Elicitor release upon flagellin glycan modification

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.

The AIM of this project is to determine how the host GH35 glycosidase contributes to immunity against the bacterial plant pathogen Pseudomonas syringae. The HYPOTHESIS is that GH35 modifies the glycan of flagellin and facilitates the release of peptide elicitors that are perceived by plants. This is IMPORTANT as nearly all plants recognise bacterial pathogens by perceiving flagellin-derived elicitors, yet the extracellular events that result in the release of the elicitors that are embedded in the flagellin structure are yet unknown. PRELIMINARY DATA show that GH35-treated bacteria release an oxidative burst in plants that is dependent on the flagellin receptor FLS2 in the plant and flagellin-encoding fliC in the pathogen. GH35 depletion increases susceptibility upon spray-inoculation, consistent with a crucial role of flagellin perception in preinvasive immunity. P. syringae flagellin is O-glycosylated at six positions with a trisaccharide consisting of two rhamnose residues and one viosamine. Genes responsible for viosamine biosynthesis and the glycosylation of flagellin have been described, and mutant strains are available. This project has three OBJECTIVES. First, we will determine the modification of glycosilated flagellin and its role in FLS2 signalling and immunity by taking advantage of the glycan mutants, the oxidative burst assay, mass spectrometry and disease assays. Second, we will investigate the broader role of GH35-mediated elicitor release by testing the release of other flagellin-derived elicitors that are perceived in rice and tomato, and testing if elicitors can be released from other strains of P. syringae and other bacterial plant pathogens and correlate flagellin protection with the presence of viosamine biosynthesis genes. Third, we will investigate flagellin processing by identifying the processing sites and responsible proteases, taking advantage of the extensive protease research tools available in the laboratory.

The primary and immediate impact of this research will be enhanced knowledge and understanding of how plant pathogens manipulate the host to cause disease, and how the host defends itself to prevent this. This knowledge will be communicated to the scientific community; commercial partners and general public in several ways (see Pathways-to-Impact).

This project will have a deep impact on plant science because we will elucidate an important mechanism in the recognition of bacterial pathogens by plants. As with many earlier discoveries involving P. syringae as a model pathogen, this project will inspire research to similar mechanisms used to thwart economically important pathogens. This project already includes studies to show that the concept also applies for crop plants (tomato, rice) and important bacterial pathogens (e.g. Xyllela, Xanthomonas, Erwinia). In addition, this project will inspire the discovery of hydrolases releasing elicitors from important fungal and oomycete pathogens. In addition, this project will underline the relevance of studying suppressed defence enzymes at the plant-pathogen interface. In conclusion, this project will deliver paradigm shifts in plant immunity research by bringing novel concepts in apoplast manipulation.
This project will also have a clear impact in glycobiology. This project will annotate a new function of the identified glycosidases: having specificity to bacterial glycans to break down flagellin. Available structural data on these glycosidases can be re-interpreted in the context of this new function, and similar glycosidase relatives may act on similar glycans that decorate other proteins or polysaccharides.

This project will have a significant impact on industry and commercial partners in several ways. First, this project will deliver novel strategies for crop protection. For instance, inhibitor-insensitive glycosidases can be introduced to reduce pathogen susceptibility. Immunity can also be enhanced using agrochemicals that block flagellin glycosylation or prevent glycosidase inhibitor production. Second, this project will stress the use of other glycan-degrading enzymes in enhancing the perception of bacterial, fungal and oomycete pathogens in crops. In conclusion, this project is likely to lead to several novel strategies in crop protection.

This project will make a substantial impact on the general public by the increased awareness of intimate relationships that pathogens engage with their host to cause disease. This project will also nicely illustrate that increased knowledge leads to new strategies for environmentally safe crop protection, stressing the relevance of basic research for the society.

The impact of this project on UK research potential resides in publications of high impact and the training of highly skilled personnel. These activities will strengthen the position of the UK to sustain its 'Knowledge Based Economy'.