Difference between revisions of "Part:BBa K3610046"

Revision as of 10:16, 19 October 2020

CORE ectodomain / YFP

This part contains the ectodomain of the plant cell surface receptor CORE from A. thaliana fused to a yellow fluorescent protein. This part lacks the natural N-terminal signal sequence but instead uses the signal sequence from the alpha-Factor from yeast.

Usage and Biology

CORE

The cold shock protein receptor (CORE) is a plant pattern recognition receptor (PRR) and as such activates host innate immunity through detection of pathogen-associated molecular patterns (PAMPs). CORE is a leucine-rich repeat receptor-like kinase with 22 LRRs, there additionally is a 6 amino acid insert at LRR 11. It consists of an extracellular domain that perceives an epitope, csp22, from the highly conserved nucleic acid binding motif RNP-1 of bacterial cold-shock proteins (CSPs), which are highly abundant proteins found in the cytosol of bacteria. Further domains are a single pass transmembrane domain and an intracellular kinase domain (The sequence encoding the kinase domain is not in this part). Interaction of CORE with brassinosteroid-associated kinase (BAK)1 is necessary for inducing an immune response in the plant. The dimerization of CORE and BAK1 depends on the csp22, the ligand of CORE. The function of CORE in S. lycopersicum has been confirmed by expressing the receptor in A. thaliana, which made the plant responsive to csp22, a PAMP that is otherwise not perceived by PRRs from A. thaliana.

CORE with YFP

In this sequence, the C-terminal domain entailing the intracellular kinase domain was replaced with the sequence coding for the yellow fluorescent protein venus, while the ectodomain and the transmembrane domain, including the juxtamembrane domain were kept. Additionally, a signal sequence native to S. cerevisiae was fused to the N-terminal sequence, which does not contain the native signal peptide. This way, the protein can be integrated into the membrane during translation and the expression can be observed as with the receptor protein, the YFP (Exλ : 515 nm, Emλ : 528 nm) gets translated as well.

Characterization

Expression of CORE ectodomain / YFP in S. cerevisiae

After successful transformation of yeast cells we checked for expression of the protein under a confocal microscope.

Imaging did not reveal increased fluorescence at the excitation and emission wavelengths for YFP.

Sequence and Features