Cysteine-rich Receptor-like Kinase 7 functions as a receptor for Wall Teichoic Acid, a MAMP specific for gram-positive bacteria with non-canonical activities in Arabidopsis thaliana
Doctoral thesis
Date of Examination:2024-09-09
Date of issue:2024-10-10
Advisor:Prof. Dr. Volker Lipka
Referee:Prof. Dr. Volker Lipka
Referee:Prof. Dr. Jörg Stülke
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Abstract
English
Plants are constantly surrounded by a plethora of microorganisms, including fungi, oomycetes, and bacteria. To sense their presence and distinguish between different kinds of microbes, plants utilize a variety of cell-surface receptors, sensing Microbe-Associated Molecular Patterns (MAMPs). MAMPs represent components indispensable for microbial fitness, like chitin derived from the fungal cell wall or flagellin originating from the bacterial flagellum. Cell-surface receptors are evolutionary diverse concerning their extracellular receptor domain. Leucine-rich repeats are predominantly used for the recognition of proteinaceous MAMPs. Lysin-motif domains and lectin-like domains are used to recognize carbohydrate- or lipid-based MAMPs. Proteins containing DUF26 domains were previously described to act as mannose-binding lectins. Therefore, DUF26- containing cell surface receptors, like Cysteine-rich Receptor-like Kinases (CRKs), are predicted to act as carbohydrate receptors. Cell-surface receptor mediated recognition of bacterial MAMPs has been intensively studied using the gram-positive model organism Pseudomonas syringae. However, our knowledge about gram-positive bacterial MAMPs remains limited. Wall teichoic acids (WTAs) are large, anionic glycopolymers specific for the cell wall of gram-positive bacteria. WTAs make up for up to 60% of the dry weight of gram-positive bacteria. They are indispensable for their fitness, which makes WTAs a suitable candidate for a gram-positive bacteria-specific MAMP. This work shows that different heat-inactivated gram-positive bacteria induce a Programmed Cell Death (PCD) reaction when infiltrated into the leaves of Arabidopsis Col-0. Using biochemical and genetic approaches, glycosylated WTA derived from the different gram-positive bacteria was identified as the PCD-inducing agent. In addition to PCD, WTA treatment induced expression of the salicylic marker gene PR1 and primed the plant's defense response towards subsequent infection with Pseudomonas syringae pv tomato DC3000. Employing a reverse genetic screen, genetic evidence was provided showing that plant perception of WTA and subsequent induction of defense responses depend on Cysteine-rich Receptor-like Kinase 7 (CRK7) and the EDS1-PAD4-ADR1 signaling hub that connects MAMP perception to ETI-like defense responses. The indispensable nature of CRK7 for WTA recognition was further supported by the complementation of the crk7-5 mutant with wild-type and fluorophore-tagged CRK7. Functional characterization of CRK7 demonstrated that CRK7 forms homo-oligomers at the plasma membrane when expressed in N. benthamiana, which was further induced in a ligand-dependent manner, a common mechanism of carbohydrate receptors. In conclusion, CRK7 likely functions as a receptor for glycosylated WTA in Arabidopsis that induces non-canonical defense responses via the EDS1-PAD4-ADR1 node.
Keywords: CRK7; wall teichoic acid; plant immunity; gram-positive bacteria