Expression and Purification of Cytosolic Domains of Cyclic Nucleotide Gated Calcium Channel (CNGC19)

Abstract

Plants have to deal with a plethora of abiotic and biotic stress during their lifetime. Insect herbivory is one such devastating biotic stress that affects plant productivity worldwide. During evolution, plants have adapted to cope up with this stress condition by perceiving insect herbivory rapidly and activating defence response. Increase in cytosolic calcium level is a key defence response against herbivore attack. Various channels involved in the exchange of calcium ions across cell membrane include Cyclic nucleotide-gated channels (CNGCs), Glutamates like receptors (GLRs) and two-pore channels (TPCs). CNGCs are an essential component of plant defence against insect attack. In Arabidopsis, CNGC19 was identified as the highly expressed upon Spodoptera herbivory. Functional characterisation of CNGC19 reveals that CNGC19 plays a key functional role in defence signalling against Spodoptera. However, the molecular mechanism of calcium flux through CNGC19 remains poorly understood mainly due to lack of three-dimensional structure and biochemical-biophysical characterization. The aim of this project is to overexpress, purify the cytosolic domains of CNGC19 and use them to determine the structure of AtCNGC19. This would help in providing a broader view to decipher the physiological role of CNGC19 and it would also help in understanding the molecular basis of calcium signalling in plant defence response. CNGC19 is known to have cyclic nucleotide monophosphates (cNMPs) binding domain and calmodulin (CaM)-binding domain. CaMs are important calcium sensors that can bind to the carboxy tail of several plant CNGCs in a Calcium-dependent manner and regulate the opening and closing of the channel. In Arabidopsis, out of all the CaM isoforms, CaM2 was highly upregulated by wounding and Spodoptera feeding and shows interaction with C-terminus of CNGC19. The objective of this project is also to overexpress and purify the CaM2 in order to study protein-protein interaction with its interacting partner CNGC19.