Leone, Maria Rosaria (2010) Stucture of macromolecules from Gram-negative bacteria involved in elicitation of plant immune system. [Tesi di dottorato] (Unpublished)

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This project was entirely focused on increasing the knowledge of the structure/activity relationships of LPS in plant immune system and their roles in instauration of important biological process like symbiosis. In the first part of the work, chemical characterization of LPSs from different Burkholderia species was presented. Firstly, we have completely characterized the LPS and the LOS produced by the endosymbiont Burhkolderia rhizoxinica strain B1 isolated from Rhizopus microsporus fungi responsible for causing rice seedling blight. The O-chain fraction from LPS was constituted by a peculiar homopolimer of gactofuranose that presented a particular biological activity since it is able to mimic the host cell wall thus avoiding the immune response. We have also reported the chemical characterization of the LPS from Burhkolderia rhizoxinica strain B4 isolated from Rhizopus microsporus var. microsporus. The O-chain domain was constituted by a mixture of two polysaccharides with different primary structure. The lipid A moiety possessed, in all analyzed Burhkolderia rhizoxinica strains, the typical carbohydrate backbone and revealed to be constituted by a mixture of penta and tetra acylated species carrying on the polar heads the non-stoichiometric presence of Ara4N. Similar data were founded also for lipid A isolated from Burkholderia cepacia strain ASP B 2D, an endophytic bacterium. In this case the lipid A has been found to possess an identical structure of lipid a from Burhkolderia rhizoxinica but with higher amount of under-acylated species and a lower amount of phosphate. Probably, the presence of under-acylated/phosphorylated species and the presence of Ara-4N residues might allow B. cepacia strain ASP B 2D to survive as an endophyte in plant host by reducing the net charge. In the last part of the project, we have reported the full chemical characterization of the LPS from Bradyrhizobium sp. BTAi1 and ORS278, peculiar strains that do not possess a canonical genes for nod factors biosynthesis. The O-chain fraction from these LPSs is formed by a homopolymer with the presence of a unique carbociclic sugar that we named Bradyrhizose; this novel monosaccharide resulted to be linked in the polymer with a-(1→7) linkage for Bradyrhizobium sp. BTAi1 O-chain, while the sugar fraction from Bradyrhizobium sp. ORS278 presented a mixture of polysaccharides constituted by a-(1→7) and a-(1→9) linked Bradyrhizose. Important structural results were found for what corcerns the lipid A fraction from both strains of Bradyrhizobium. The lipid A resulted manly composed of a mixture of hexa- and penta-acylated species with asymmetrical distribution of the acyl chains (arrangement 4+2). Notable is the presence of lipid A species carrying an additional fatty acid (D m/z 512) not yet identified but likely present as terziary fatty acid linked to the o-1 position of the long fatty acids and resulting in an unusual 5+2 arrangement of the acyl chains on the saccharide backbone. This lipid A structure is innovative since it is among the first Bradyrhizobium structures characterized so far. Preliminary biological assays demonstrated that the LPS from both strains of Bradyrhizobium showed a very reduced immune response (tested with callose formation) in Arabidopsis thaliana.

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