Figure 3: FASS interacts with both TON1 and TRMs.

(a,b) Interaction between TON1 and FASS. FASS interacts with TON1 in yeast two-hybrid experiments (a) and BiFC experiments in Nicotiana benthamiana abaxial epidermal leaf cells (b). (a) Full-length, N- or C-terminal fragments of FASS and TON1 were tested for interaction in yeast two-hybrid. (+) and (−) indicates positive and negative results. Absence of interaction between TON1 full-length protein and FASS might result from improper folding in yeast or from the full-length protein being locked into a ‘closed’ conformation, masking potential interaction domains. Interactions with FASS^186–480 were meaningless due to strong self-activation of this construct. The position of the two A subunit-binding domains of FASS (ASBD1 and ASBD2) and the TOF, LisH-PLL, and the serine-rich (S) domains of TON1 are indicated. TON1 fragments were fused to the GAL4 activation domain and FASS to the LEX-binding domain. (b) Transient expression assays in Nicotiana benthamiana leaf epidermal cells. When overexpressed in this system, GFP-TON1 and GFP-FASS accumulate in the cytoplasm, which is restricted to the cell’s periphery in these epidermal jigsaw puzzle cells. Co-expression of BiFC constructs YFP^N-FASS and YFP^C-TON1 gave a clear cytoplasmic YFP fluorescence signal, revealing interaction between FASS and TON1. Negative controls correspond to co-expression of YFP^C-TON1 with the unrelated YFP^N-GLOBOSA protein¹¹ (YFP^N-GLO). Scale bars, 20μm. (c) The M3 motif of TRMs is involved in direct interaction between TRMs and FASS. A schematic representation of TRMs fragments tested for interaction with FASS in yeast two-hybrid is shown. Coloured boxes correspond to the position of the motifs present in the TRM proteins. (+) and (−) indicates positive and negative results. (d) Schematic view of the interactions between TON1, FASS and TRM proteins. The M2 and M3 motifs of TRMs are involved in interaction with TON1 and FASS, respectively.