Detection of in vivo protein interactions between Snf1-related kinase subunits with intron-tagged epitope-labelling in plants cells

Plant orthologs of the yeast sucrose non-fermenting (Snf1) kinase and mammalian AMP-activated protein kinase (AMPK) represent an emerging class of important regulators of metabolic and stress signalling. The catalytic α-subunits of plant Snf1-related kinases (SnRKs) interact in the yeast two-hybrid system with different proteins that share conserved domains with the β- and γ-subunits of Snf1 and AMPKs. However, due to the lack of a robust technique allowing the detection of protein interactions in plant cells, it is unknown whether these proteins indeed occur in SnRK complexes in vivo. Here we describe a double-labelling technique, using intron-tagged hemagglutinin (HA) and c-Myc epitope sequences, which provides a simple tool for co-immunopurification of interacting proteins expressed in Agrobacterium-transformed Arabidopsis cells. This generally applicable plant protein interaction assay was used to demonstrate that AKINβ2, a plant ortholog of conserved Snf1/AMPK β-subunits, forms different complexes with the catalytic α-subunits of Arabidopsis SnRK protein kinases AKIN10 and AKIN11 in vivo.