Medicago truncatula syntaxin SYP132 defines the symbiosome membrane and infection droplet membrane in root nodules
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- Catalano, C.M., Czymmek, K.J., Gann, J.G. et al. Planta (2007) 225: 541. doi:10.1007/s00425-006-0369-y
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Symbiotic association of legume plants with rhizobia bacteria culminates in organogenesis of nitrogen-fixing root nodules. In indeterminate nodules, plant cells accommodate rhizobial infection by enclosing each bacterium in a membrane-bound, organelle-like compartment called the symbiosome. Numerous symbiosomes occupy each nodule cell; therefore an enormous amount of membrane material must be delivered to the symbiosome membrane for its development and maintenance. Protein delivery to the symbiosome is thought to rely on the plant secretory system; however, the targeting mechanisms are not well understood. In this study, we report the first in-depth analysis of a syntaxin localized on symbiosome membranes. Syntaxins help define a biochemical identity to each compartment in the plant secretory system and facilitate vesicle docking and fusion. Here, we present biochemical and cytological evidence that the SNARE MtSYP132, a Medicago truncatula homologue of Arabidopsis thaliana Syntaxin of Plants 132, localizes to the symbiosome membrane. Using a specific anti-MtSYP132 peptide antibody, we also show that MtSYP132 localizes to the plasma membrane surrounding infection threads and is most abundant on the infection droplet membrane. These results indicate that MtSYP132 may function in infection thread development or growth and the early stages of symbiosome formation.
KeywordsInfection threadMedicagoNoduleSymbiosome membraneSyntaxinSNARE
Enzyme-linked immunosorbent assay
Keyhole limpet hemocyanin
Medicago truncatula syntaxin
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Soluble N-ethylmaleimide-sensitive factor adaptor protein receptors
Tris buffered saline
TBS with Tween-20
Nitrogen-fixing root nodules are specialized organs that form on the roots of legume plants during successful symbiotic interaction with the soil bacteria rhizobia (for review, see Gage 2004). Nodules shelter thousands of mature bacteria (bacteroids) that convert atmospheric nitrogen into bioavailable nitrogen for the host plant. Within an infected nodule cell, each bacteroid is enclosed by a plasmalemma-derived membrane called the symbiosome membrane. Collectively the symbiosome membrane, bacteroid, and the space between the two (the symbiosome space) define the organelle-like compartment called the symbiosome (Roth et al. 1988). The symbiosome develops after a series of intricate host–microbe interactions whereby rhizobia enter the plant by an infection structure called an infection thread. The infection thread arises from a site of rhizobial attachment to the root hairs beginning with localized cell wall hydrolysis and plasma membrane ingrowth; this is followed by subsequent growth of plasma membrane and cell wall material and division of the rhizobia until the infection thread reaches the nodule primordium (VandenBosch et al. 1989; Rae et al. 1992; Brewin 2004). Within the nodule primordium, rhizobia are released into the host cell cytoplasm by an endocytic process from unwalled infection droplets. Symbiosomes result from coordinated division of the bacteria and the surrounding membrane, the symbiosome membrane. The symbiosome membrane, which was originally derived from the plant plasma membrane, becomes biochemically distinct from its parent membrane by enrichment with additional protein and lipid material. In a single infected nodule cell, symbiosome membrane surface area is estimated to be 100× that of the plasma membrane (Brewin et al. 1988); therefore, a massive supply of membrane material must be targeted to the symbiosome membrane.
