MPMI Vol. 21, No. 4, 2008, pp. 404–410. doi:10.1094/ MPMI -21-4-0404. © 2008 The American Phytopathological Society e-Xtra* Transcription of ENOD8 in Medicago truncatula Nodules Directs ENOD8 Esterase to Developing and Mature Symbiosomes Laurent Coque,1 Purnima Neogi,1 Catalina Pislariu,1 Kimberly A. Wilson,1 Christina Catalano,2 Madhavi Avadhani,2 D. Janine Sherrier,2 and Rebecca Dickstein1 1University of North Texas, Department of Biological Sciences, Chestnut and Avenue C, Denton 76203-5220, U.S.A.; 2Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark 19711, U.S.A. Submitted 25 October 2007. Accepted 5 December 2007. In Medicago truncatula nodules, the soil bacterium Sino- attaching to root hairs, reorienting root hair cell wall growth, rhizobium meliloti reduces atmospheric dinitrogen into and forming a bacterial colony within a tightly curled root hair, nitrogenous compounds that the legume uses for its own rhizobia enter plant roots through plant-derived infection threads growth. In nitrogen-fixing nodules, each infected cell con- that traverse several cell layers. Ultimately, the rhizobia are de- tains symbiosomes, which include the rhizobial cell, the posited in nodule primordium cells in a process that resembles symbiosome membrane surrounding it, and the matrix endocytosis or phagocytosis. between the bacterium and the symbiosome membrane, A plasma membrane-derived membrane surrounds each bac- termed the symbiosome space. Here, we describe the local- terium and results in a new organelle-like compartment in the ization of ENOD8, a nodule-specific esterase. The onset of infected nodule cells termed the symbiosome. Within symbio- ENOD8 expression occurs at 4 to 5 days postinoculation, somes, coordinated division of rhizobia and the symbiosome before the genes that support the nitrogen fixation capa- membrane occurs. Eventually, rhizobial division ceases and bilities of the nodule. Expression of an ENOD8 promoter– rhizobia differentiate into bacteroids, a form recognized as gusA fusion in nodulated hairy roots of composite trans- being capable of nitrogen fixation (Vasse et al. 1990). The ma- formed M. truncatula plants indicated that ENOD8 is ex- trix that surrounds the bacteroid is called the symbiosome pressed from the proximal end of interzone II to III to the space. In some literature, the symbiosome membrane and the proximal end of the nodules. Confocal immunomicroscopy symbiosome space are termed the peribacteroid membrane and using an ENOD8-specific antibody showed that the peribacteroid space, respectively (Hirsch 1992). Both the sym- ENOD8 protein was detected in the same zones. ENOD8 biosome space and the symbiosome membrane become spe- protein was localized in the symbiosome membrane or cialized during nodule development concomitantly with bac- symbiosome space around the bacteroids in the infected teroid differentiation, and these changes eventually enable nodule cells. Immunoblot analysis of fractionated symbio- nitrogen fixation and transport. somes strongly suggested that ENOD8 protein was found In mature indeterminate nodules, such as the type formed by in the symbiosome membrane and symbiosome space, but Medicago truncatula, nodule invasion occurs immediately ad- not in the bacteroid. Determining the localization of jacent to the nodule meristem, in zone II (Vasse et al. 1990). ENOD8 protein in the symbiosome is a first step in under- Rhizobial division and differentiation occur within zone II. standing its role in symbiosome membrane and space dur- Starch accumulation marks the interzone II to III, proximal to ing nodule formation and function. zone II, where the final stages of bacteroid differentiation occur. Nitrogen fixation occurs in zone III. In older nodules, a senescent zone IV also occurs (Hirsch 1992). Nitrogen-fixing nodules form on the roots of legumes as the The symbiosome membrane serves as a physical barrier and culmination of a successful interaction between the plant and a mediator of metabolite exchange between the bacteroid and soil bacteria called rhizobia. Nodule development is a complex the plant host cell cytosol (Udvardi and Day 1997). In nitrogen- process that involves differentiation of both symbiotic partners fixing nodule cells, the symbiosome membrane resembles both (Brewin 2004; Gage 2004; Hirsch 1992; Jones et al. 2007). After the plasma membrane and the vacuole membrane with respect to its substituent proteins (Brewin 1991; Mylona et al. 1995). Current address of L. Coque: Department of Psychiatry, University of Previous studies have focused on the biochemical composition Texas Southwestern Medical Center, Dallas 75390-9070, U.S.A. of the symbiosome membrane and how it differs from other cellular membranes, how it changes