA sequence of interest and the resulting N-terminal or C-terminal fusions used for in vivo studies on vesicular trafficking, protein localization and cellular compartmenta- tion in plants [20]. Potential problems using GFP fusions are conformational changes in the attached protein, which could activate localization signals that are normally seques- tered in the absence of GFP, and improper folding or instability of the encoded chimeric GFP, so that little or no fluorescence is detectable. Negative results of this type are rarely described in the literature [21]. Using this reporter system, simple and rapid transient assays have been developed to test the GFP fusion proteins before stable transformation. For example, the onion epidermal cell bombardment protocol described by Scott et al. [13] is very suitable for testing GFP fusion proteins before stable transformants are attempted. Onion epidermis, which has large, living and transparent cells in a single layer, appears to be particularly useful material for visualizing GFP in transient assays. In the work presented here we have developed an alternative to the onion cell system for studying protein localization in relation to cell growth, absent in the onion cells. This protocol is based on the production of transgenic hairy root on wounded hypocotyls of young seedlings of M. truncatula, a diploid autogamous legume that is cur- rently being developed as a model plant for the study of root endosymbiotic associations [22]. Due to their fast and hormone-independent growth, hairy root cultures represent a material of choice to study roots and they have been obtained from more than 100 different species [23]. This transformation system is faster and cheaper than complete plant transformation and has the advantages of stable transgenic material over transient assays, in which damage often occurs during DNA incorporation and for which there is variability in the amount of DNA delivered. All the plant PGs cloned to date have a N-terminal hydrophobic signal sequence that targets the protein to the lumen of the ER. The presence of a 22 amino acid hydrophobic N-terminal section in MsPG3, displaying the properties of a signal peptide [24], strongly suggests post- translational cleavage of the protein and secretion of the mature protein. In this work we have used two different controls: a cytoplasmic GFP (cyt-GFP) expressed from the CaMV35S promoter fusion, that showed the previously reported [20] cytoplasmic and nuclear localization of the fluorescence, and a modified GFP with a target sequence that keeps the protein in the lumen of the ER [19]. This second control helped us to explain part of our results. The localization in our work of the PGSP-GFP fusion (MsPG3 signal peptide fused to GFP) in the ER, and of the PGWSP- GFP fusion in the cytoplasm and in the nucleus suggest, as expected, that the predicted MsPG3 signal peptide is enough to target GFP to the ER but is not enough for cell wall localization. When the pgc-gfp fusion, containing the complete MsPG3, was expressed in onion cells, we observed only a weak GFP fluorescence around the nucleus, probably corresponding to the ER. The GFP fluorescence is pH dependent, with fluorescence intensity decreasing at low pH [25]. Thus the lack of visualization of GFP in the cell wall can be attributed to the low pH of this compartment. Scott et al. [13] showed indeed that GFP fluorescence appeared in the cell wall 4 h after buffering the cells at pH 7.0. As the development of the GFP fluorophore takes approximately 4 h [26], it was thus suggested that newly synthesized GFP was exported to the cell wall. Because PGs are supposed to be localized within the cell wall, we bathed onion peels transformed with this fusion overnight at pH 7.0, and fluorescence appeared associated with the border of the cell. The pattern of fluorescence did not change when cells expressing the cytoplasmic GFP underwent the same treatment (data not shown). Thus, our results suggest that GFP is targeted to the cell wall when fused to the complete MsPG3 peptide. Interestingly, when the pgte-gfp fusion corresponding to the two first exons of the gene was expressed in onion cells, ER localization was also observed, but in addition we detected fluorescence associated with transport vesicles Fig. 4. Confocal scanning microscopy images of roots transformed with GFP. (A) Root transformed with the cyt-GFP construct. No preferential accumulation of fluorescence in the root hair tip was observed. (B,C) Roots transformed with the PGTE-GFP construct. Preferential localization of the fluorescence was observed in the region of the emerging root hairs (ERH), both in a hair in the bulge stage (B) and in a growing root hair in the phase of organelle zonation (C). Images 1–4 are sequential confocal images of the same cells. ERH, emerging root hair; N, nucleus. Bars ¼ 10 lm. Images have been coloured. Ó FEBS 2003 Tip localization of MsPG3-GFP fusions (Eur. J. Biochem. 