Technische Universität München Lehrstuhl für Phytopathologie Lipopolysaccharide of plant-associated bacteria and their role in innate immunity of Arabidopsis thaliana

Lipopolysaccharide (LPS), a glycolipid of unique molecular composition, is the main component of the outer membrane of Gram-negative bacteria. It protects the bacterial cell from external stresses and is involved in various interaction processes between the bacteria and their environment. LPS can be divided into three substructures the lipid A moiety, the core oligosaccharide (core-OS) and the O-polysaccharide (OPS) which fulfill different functions and thus contribute to the properties of the molecule. The role of LPS during plant-bacteria interactions, however, remains unclear and genetic information about LPS synthesis in plant-associated bacteria is largely missing. As part of this thesis, genomes of Pseudomonas spp. were analysed for genes involved in LPS synthesis using Pseudomonas aeruginosa as reference. While homologs of most of the lipid A and core-OS synthesis genes could be identified, OPS gene clusters were not conserved in most plant-associated Pseudomonas strains. However, single homologs of genes involved in OPS synthesis were found. Gene homologs of the glycosyltransferase WbpL which initiates OPS synthesis in P. aeruginosa are present in all analyzed strains. Deletion of the respective gene in the plant pathogens Pseudomonas syringae pv. tomato DC3000 (Pst) and Pseudomonas cichorii ATCC10857/DSM50259 disrupts OPS synthesis and leads to the production of OPS-deficient LPS. These ∆wbpL knockout strains display a reduced motility and are impaired in their ability to infect host plants. This indicates that OPS contributes to host colonization processes and therefore is an important virulence factor of plant-pathogenic Pseudomonas species. Elucidation of the Pst core-OS revealed a relatively high degree of phosphorylation. Together with the conservation of core-OS kinases in the analyzed Pseudomonas genomes the results suggest that a highly phosphorylated core-OS might be a common feature of the genus Pseudomonas. LPS is considered to be a microbe-associated molecular pattern (MAMP) which is perceived by the plant immune system. In A. thaliana, immune responses after the treatment with LPS preparations from Pseudomonas spp. and Xanthomonas spp. require the receptorlike kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE). Screens with various LPS preparations and structurally similar compounds were conducted to identify the minimal MAMP-active motif of LPS. The results show that not LPS, but free medium chain 3-hydroxy (mc-3-OH) fatty acids are sensed by A. thaliana in a LORE-dependent manner. The strongest immune responses were elicited by 3-hydroxy decanoic acid (3-OH-C10). Bacterial compounds which comprise 3-OH-C10-acyl moieties with blocked 3-OHor COOHgroup such as LPS, rhamnolipids, lipopeptides, and N-acylhomoserine-lactones are not inducing LORE-dependent immune responses. However, free mc-3-OH fatty acids could be released during synthesis of these compound or in other metabolic processes. The data therefore suggests A. thaliana senses microbial metabolites rather than complex bacterial compounds to trigger LORE-dependent immune responses.