Triplin, a small molecule, reveals copper ion transport in ethylene signaling from ATX1 to RAN1
Fig 5
The copper chaperone ATX1 interacts with RAN1.
(A) atx1-1 and atx1-2 are hypersensitive to triplin. The phenotypes of 3-day-old, dark-grown Col-0, atx1-1 and atx1-2 seedlings treated with 20 μM triplin are shown. Scale bar represents 1 mm. (B) Hypocotyl lengths of 3-day-old, dark-grown seedlings of Col-0, atx1-1, atx1-2, ran1-2, atx1-1 ran1-2 and two 35S:ATX1-GFP (atx1-1) transgenic lines treated with different doses of triplin are shown. Each experiment was repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. (C) Subcellular co-localization of ATX1 and RAN1. ATX1-RFP and RAN1-GFP were transiently expressed in N. benthamiana leaves and observed and imaged under a confocal microscope. Scale bars represent 20 μM. (D) ATX1 interacted with RAN1 in yeast two-hybrid assay. ATX1 was fused to a GAL4 DNA-binding domain (BD) and RAN1-N (289 amino-terminal amino acids of RAN1) was ligated to a GAL4 activation domain (AD). The protein interactions were examined on cells grown on synthetic dropout (-Leu/-Trp/-His/-Ade) medium plus X-α-Gal (50mg/L) plates for 3 days. (E) Bimolecular fluorescence complementation assays showed interaction between ATX1 and RAN1 using the split luciferase system. Nicotiana benthamiana leaves were infiltrated with agrobacteria containing different construct combinations harboring both the C- and N-terminal of the luciferase fused to either ATX1 and RAN1-N or just one of them (controls). (F) Co-Immunoprecipitation assays showed interaction between ATX1 and RAN1. Nicotiana benthamiana leaves were infiltrated with agrobacteria containing ATX1-GFP/FLAG and RAN1-N-FLAG/GFP or just one of them (controls). The protein extracts were immunoblotted with anti-FLAG antibody or anti-GFP antibody.