Final Report Summary - CASINO (Carbohydrate signals controlling nodulation)
Research on the role of rhizobial exopolysaccharides as signal molecules in plant-microbe interaction was a highlight of the CASINO research. Detailed studies of the infection of the model legume Lotus japonicus suggested that legume host plants possessed a perception system enabling them to distinguish compatible from incompatible bacterial exopolysaccharides. Bacterial surface exopolysaccharides were previously suggested to be important for biofilm formation, protection against reactive oxygen species synthesized by the host plant or as osmoprotectants. They were also considered as “stealth compounds” that allowed rhizobia to avoid the plant´s innate immune response. Combining plant and microbial genetics with carbohydrate chemistry and biochemistry, a direct function of an octameric oligosaccharide unit of exopolysaccharide as a compatible exopolysaccharide and a truncated pentamer as an incompatible exopolysaccharide was demonstrated. Following genetics based identification of the exopolysaccharide receptor gene, Epr3, direct binding of an octameric oligosaccharide unit by purified EPR3 ectodomains carrying LysM domains (Kd in the µM range) further demonstrated that LysM protein domains bind carbohydrate ligands lacking N-acetylglucosamine (GlcNAc) moieties. This observation debunks the dogma that LysM only binds ligands containing N-acetylglucosamine such as chitin, lipo-chitooligosaccharides (Nod-factor) and peptidoglycans. Interdisciplinarity was key to these accomplishments and similar approaches have been central for redefining research on chitin and lipo-chitoligosaccharide as ligands for pattern recognition receptors controlling plant immunity and symbiosis.
Another highlight was the successful demonstration of direct receptor binding of lipo-chitooligosaccharides (Nod-factor) by the Nod Factor Receptors (NFR) from Lotus japonicus. Both NFR1 and NFR5 LysM receptors, the two receptors that are critical for triggering the earliest physiological responses in plant roots, bind Nod factor with Kd values in the nanomolar range. This concentration corresponds to the Nod factor concentrations triggering physiological responses, for example calcium spiking, and is clearly different from chitin binding. These results were obtained by developing several novel methodologies for protein expression and purification of the intact and active receptor proteins/ectodomains and technology for labelling of Nod factor, chitin and other carbohydrates.
Kawaharada Y., Kelly S., Wibroe Nielsen M., Hjuler C.T Gysel K., Muszyński A., Carlson, RW. Thygesen, M.B. Sandal N., Asmussen M.H. Vinther M. , Andersen S.U. Krusell L., Thirup, S. , Jensen K.J. , Ronson C.W. Blaise M. , Radutoiu S. & Stougaard J. (2015) Receptor-mediated exopolysaccharide perception controls bacterial infection. Nature 523, 308-312.
Kelly, S.J. Muszynski, A., Kawaharada, Y., Hubber, A.M. Sullivan, J.T. Sandal, N., Carlson, R.W. Stougaard, J. and Ronson, C.W. (2013) Requirement for Exopolysaccharide in the Mesorhizobium-Lotus Symbiosis. MPMI 26, 319-329.
Another highlight was the successful demonstration of direct receptor binding of lipo-chitooligosaccharides (Nod-factor) by the Nod Factor Receptors (NFR) from Lotus japonicus. Both NFR1 and NFR5 LysM receptors, the two receptors that are critical for triggering the earliest physiological responses in plant roots, bind Nod factor with Kd values in the nanomolar range. This concentration corresponds to the Nod factor concentrations triggering physiological responses, for example calcium spiking, and is clearly different from chitin binding. These results were obtained by developing several novel methodologies for protein expression and purification of the intact and active receptor proteins/ectodomains and technology for labelling of Nod factor, chitin and other carbohydrates.
Kawaharada Y., Kelly S., Wibroe Nielsen M., Hjuler C.T Gysel K., Muszyński A., Carlson, RW. Thygesen, M.B. Sandal N., Asmussen M.H. Vinther M. , Andersen S.U. Krusell L., Thirup, S. , Jensen K.J. , Ronson C.W. Blaise M. , Radutoiu S. & Stougaard J. (2015) Receptor-mediated exopolysaccharide perception controls bacterial infection. Nature 523, 308-312.
Kelly, S.J. Muszynski, A., Kawaharada, Y., Hubber, A.M. Sullivan, J.T. Sandal, N., Carlson, R.W. Stougaard, J. and Ronson, C.W. (2013) Requirement for Exopolysaccharide in the Mesorhizobium-Lotus Symbiosis. MPMI 26, 319-329.