The ability of nodule cells to control protein trafficking and targeting to the symbiosome is essential for nodule development and function. The mechanism(s) by which proteins are targeted to this novel subcellular compartment to accommodate its demand for membrane proteins is largely unknown; however, four pathways have been hypothesized (Catalano et al. 2004). Protein targeting to the symbiosome relies on the plant secretory system. In eukaryotic cells, proteins that are destined for the plasma membrane or endosomal organelles travel through the secretory system and are targeted to their final destination in cargo vesicles. Each target organelle in the secretory system must maintain unique biochemical properties for accurate targeting of the cargo vesicle from the trans Golgi. In one possible mechanism, integral membrane proteins called SNARES (soluble N-ethylmaleimide-sensitive factor adaptor protein receptors) and their interacting proteins confer “biochemical identity” to each compartment of the secretory system and are essential for correct docking of cargo vesicles at the proper target membrane (Sanderfoot and Raikhel 1999; McNew et al. 2000; Sanderfoot et al. 2000). In the “SNARE hypothesis”, vesicle fusion occurs by the selective interaction of a target membrane SNARE (t-SNARE) and a vesicle SNARE (v-SNARE) along with interacting proteins such as GTPases and Rab proteins (Sanderfoot and Raikhel 1999; McNew et al. 2000). One of the four proteins located at the target membrane SNARE complex is syntaxin (Jahn et al. 2003). Syntaxin proteins are a conserved family of proteins in eukaryotes that reside on organelles of the secretory system. Syntaxins have been studied extensively in yeast and mammalian cells. Less is known about plant syntaxins; however, plant SNARE proteins have been identified by functional complementation studies of analogous mutations in yeast (Blatt et al. 1999). With the completion of the Arabidopsis genome sequencing project, it is apparent that Arabidopsis contains 24 syntaxins, including novel syntaxins and orthologs to syntaxins in yeast and mammals (Sanderfoot et al. 2000, 2001). Besides the conventional housekeeping roles of syntaxin proteins, plant syntaxins also have been implicated in other roles such as signaling, abiotic stress response, pathogen response, cytokinesis, gravitropism, and others (for reviews see Carter et al. 2004; Pratelli et al. 2004; Surpin and Raikhel 2004).
In a previous proteomic study we identified a syntaxin, MtSYP132, as a component of the symbiosome membrane from M. truncatula root nodules (Catalano et al. 2004). MtSYP132 is highly homologous to one of the nine-member SYP1 group of Arabidopsis syntaxins of unknown function, AtSYP132 (Sanderfoot et al. 2000). This group has highest homology with Sso1/2p of yeast and mammalian Syntaxin1, both of which reside on the plasma membrane and aid in vesicle docking (Aalto et al. 1993; Bennett et al. 1993). Our previous work was the first report of a syntaxin protein in nitrogen-fixing root nodules. It is still unknown at which developmental stage MtSYP133 is present on symbiosomes and if MtSYP132 localizes to other cellular compartments in nodule tissues.
In this study, we present biochemical and cytological evidence that MtSYP132 is a symbiosome membrane protein and also localizes specifically to the plasma membrane surrounding the infection thread and the infection droplet membrane. In total, these results provide intriguing insights on the function of MtSYP132 within nitrogen fixing root nodules
Materials and methods
The nucleotide and open reading frame of MtSYP132 can be accessed through the TIGR Medicago truncatula Gene Index database (http://www.tigr.org/tdb/tgi/mtgi) under TC86779 (Lee et al. 2005). The primary structure of MtSYP132 was analyzed using Swiss-Prot (Gasteiger et al. 2003) prediction tools. Coiled-coil domains were predicted using MultiCoil (Wolf et al. 1997). The peptide selected for antibody generation was analyzed using NCBI blastp (http://www.ncbi.nlm.nih.gov/BLAST/) (Altschul et al. 1997).
MtSYP132 antibody production
Polyclonal antibodies to a unique peptide of the MtSYP132 protein were synthesized by Open Biosystems (Huntsville, AL, USA). The peptide (KKFRDLMTEFQTLRQR) was selected for synthesis based upon its divergence with other protein family members, using hydropathy and antigen profiling. Cross reactivity with other proteins was checked by performing BLASTp searches against known sequences in the NCBI database (http://www.ncbi.nlm-nih.gov/). The peptide was synthesized using Fmoc solid phase chemistry by Open Biosystems. MtSYP132 antibodies were generated using White New Zealand female rabbits with a 90-day immunization protocol. Preimmune serum was collected before immunization. Serum was titered by indirect ELISA using passively bound peptide.
Plant growth and nodulation
Medicago truncatula line A-17 (Jemalong) were inoculated with Sinorhizobium meliloti 2011 (Meade et al. 1982) to induce nodule formation in an aeroponic growth system as described by Catalano et al. (2004). Initial seed stocks were provided by Dr. K. A. VandenBosch, University of Minnesota, St. Paul and Dr. R. Dickstein, University of North Texas, Denton. Root nodules for biochemistry and for immunomicroscopy were harvested 10–14 days post-inoculation.
