Yeast two‐hybrid‐sequencing and bifluorescence complementation resources for assessing protein–protein interactions in arbuscular mycorrhizal roots: <scp>CKL2</scp> as a case study

Reverse genetics, facilitated by CRISPR technologies and comprehensive sequence-indexed insertion mutant collections, has advanced the identification of plant genes essential for arbuscular mycorrhizal (AM) symbiosis. However, a mutant phenotype alone is generally insufficient to reveal the specific role of a protein in AM symbiosis, and in many cases, identifying interacting partner proteins is useful. To enable the identification of protein-protein interactions during AM symbiosis, we established a Medicago truncatula-Diversispora epigaea yeast two-hybrid (Y2H) library which, through Y2H-sequencing (Y2H-seq) screening, can provide a rank-ordered list of candidate interactors of a protein of interest. We also developed a vector system to facilitate bimolecular fluorescence complementation assays (BiFC) in mycorrhizal roots so that protein interactions can be assessed in their native cell types and subcellular locations. We demonstrate the utility of a Y2H-seq screen coupled with BiFC in mycorrhizal roots, with a search for proteins that interact with CYCLIN-DEPENDENT KINASE-LIKE 2 (CKL2), a kinase essential for AM symbiosis. The Y2H-seq screen identified three 14-3-3 proteins as the highest ranked CKL2 interacting proteins. BiFC assays in mycorrhizal roots provided evidence for a CKL2-14-3-3 interaction at the periarbuscular membrane (PAM) in colonized root cells. Downregulation of 14-3-3 by RNA interference provides initial evidence for a function in AM symbiosis. Thus, CKL2 may utilize 14-3-3 proteins to direct signaling from the PAM. The Y2H and BiFC resources will accelerate understanding of protein functions during AM symbiosis.