Tomato ethylene receptor-CTR interactions: visualization of NEVER-RIPE interactions with multiple CTRs at the endoplasmic reticulum

In the model plant Arabidopsis, members of a family of two-component system His kinase-like ethylene receptors have direct protein-protein interactions with a single downstream Ser/Thr kinase CTR1. These components of the ethylene signalling network found in Arabidopsis are conserved in the climacteric fruit tomato, but both the ethylene receptors and CTR1-like proteins (LeCTRs) in tomato are encoded by multigene families. Here, using a yeast two-hybrid interaction assay, it is shown that the tomato receptors LeETR1, LeETR2, and NEVER-RIPE (NR) can interact with multiple LeCTRs. In vivo protein localization studies with fluorescent tagged proteins revealed that the ethylene receptor NR was targeted to the endoplasmic reticulum (ER) when transiently expressed in onion epidermal cells, whereas the four LeCTR proteins were found in the cytoplasm and nucleus. When co-expressed with NR, three LeCTRs (1, 3, and 4), but not LeCTR2, also adopted the same ER localization pattern in an NR receptor-dependent manner but not in the absence of NR. The receptor-CTR interactions were confirmed by biomolecular fluorescence complementation (BiFC) showing that NR could form a protein complex with LeCTR1, 3, and 4. This suggested that ethylene receptors recruit these LeCTR proteins to the ER membrane through direct protein protein interaction. The receptor-CTR interactions and localization observed in the study reinforce the idea that ethylene receptors transmit the signal to the downstream CTRs and show that a single receptor can interact with multiple CTR proteins. It remains unclear whether the different LeCTRs are functionally redundant or have unique roles in ethylene signalling.