Preparation of nodule fraction proteins
Symbiosome membrane, symbiosome space, and bacteroid protein fractions were purified as described in Catalano et al. (2004). Protein concentrations of all fractions were determined using the BioRad DC protein assay (Bio-Rad) according to the manufacturer’s instructions.
Total nodule, total nodule membrane, symbiosome membrane, symbiosome space, and bacteroid proteins were resolved on SDS-PAGE gels for Western-blot analyses as in Catalano et al. (2004). Proteins were transferred to 0.45 μm nitrocellulose (Osmonics, Inc.) following an established protocol (Towbin et al. 1979). MtSYP132 protein bands were detected on Western blots using enhanced chemiluminesence. Blots were blocked in 2% (w/v) nonfat dry milk, 0.05% (w/v) Tween-20 in 1× Tris-buffered saline (TBS) pH 7.4 overnight at 4°C with rotation, or at room temperature for 2 h with rotation. Blocking buffer was removed and the blot incubated in anti-MtSYP132 at a dilution of 1:5,000 in blocking buffer. Blots were incubated for 2 h at room temperature with gentle rotation in primary antibody solution. Blots were then rinsed three times 15 min with TBS, 0.05% Tween-20 (TBST). Blots were then incubated in goat anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (Sigma Chemicals, St. Louis, MO, USA) at a dilution of 1:10,000 in blocking buffer for 1 h at room temperature with rotation. Blots were then rinsed three times 15 min with TBST and protein bands were visualized by enhanced chemiluminesence.
Nodule tissue was sectioned using a double-edged razor blade into 80 mM Pipes buffer pH 7.0 and then transferred to 4% formaldehyde in 80 mM Pipes, pH 7.0. Following vacuum infiltration to promote optimal fixative infiltration, the solution was replaced with fresh 4% formaldehyde in 80 mM Pipes, pH 7.0. Nodule sections were rinsed three times for 10 min with 80 mM Pipes. Samples were blocked in 2% (w/v) nonfat dry milk in TBS, pH 7.4 for 2 h with rotation. Samples were incubated in anti-MtSYP132 at a dilution of 1:500 or MtSYP132 preimmune serum at a dilution of 1:500 overnight at 4°C with rotation. Samples were then rinsed three times for 10 min with TBS and incubated in Alexa Fluor® 546 goat anti-rabbit IgG (H + L, highly cross absorbed) at a dilution of 1:50 in blocking buffer (Molecular Probes, Eugene, OR, USA). Sections were also counterstained in SYTO®13 (Molecular Probes) to visualize nucleic acid (Haynes et al. 2004). Sections were rinsed three times for 10 min with TBS. Confocal images were acquired on a Zeiss Axiovert 200 M equipped with a LSM 510 NLO laser-scanning microscope (Carl Zeiss, Inc., Germany) using a Zeiss 10× Plan-Apochromat lens (NA 0.45), 20× Plan-Apochromat lens (NA 0.75) or 40× C-Apochromat (NA 1.2) objective lens. Multi-channel images of SYTO®13 and Alexa Fluor® 546 were acquired in fastline-switch mode using the 488 nm laser line of a 25 mW Argon laser (LASOS, Germany) with the 500–550 band pass and 543 nm laser line of a 1 mW h–n laser with the 560 long pass emission filters, respectively. Images were acquired as individual optical sections or as a z-series of optical sections. For 3-D renderings, data sets were generated using Zeiss LSM software v3.2 and displayed as single maximum intensity projections.
