Identify the signalling components downstream of the Cf-4/SOBIR1 complex
How is the specificity of the interaction between cytoplasmic signalling partners (RLCKs) and downstream MAP kinases, and between the RLCKs and the enzyme taking care of the production of reactive active oxygen species (RBOH), regulated? Which RLCKs bridge the gap between the activated Cf-4/SOBIR1 complex and the MAP kinases?
Plants deploy numerous cell surface-localized immune receptors to perceive microbe-derived effectors. The Cf protein Cf-4 and its co-receptor SOBIR1 constitutively interact at the plasma-membrane and this Cf-4/SOBIR1 complex acts like a two-component receptor-like kinase (RLK).
When the effector Avr4 from invading C. fulvum comes in, the co-receptor BAK1 is recruited to the Cf-4/SOBIR1 complex. Avr4 might directly bind to the extracellular domain of Cf-4 and might act as a “bridge” for BAK1 binding. Trans-phosphorylation events have been shown to take place between the kinase domains of BAK1 and SOBIR1, and probably various downstream RLCKs will be phosphorylated too. Subsequently, these activated RLCKs are probably released from the complex, and in their turn phosphorylate the RBOH enzyme, thereby activating it and inducing ROS production. The defence signal is amplified through a MAP kinase cascade and possibly also by the activation of so-called calcium-dependent protein kinases (CDPKs) that is caused by a strong Ca2+ influx, which then activates hypersensitive cell death. It is clear that RLCKs and MAP kinases play very important roles in plant immunity, but it is not known which MAP kinases and RLCKs are actually involved and how they perform downstream signalling.
- Identification of the RLCKs that bridge the gap between the activated Cf-4/SOBIR1 complex and downstream MAP kinase cascade by multiplex CRISPR/Cas technology
- Search for the MAP kinases involved in Cf-4 immune signalling by proximity-dependent labelling of RLCKs, followed by mass spectrometry
- Identify important RLCKs by performing proximity-dependent labelling with candidate MAP kinases known to play a role downstream of Cf-4
- • Identification of the various RLCK/MAPK signalling pathways that are involved downstream of the Cf-4/SOBIR1 complex
- HR test by red light imaging
- MAPK activation studies by western blotting
- CRISPR-Cas9 mediated gene knock-out
- Proximity-dependent labelling
- Analysis of proteome data obtained by mass spectrometry
- In vivo and in vitro phosphorylation studies