270) 267 probably representing Golgi stacks, as suggested by the BFA treatment. BFA is a drug that induces reorganization of the Golgi apparatus and blocks protein exportation [27]. Finally, cell wall localization of GFP fluorescence was revealed when cells expressing this fusion were bathed at pH 7.0 as found for the full length fusion. Thus, this construct was useful because it allowed us to detect the various steps of the exportation process followed by the MsPG3 protein. These results indicate either that the presence of the entire MsPG3 peptide allowed a more complete or faster export of the protein to its final location, or that this shorter fusion may not contain all the information (such as glycosylation sites) necessary for the efficient targeting of the protein. Another possibility is that the conformation of the hybrid protein does not allow its proper exportation, resulting in its partial retention in the different compartments (ER, Golgi), or finally that the shorter fusion is more fluorescent that the longer one and allows a better detection. In conclusion, the fusion including the signal peptide and the two first exons of the MsPG3 protein is sufficient and necessary to detect the protein along the exportation pathway and to localize it to the cell wall. We took advantage of this construct to study the localiza- tion of the MsPG3 protein in developing root hairs. In M. truncatula root cells that were developing to form a root hair, the PGTE-GFP fusion was detected in the ER apparatus as well as inside the cell at the site of the emerging root hair at bulge stage and at the apical part of a growing hair. The apical region in a growing hair corresponds to the transport vesicles-rich region [5]. Similarly, this fusion was specifically detected at the tip of the mature root hairs, rich in secretory vesicles containing cell-wall components. The result observed in roots transformed with the GFP-ER construct helped us to understand which part of the pattern of fluorescence detected in PGTE-GFP expressing roots is related to the localization of the fusion protein in the ER and which one represents a more specific targeting. In root hairs expressing the GFP-ER fusion, the tip localization of the fluorescence described in PGTE-GFP expressing root hairs could not be observed, suggesting further exportation of MsPG3 protein in the developing root hair. The fluorescent pattern in roots transformed with the PGTE- GFP construct might result from a specific localization of the MsPG3 protein to the tip of these cells, but we can not exclude the possibility that it also represents the localization of all proteins that are excreted following the secretion pathway in the root hairs and thus is a reflection of the cell biology of a developing root hair. In all cases, it indicates that the MsPG3 protein can be exported to the root hair tip and thus can, by its enzymatic activity, participate to the tip growth processes during symbiosis. In addition to giving us information about the localiza- tion of the MsPG3 protein, this fusion protein turned out to be a useful tool to visualize protein trafficking and localization in developing root hairs. Thus, the experimental system described in this work may be used to study in vivo and at the cellular level different aspects of root hair tip growth. Because these hairy roots are suitable for hormone or drug treatments, the system could be used to study the secretory system activity in different physiological, bio- chemical and developmental contexts. The transformation protocol described allows the generation of composite plants, consisting of transgenic roots on M. truncatula untransformed shoots, which can be nodulated successfully by their symbiotic partner. Recently, Boisson-Dernier et al. [28] described a protocol for hairy root production in M. truncatula that is probably faster than our one, making this kind of study even easier. Thus our fusions can also be used to study localization of proteins involved in infection thread formation during symbiosis, another tip growth based process. Finally, the protein fusions used in this work could also be used as a tool to examine the secretory system in other contexts, such as pollen tubes, wound sites or abcission zones. 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Plant-Microb. Interac. 14, 695–700. Ó FEBS 2003 Tip localization of MsPG3-GFP fusions (Eur. J. Biochem. 270) 269 . Expression of MsPG3-GFP fusions in Medicago truncatula Ôhairy rootsÕ reveals preferential tip localization of the protein in root hairs Ignacio D. Rodrı ´ guez-Llorente 1 ,. Here, Ôhairy rootsÕ were produced in 30% of the infected plants, indicating that the transformation efficiency in these experiments is independent of the construct used. None of the control roots. at either the 5¢ or 3¢ end of the coding region of a DNA sequence of interest and the resulting N-terminal or C-terminal fusions used for in vivo studies on vesicular trafficking, protein localization