To label individual symbiosomes and infection threads optimally, some nodule tissue was prepared and cryosectioned according to Reed et al. (2001). Briefly, nodule tissue was hand sectioned as described above, fixed in 4% formaldehyde, 80 mM Pipes pH 7.0, vacuum infiltrated, and allowed to incubate in fixative solution for 2 h with constant rotation. Nodule tissue was then embedded in gelatin containing 10% (w/v) gelatin and 2% sucrose and allowed to solidify overnight at 4°C. Nodule sections were cryosectioned using a Leica CM 3050 Cryostat with a CE knife holder and frozen nodule sections were mounted on Colorfrost/Plus Microscope Slides (Fisherbrand). For immunolabeling of cryosections, tissue was incubated in blocking buffer (2% (w/v) nonfat dry milk, 0.05% Tween-20 in TBS pH 7.4) for 1 h in a humid chamber. Block was discarded and the sections incubated in anti-MtSYP132 or MtSYP132 preimmune serum at a dilution of 1:5,000 for 2 h. Sections were rinsed three times for 10 min with TBST. Sections were incubated in Alexa Fluor® 546 goat anti-rabbit IgG (H + L, highly cross absorbed) at a dilution of 1:50 in blocking buffer (Molecular Probes, Eugene, OR, USA). Sections were also counterstained in SYTO®13 (Molecular Probes) to visualize nucleic acid. Sections were rinsed three times 10 min with TBST. Images were acquired as described in the previous section.
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Generation of a specific MtSYP132 polyclonal antibody probe
MtSYP132 was present in the symbiosome membrane protein fraction
MtSYP132 localized to infected nodule cells and the symbiosome membrane
Individual symbiosomes were evaluated at higher magnification, using confocal-immuno-microscopy to determine the specific location of MtSYP132. Individual, isolated symbiosomes exhibited intense MtSYP132 label surrounding the bacteroid outer membrane (Fig. 3c). The MtSYP132 label did not overlap with the bacteroid, indicating that MtSYP132 does not reside on the bacterial membrane (Fig. 3c). These data suggested that MtSYP132 localized to a membrane surrounding the bacteroid outer membrane. To provide additional experimental evidence of the localization of MtSYP132 in nodule tissues, resin embedded nodule tissues were probed with anti-MtSYP132 peptide antibodies and visualized with the transmission electron microscope. Unfortunately, after numerous and varied approaches to EM localization, the results were inconclusive.
Taken together with the biochemical analysis that demonstrated the absence of MtSYP132 in the symbiosome space or bacteroid protein fractions, the evidence indicated that MtSYP132 localized to the symbiosome membrane.
MtSYP132 localized to the infection thread and infection droplet membrane
In this study we have reported the identification and subcellular distribution of a syntaxin, MtSYP132, in symbiotic nodules of M. truncatula, providing insight into the role of MtSYP132 in nodule development. MtSYP132 is most closely related to the SYP1 group of syntaxins that contains three subgroups, SYP11–SYP13. Syntaxins within the SYP1 group are most homologous to Sso1/2p from yeast and Syntaxin1 from mammalian cells. Sso1/2p and Syntaxin1 are both plasma membrane syntaxins (Aalto et al. 1993; Bennett et al. 1993; Sanderfoot et al. 2000). Using a peptide-specific antibody against a unique antigen of MtSYP132, the cellular and subcellular distribution of MtSYP132 in nodules were visualized. MtSYP132 is a symbiosome membrane protein and is also located at the plasma membrane that surrounds infection threads and infection droplets in nodules. Interestingly, MtSYP132 was not distributed on the plasma membrane of uninfected nodule cells nor on the non-infection thread plasma membrane of infected cells. This is the first report showing detailed biochemical analysis of a syntaxin protein residing at two specialized membranes within M. truncatula root nodules.
Site-directed vesicle trafficking in plant cells is essential for plant growth and development (Homann 1998; Thiel and Battey 1998). Until recently, syntaxins were thought to be involved primarily in the traditional housekeeping roles associated with vesicle trafficking (for review, see Sanderfoot et al. 2000). Plant-unique roles attributed to syntaxins include regulation of ion channels in guard cell movement and signaling, plant pathogen response, cytokinesis, and vesicle fusion at the forming cell plate. NtSyr1, a plasma membrane t-SNARE syntaxin in tobacco, is involved in ABA responses in guard cells, possibly by regulating potassium and chloride ion channels (Leyman et al. 1999). The inability of these channels to respond to abscisic acid when the NtSyr1 gene is disrupted shows that this gene may have a role in signaling, rather than a classical targeting role (Leyman et al. 1999). Similarly OSM1/SYP61, another t-SNARE syntaxin located at the trans Golgi network and the prevacuolar compartment, also has been implicated in controlling ABA and non-ABA responses to salt and osmotic stress in addition to regulating guard cell movement (Zhu et al. 2002). Secondly, genetic screens in Arabidopsis to identify resistance to fungi have identified the plasma membrane syntaxin AtSyp121 (PEN1) (Collins et al. 2003). PEN1 is essential for non-host resistance to powdery mildew, and acts in a specialized defense-related function to delay the formation of fungal papillae (Assaad et al. 2004). Thirdly, syntaxins have been shown to function in cytokinesis and cell plate formation (Lauber et al. 1997; Assaad et al. 2001; Muller et al. 2003). AtSYP111, also referred to as KNOLLE, accumulates in the phragmoplast and is essential in trafficking Golgi vesicles to the cell plate during cytokinesis (Lauber et al. 1997). Lastly, syntaxin proteins may assume other plant-unique roles including a role in vacuole biogenesis (Rojo et al. 2003; Surpin and Raikhel 2004), fusion of ER bodies with the lytic vacuole tonoplast, and plant cell expansion (Carter et al. 2004).
