Transcription of ENOD8 in Medicago truncatula Nodules Directs ENOD8 Esterase to Developing and Mature Symbiosomes

Abstract

In Medicago truncatula nodules, the soil bacterium Sinorhizobium meliloti reduces atmospheric dinitrogen into nitrogenous compounds that the legume uses for its own growth. In nitrogen-fixing nodules, each infected cell contains symbiosomes, which include the rhizobial cell, the symbiosome membrane surrounding it, and the matrix between the bacterium and the symbiosome membrane, termed the symbiosome space. Here, we describe the localization of ENOD8, a nodule-specific esterase. The onset of ENOD8 expression occurs at 4 to 5 days postinoculation, before the genes that support the nitrogen fixation capabilities of the nodule. Expression of an ENOD8 promoter-gusA fusion in nodulated hairy roots of composite transformed M. truncatula plants indicated that ENOD8 is expressed from the proximal end of interzone II to III to the proximal end of the nodules. Confocal immunomicroscopy using an ENOD8-specific antibody showed that the ENOD8 protein was detected in the same zones. ENOD8 protein was localized in the symbiosome membrane or symbiosome space around the bacteroids in the infected nodule cells. Immunoblot analysis of fractionated symbiosomes strongly suggested that ENOD8 protein was found in the symbiosome membrane and symbiosome space, but not in the bacteroid. Determining the localization of ENOD8 protein in the symbiosome is a first step in understanding its role in symbiosome membrane and space during nodule formation and function.

Multiple Domains in MtENOD8 Protein Including the Signal Peptide Target It to The Symbiosome

“…2, A and B, arrows). Similar to endogenous MtENOD8, observed using immunolocalization (Coque et al, 2008), MtENOD8-GFP is found in proximal symbiosomes, but not in those in the distal zones of the nodule (Supplemental Fig. S1).…”

“…1 and 2; Coque et al, 2008). Normally, the MtENOD8 protein is found highly expressed in some, but not all symbiosomes: Mature symbiosomes with elongated bacteroids contain MtENOD8, whereas younger, distal, infected nodule cells do not express it; this occurs because MtENOD8 is expressed in the proximal parts of nodules (Coque et al, 2008).…”

“…In previous studies, using a combination of immunolocalization and subcellular fractionation, MtENOD8 protein was shown to be localized primarily to the SymS, with a small amount associated with the SymM (Coque et al, 2008). We tagged MtENOD8 protein at its C-terminal (CT) end with GFP, connected by a 10-Ala linker, and put the construct under MtENOD8 promoter control (Fig.…”

“…Comparatively less is known about the proteins and biochemistry of the SymS. In Medicago truncatula, members of the calmodulin-like MtCaML1-6 family of proteins are found in the SymS (Liu et al, 2006), as are MtNOD25 (Hohnjec et al, 2009) and M. truncatula EARLY NODULIN8 (MtENOD8) (Coque et al, 2008). In pea (Pisum sativum), PsNLEC-1 and PsCYP15A have been immunolocalized to symbiosomes and vacuoles and symbiosomes, vesicles and vacuoles, respectively (Dahiya et al, 1997;Vincent and Brewin, 2000).…”

Identification and Crystallographic Analysis of a New Carbohydrate Acetylesterase (SmAcE1) from Sinorhizobium meliloti

“…Among these proteins, carbohydrate esterases (CEs) play key roles in making other CAZymes access their carbohydrate substrates by removing acetyl groups from substituted saccharides with various chain lengths [5,6]. In the physiological aspect, CEs are considered to be involved in the formation of cell walls, interactions between host and microbes, and energy metabolism pathways [7][8][9][10][11][12][13][14][15]. CEs are also important for biotechnological application, as modifications of carbohydrates are necessary for the preparation of biofuel, drug carriers, and cosmetics [16][17][18].…”