Title: Identification of LEFKOTHEA/PORR protein partners

LEFKOTHEA (At5g62990) is a nuclear gene encoding an RNA-binding protein that carries a transit peptide (TP) and monopartite or bipartite nuclear localization signals (NLS). These motifs result to dual-targeting of LEFKOTHEA to the nucleus and chloroplasts. To identify the chloroplast and nuclear protein interactors of LEFKOTHEA, co-immunoprecipitation coupled to mass spectrometry (co-IP/MS) analysis was performed. The construct of LEFKOTHEA gene, expressed under the control of the endogenous promoter, with a FLAG tag at the C-terminus was introduced in Arabidopsis lefko2 mutant plants using the Agrobacterium system. The proteins were extracted under native conditions and incubated with anti-FLAG M2-agarose affinity gel. High salt concentrations and 3X FLAG peptide were applied to the beads for protein elution. The eluted protein fractions were subjected to Ultra High Pressure nanoLC-MS/MS. The LEFKOTHEA protein partners were identified using the MaxQuant software (version 1.5.3.30) against the complete Uniprot proteome of Arabidopsis thaliana (version of 19/1/2016) and a common contaminants database by the Andromeda search engine. Protein abundance was calculated on the basis of the normalized spectral protein intensity as label free quantitation (LFQ intensity). The statistical analysis was performed using Perseus (version 1.5.3.2).