Identification of receptor complex components and receptor activation mechanisms in plant innate immunity (2011)

Plants rely on an innate immune system which successfully recognizes and restricts pathogenic microbes. The key for this defense is the detection of pathogen derived non-self signatures and endogenous elicitors released during a microbial attack.&#13;\nHere we report the identification of PEPR2, a new receptor for endogenous elicitors in Arabidopsis (chapter 1). Together with its homologue PEPR1 it functions redundantly in the recognition of AtPep1, a plant derived peptide released during wounding and pathogen defense. Our analysis showed that the defense signaling triggered upon AtPep1 stimulation exhibits strong similarity to the response to microbe derived elicitors.&#13;\nFor detection of pathogen derived elicitors the flagellin perception through the receptor FLS2 evolved as model system in plants. FLS2 is known to function together with an associated receptor-like kinase referred to as SERK3/BAK1. In an in vitro analysis of the FLS2-kinase and the BAK1-kinase we were able to show, that FLS2 is a substrate for the BAK1-kinase. This indicates that BAK1 acts as upstream kinase, which phosphorylates and activates the receptor upon dimerization (chapter 2). Using a mass spectrometric analysis on immunopurified FLS2 protein we identified one elicitor independent and one elicitor dependent putative phosphorylation site. The position of both sites suggests a role for phosphorylation in the regulation of ubiquitination and endocytosis.&#13;\nWe further analyzed the impact of receptor kinase activity by a characterization of a kinase inactive version of the EF-Tu receptor EFR (chapter 3). This analysis verified that also EFR functions through BAK1 and demonstrated that kinase activity of the receptor is not required for formation of the EFR/BAK1 complex. Strikingly, kinase inactive EFR was able to initiate an elicitor dependent ethylene accumulation and conferred partial resistance to Agrobacterium tumefaciens, while other signaling events were absent. This finding revealed a diverging signaling network in which not all pathways require receptor kinase activity to get activated.&#13;\nBy immunopurification and subsequent mass spectrometric analysis of FLS2 protein we further explored this signaling system and its components. Importantly we found not only BAK1, but also its paralogues SERK1, SERK2, SERK4 and SERK5 to co-purify with the flagellin receptor, which indicates a redundant function of these proteins (chapter 4). We also identified several isoforms of the family of 14-3-3 general regulating factors. This is in line with an in silico analysis of the FLS2 sequence, which predicted the putative phosphorylation site S 1078 to operate as 14 3 3 protein binding site. Another group of proteins which co-purified with FLS2 in an elicitor dependent manner comprises RAB-GTPases and SNARE proteins. These protein factors are known to control vesicle fusion events. Since bacterial infections trigger focal secretion, we speculate that the elicitor activated FLS2 complex might lead secretory vesicles directly to the site of infection.&#13;\nTaken together this works provides new insight into different levels of plant immunity. This includes not only the identification of a new receptor and receptor associated proteins, but also adds new aspects to our understanding of receptor activation and downstream signaling. Therefore these results provide a basis to further investigate plant innate immunity on the whole.