Fig 2. The EAR motif of RxL21 interacts with the CTLH domain of TPL.

(A) RxL21 co-localizes with TPL in the nucleus. GFP:RxL21 and RFP:TPL were expressed transiently in N. benthamiana. Scale bars are 10 μm. (B) TPL Interacts with the EAR domain of RxL21 in yeast. RxL21 was cloned without the signal peptide and lacking the EAR motif (RxL21ΔEAR1; Δ402–409 and HaRxL21ΔEAR2; Δ360–409), without the N-terminus (RxL21ΔN; Δ1–36) and RxLR-DEER motif (RxL21ΔRxLR; Δ1–51) and combinations of the above. Amino acid locations of deletion constructs are indicated. Deleting the EAR motif at the C-terminus of RxL21 abolishes interaction with TPL by Y2H. Successful mating is indicated by growth on media lacking Leucine and Tryptophan (-LW). Growth on media additionally lacking Histidine (-LWH) indicates GAL1::HIS3 reporter gene activation and protein-protein interaction. 3AT is used to increase stringency in a concentration dependent manner. Growth on media additionally lacking Adenine (-LWHA) indicates activation of the GAL2::ADE2 reporter gene. AD; activation domain. DB; DNA binding domain. (C) Deleting the CTLH domain of TPL (TPLΔCTLH; Δ25–91) abolishes interaction with RxL21 in yeast. Interaction (indicated by growth on–LWH +1mM 3AT) was observed between RxL21 and TPL but not between RxL21 and TPLΔCTLH. All Y2H was repeated at least twice with similar results. (D) TPL interacts with RxL21 and the interaction requires the EAR motif in planta. TPL:HA, TPLΔCTLH:HA together with RxL21 and RxL21 EAR motif mutants RxL21ΔEAR, RxL21-FMFTF and RxL21-AMATA (under control of an estradiol-inducible promoter and N-terminal Myc tag) were transiently expressed in N. benthamiana leaves. RxL21 expression was induced by 30μM β-estradiol 24 hr prior to harvesting. c-myc beads were used for immunoprecipitation (IP). HA antibody was used for TPL and TPLΔCTLH immunoblots (IB) and c-myc antibody was used to detect RxL21, RxL21ΔEAR and the respective EAR motif mutants.