Project
Regulation and activation of the Cf-4/SOBIR1/BAK1 complex
Deciphering of how the Cf/SOBIR1/BAK1 complex is assembled, and which domains of these different proteins are essential for complex assembly and subsequent initiation of the downstream cytoplasmic immune response, is important to understand how the first line of defense in plant immunity fundamentally functions. We aim to understand how the activity of the Cf/SOBIR1/BAK1 complex is regulated.
Background
Plants only have an innate immune system to protect themselves against microbial infection. The first layer of defense is mediated by extracellular plasma membrane-associated receptors. These cell surface receptors perceive extracellular immunogenic patterns and trigger the initiation of downstream defense signaling, which finally leads to extracellularly-triggered immunity.
Cf resistance proteins of tomato that act against the fully extracellular pathogenic fungus Cladosporium fulvum are so-called receptor-like proteins (RLPs) that localize at the cell surface. Cf proteins require two co-receptors for the activation of downstream cellular responses, because of the lack of a cytoplasmic kinase domain. In the resting state, the Cf-4 protein constitutively interacts with the receptor-like kinase (RLK) SUPPRESSOR OF BIR1 (SOBIR1), whereas upon recognition of the Avr4 effector of C. fulvum by Cf-4, the RLK BRI1-ASSOCIATED KINASE 1 (BAK1) is recruited by the Cf-4/SOBIR1 complex.
Subsequent trans-phosphorylation of the kinase domains of the RLKs SOBIR1 and BAK1 then initiates downstream signaling, which results, amongst others, in a swift production of reactive oxygen species (ROS) by the respiratory burst oxidase homolog protein (RBOH) that gets phosphorylated, the activation MAP kinase (MAPKs) cascades and eventually the activation of hypersensitive cell death and resistance. In their turn, receptor-like cytoplasmic kinases (RLCKs) are thought to play a role in downstream signaling to initiate the ROS burst and connect immune signaling triggered by the cell surface receptors with the cytoplasmic signaling machinery.
Project description
Studying the roles of specific domains of BAK1, SOBIR1 and Cf-4 in the assembly of the BAK1/Cf-4/SOBIR1 complex and their participation in the initiation of downstream signaling; studying possible negative regulation of the activated BAK1/Cf-4/SOBIR1 complex by BAK1-INTERACTING RECEPTOR-LIKE KINASES (BIRs); studying how specific recruitment of BAK1 by the Cf-4/SOBIR1 complex takes place upon Avr4 perception; determining whether a direct or indirect interaction between the LRR domain of Cf-4 and Avr4 takes place and studying how immune signaling is actually initiated.
Used techniques
- Molecular cloning
- Agrobacterium-mediated transient transformation assays
- Immunoprecipitation
- Co-immunoprecipitation
- Western Blotting
- Proximity-dependent labeling
- Split-luciferase assays
- Confocal microscopy
- Luminol-based measurement of ROS production