during symbiosome and Current address of C. Pislariu: The Samuel Roberts Noble Foundation, Ardmore, OK 73401, U.S.A. bacteroid maturation, and functions during nodule develop- ment and nitrogen fixation (Catalano et al. 2004, 2007; Panter Current address of K. A. Wilson: Departments of Pediatrics and Bio- et al. 2000; Wienkoop and Saalbach 2003). chemistry, University of Texas Southwestern Medical School, Dallas, TX Proteomic analysis indicates that approximately 200 proteins 75390-9063, U.S.A. exist in the symbiosome space in M. truncatula (Catalano et al. Corresponding author: Rebecca Dickstein; 2004) and in pea (Saalbach et al. 2002). Immunolocalization E-mail: [email protected]; Fax +1.940.565.3821. studies have shown that a lectin protein (Dahiya et al. 1997; *The e-Xtra logo stands for “electronic extra” and indicates that a sup- Kardailsky et al. 1996) and a cysteine protease are in the sym- plemental figure is published online. biosome space (Vincent and Brewin 2000) in addition to sev- 404 / Molecular Plant-Microbe Interactions eral members of a calcium-binding protein family (Liu et al. was some variability of staining from nodule to nodule; most of 2006). However, the functional significance of many of the the nodules examined came from independent transformation proteins in the symbiosome space is largely unknown. events. The nodule shown in Figure 1 represents the staining In this report, we describe the localization of ENOD8. In pattern found in most pMtENOD8-gusA-transformed nodules. M. truncatula, ENOD8 is a member of a duplicated gene family of which only ENOD8 is highly active in root nodules Localization of ENOD8 protein at the tissue level. (Dickstein et al. 2002). ENOD8 belongs to the GDSL family An ENOD8 antiserum was used to localize ENOD8 protein encoding hydrolytic enzymes (Akoh et al. 2004; Upton and in fixed nodule sections. This antibody that was raised against Buckley 1995). This gene family, consisting of plant and bac- the oligopeptide CKNPSTRITWDGTHYTEAA was reported terial genes, encodes hydrolytic proteins, some of which have previously (Dickstein et al. 2002; Pringle and Dickstein 2004). esterase, lipase, or carbohydrate hydrolase activity. Of the This sequence corresponds to amino acids 336 to 354 of the GDSL plant enzymes that have been biochemically character- M. sativa ENOD8 putative polypeptide sequence (GenBank ized, lanatoside 15′-O-acetylesterase (LAE) from Digitalis accession AAB41547) and an 89% identical sequence in M. lanata is a close homolog to ENOD8, with 44% sequence truncatula’s predicted ENOD8 protein (GenBank accession identity at the amino acid level (Kandzia et al. 1998). LAE AAC26810). This antiserum has high specificity for ENOD8 deacetylates the substrate lanatoside A, an acetylated glycosy- protein from both M. sativa and M. truncatula (Dickstein et al. lated steroid, to purpureaglycoside A in cardenolide metabo- 2002). In conditions tested previously, the antiserum was not lism. Another close homolog of ENOD8 (47% identical) en- an effective ENOD8 protein localization tool (Pringle and codes an exopolygalacturonase from Daucus carota, carrot Dickstein 2004). After trial and error, a protocol was found (Tanaka et al. 2002). The exopolygalacturonase is 97% identi- that recovers antigen signal loss during fixation (see Methods). cal to iEP4 (Bertinetti and Ugalde 1996) and EP4, found Additionally, and in confirmation of staining patterns obtained, extracellularly (van Engelen et al. 1995). Purified M. sativa we raised another ENOD8 antiserum to a recombinant mal- ENOD8 proteins have esterase activity that is more active on tose-binding protein (MBP)-ENOD8 fusion protein and used it shorter-chain esters than on longer-chain esters (Pringle and instead of the ENOD8 oligopeptide antiserum in additional Dickstein 2004). ENOD8 protein was shown to be completely immunolocalization experiments not reported here. In every soluble in aqueous solutions (Pringle and Dickstein 2004). tested case, identical localization patterns were observed when Curiously, ENOD8 protein also was identified in purified sym- the MBP-ENOD8 fusion protein antiserum were used (data biosome membrane fractions (Catalano et al. 2004). To date, not shown), instead of the ENOD8 oligopeptide antiserum. however, there has been no comprehensive study of the distri- ENOD8 antibodies were purified from the ENOD8 oligo- bution of ENOD8 protein localization. peptide antiserum by immunoaffinity chromatography and were Here, we report on the spatial localization of a pENOD8- used in immunolocalization experiments. To assess the utility gusA transgene in M. truncatula nodules. We also report on the of the ENOD8 antibodies in localization, they were immuno- localization of the ENOD8