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Abstract Reverse genetics, facilitated by CRISPR technologies and comprehensive sequence-indexed insertion mutant collections, has advanced the identification of plants genes essential for arbuscular mycorrhizal (AM) symbiosis. However, a mutant phenotype alone is generally insufficient to reveal the specific role of the protein in AM symbiosis and in many cases, identifying interacting partner proteins is useful. To enable identification of protein:protein interactions during AM symbiosis, we established aMedicago truncatula -Diversispora epigaeayeast-two-hybrid (Y2H) library which, through 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 sub-cellular 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 LIKE KINASE 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. Down-regulation 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.