Elicitor release upon flagellin glycan modification

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Immunity to pathogens in plants is essential for agriculture and even for life on earth. Pathogen recognition is the first crucial step in plant immunity. Most plants recognise bacterial pathogens by their flagella, which bacteria use to move. Different plant species can recognise different fragments of the flagellin proteins, though most seed plants recognise a conserved, 22 amino acid region, known as flg22, the major recognition element known for bacterial pathogens. Recognition of flg22 at the cell surface by the FLS2 receptor-like kinase is very well studied. Flg22 perception elicits immune responses in most plants, including the model plant Arabidopsis thaliana and tobacco relative Nicotiana benthamiana. These immune responses include an oxidative burst, MAP kinase signalling and transcriptional reprogramming, mounting a defence response that includes cell wall strengthening and the secretion of toxic metabolites and harmful proteins. The relevance of flg22-triggered immunity is stressed by the fact that adapted bacterial pathogens all use effectors to block flg22-induced signalling. Currently, flg22-FLS2 signalling is the best understood and most important recognition system of plant pathogens. A crucial step, however, is still unresolved. Flagellin-derived elicitors like flg22 are embedded deep within the structure of flagellin protein and reside inside the flagellar rod. How can these buried elicitors bind to the FLS2 receptor? It seems obvious that they must be released by extracellular hydrolase activities, but elicitor-releasing hydrolases and even the naturally-released elicitor have not yet been identified. One may expect that bacteria suppress these elicitor-releasing hydrolases during infection to prevent their recognition, so it is very likely that flagellin hydrolysis represents an important battlefield at the plant-pathogen interface. Importantly, we discovered an extracellular galactosidase-like GH35 glycosidase that releases flagellin-derived elicitors from bacteria. The relevance of this GH35 enzyme was indicated by a suspicious cover-up: GH35 is specifically inhibited during bacterial infection by a bacterial metabolite. Importantly, GH35 treatment of the model bacterial pathogen Pseudomonas syringae triggers an oxidative burst in Nicotiana benthamiana and Arabidopsis thaliana. This oxidative burst is dependent on the FLS2 receptor in the plant, and the flagellin-encoding fliC gene in the pathogen. Mutant N. benthamiana lacking the GH35 enzyme are more susceptible for P. syringae, confirming their role in immunity.Flagella are glycosilated with a unique trisaccharide glycan. We HYPOTHESISE that GH35 acts on these glycans and that this modification, in concert with other hydrolases, results in the release of flagellin-derived elicitors that bind to FLS2. Using well described P. syringae mutants with altered flagellin glycosylation and Nicotiana benthamiana as a host, we are in the unique position to test how GH35 contributes to immunity. The AIM of this proposal is to elucidate how flagellin-derived elicitors are released by GH35 and other hydrolases and to investigate how common this mechanism is in plant-bacteria interactions. The OBJECTIVES are to: i) elucidate the role and mechanism of GH35 modification of flagellin; ii) investigate the broader role of GH35-mediated elicitor release from flagellin in crop plants and from other pathogens; and iii) elucidate protein processing of flagellin upon GH35 treatment.This project will lead to the elucidation of an important novel mechanism in bacterial pathogen recognition by plants that is probably universal in the plant kingdom. Similar hydrolase-driven elicitor release is expected for the recognition of filamentous pathogens. These discoveries will inspire new crop protection strategies, including the introduction of inhibitor-resilient hydrolases and agrochemicals blocking flagellin glycosylation or GH35 inhibitor biosynthesis.