The C-terminal domain of the LjSYMREM1 protein mainly contributed to protein interactions.

<p>The yeast split-ubiquitin assay was used to test interactions between the LjSYMREM1 variants and the RLKs NFR1, NFR5 and SYMRK. The coding regions were fused to the C-terminal half (Cub) and the N-terminal half (NubG) of ubiquitin and interaction was tested on an individual basis. Yeast growth on medium lacking leucine and tryptophan (−LW) indicates presence of both constructs. Interaction was tested on medium additionally lacking histidine (−LWH) that was supplemented with 15 mM 3-amino-1,2,4-triazole (3-AT) to suppress residual levels of endogenous histidine biosynthesis. The yeast resident ER protein Alg5 was used as negative control (Alg5:NubG and Alg5:Cub). Yeast growth was sustained on –LWH medium indicating strong interaction of the RLKs and Remorins variants with LjSYMREM1_C (A). Weak interaction of LjSYMREM1_N with the RLKs and Remorins variants indicates minor or transient contribution of the N-terminal region to protein interactions (B). Pigmentation of yeast indicates severe adenine deficiency as a consequence of lacking interaction. A series of three dilutions (non-diluted, 10⁻¹ and 10⁻²) are shown in each panel from left to right).