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Investigating Genetic Components of the Symbiosis-associated Glycocalyx in Medicago truncatula


Plants form a variety of endosymbiotic relationships with bacteria and fungi that promote plant growth and fitness through reciprocal nutrient exchange. Central to the development and function of endosymbiotic relationships is the synthesis of a specialized host-derived membrane, which compartmentalizes the endosymbiont inside plant cells, and creates a dynamic interface for the exchange of nutrients and information. The development of these interfaces is dependent upon bidirectional signaling between the plant and microorganism, and is highly coordinated with the morphological differentiation of the endosymbiont within plant cells. Previous studies using glycan-directed monoclonal antibodies indicated that glycoproteins, glycolipids, and pectic polysaccharides localize to periarbuscular and symbiosome membranes. Among these epitopes were arabinogalactan proteins (AGPs) and glycosyl inositolphophorylceramides (GIPCs), which have potent signaling properties in plants. These epitopes appeared to be developmentally regulated, which led to the hypothesis that a membrane-associated glycocalyx of glycoproteins and glycolipids might be important for mediating interactions through these membrane interfaces. However, AGPs have not been well documented outside of Arabidosis thaliana and are intrinsically disordered, which make them difficult to identify and study in plants species capable of forming endosymbiosis. Here, we have developed a new bioinformatic search tool that identifies AGP-encoding genes based on the noncontiguous hydroxyproline motifs that direct AGP glycosylation. We used this tool to identify all putative AGP-encoding genes in the Medicago truncatula genome, which were cross-referenced to transcriptomic studies of roots engaged in symbiosis with Sinohizobium meliloti and the arbuscular mycorrhizal (AM) fungus Rhizohagus irregularis. Using this approach we identified a small three-member family of tandemly duplicated SYMBIOSIS-ASSOCIATED ARABINOGALACTAN PEPTIDES (SAPs) that were differentially expressed in root nodules and AM colonized roots. SAPs localized to symbiotic membranes and knockdown of SAP expression using RNAi-mediated gene silencing impaired the growth and differentiation of Sinorhizobium meliloti and Rhizphagus irregularis within these compartments. In parallel we also identified a glycosyltransferase gene highly expressed in root nodules and AM colonized roots as GIPC GLUCOSAMINE TRANSFERASE 1 (GINT1), and showed that the corresponding GINT1 enzyme functions in the synthesis of HexN(Ac) decorated GIPCs in planta. Silencing of GINT1 using RNAi impaired the development of symbiotic membranes, which resulted in the senescence of symbiosomes and arbuscules. Taken together these results provide genetic evidence to support that reprogramming of the membrane-associated glycocalyx with specific AGPs and GIPCs is necessary for endosymbiosis.

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