As is evident in animals and in plants, syntaxins are important for formation of discrete subcellular organelles such as the phagosome and vacuole, respectively (Hackman et al. 1996; Collins et al. 2002; Surpin and Raikhel 2004). Our results, which indicate that MtSYP132 is a symbiosome membrane protein, support a role for syntaxins in specialized organelle biogenesis. Symbiosomes within nodules are unique, subcellular, organelle-like units that are specialized to harbor symbiotic bacteria. The symbiosome membrane, a plasma membrane-derived membrane that surrounds each bacteroid, becomes biochemically specialized from its parent membrane during maturation (Catalano et al. 2004). Formation of the symbiosome membrane occurs when bacteria are released from the infection thread into the host cell cytoplasm. Another subcellular domain that is sheathed in plasma membrane material is the infection thread. The infection thread proliferates by selective targeting of plasma membrane and cell wall material to its growing apex, and releases bacteria in an unwalled outgrowth, the infection droplet (for review, see Brewin 2004). This study shows that MtSYP132 is localized specifically on the plasma membrane surrounding the infection thread and droplet and not on other plasma membrane within the same cell. These results indicate that there is a discrete developmental stage, namely the formation of symbiosomes, at which MtSYP132 is important for nodule development. Therefore, we suggest that protein changes in the plasma membrane surrounding the infection thread are important for defining the region of the plasma membrane that becomes a functional infection droplet.
Does MtSYP132 have mulitple distinct functions, one function at the plasma membrane surrounding the infection thread and another at the symbiosome membrane, or is MtSYP132 functioning similarly at both locations? Does MtSYP132 exhibit a symbiotic-specific function in nodules or does it perform the same function in other plant tissues? The plasma membrane surrounding the infection thread is a distinct biochemical compartment that is different from other cellular plasma membrane and the symbiosome membrane. MtSYP132 may fulfill the non-symbiotic, traditional role of a plasma membrane t-SNARE functioning in site-specific vesicle fusion for the delivery of cargo vesicles to the plasma membrane around the thread, the infection droplet membrane, and the symbiosome membrane. Conversely, this protein may fulfill a unique symbiotic role by contributing to the specialization of the plasma membrane material around the infection thread and specialization of infection droplet and the symbiosome membrane. This specialization may promote selective targeting of proteins to these two distinct nodule subdomains. Other functions of syntaxins have been elucidated and include a direct role in signaling in plant cells, particularly the signaling cascade regulating intercellular ion flux. During nodule formation and function, regulation of ion flux between the symbiosome and host cell is critical for bacteroid maintenance and survival. Association of symbiosome membrane syntaxin proteins with ion channels may be one mechanism that regulates ion transport into the symbiosome. Further experiments on MtSYP132 using genetic approaches will be helpful in determining if this gene product contributes to a symbiotic-specific vesicle targeting mechanism in root nodules or if the protein functions as part of a signal transduction cascade.
The MS/MS spectra identified for MtSYP132 in Supplemental Figure 1 are provided courtesy of William S. Lane, Harvard University Microchemistry and Proteomics Analysis Facility. This work was supported by USDA NRI CGP awards 2005-35318-16215, 2001-35318-10915, and 2001-35311-10161 to DJS.