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-componentsystem His kinase-like ethylene receptors have direct protein–proteininteractions with a single downstream Ser/Thr kinase CTR1. Thesecomponents of the ethylene signalling network found in Arabidopsisare conserved in the climacteric fruit tomato, but both theethylene receptors and CTR1-like proteins (LeCTRs) in tomatoare encoded by multigene families. Here, using a yeast two-hybridinteraction 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 taggedproteins revealed that the ethylene receptor NR was targetedto the endoplasmic reticulum (ER) when transiently expressedin onion epidermal cells, whereas the four LeCTR proteins werefound in the cytoplasm and nucleus. When co-expressed with NR,three LeCTRs (1, 3, and 4), but not LeCTR2, also adopted thesame ER localization pattern in an NR receptor-dependent mannerbut not in the absence of NR. The receptor–CTR interactionswere 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 theseLeCTR proteins to the ER membrane through direct protein–proteininteraction. The receptor–CTR interactions and localizationobserved in the study reinforce the idea that ethylene receptorstransmit the signal to the downstream CTRs and show that a singlereceptor can interact with multiple CTR proteins. It remainsunclear whether the different LeCTRs are functionally redundantor have unique roles in ethylene signalling. Key words: BiFC, endoplasmic reticulum, Ser/Thr kinase, tomato ethylene signalling, two-component system His kinase