Journal: Horticulture Research
Article Title: Scaffold protein RhCASPL1D1 stabilizes RhPIP2 aquaporins and promotes flower recovery after dehydration in rose ( Rosa hybrida )
doi: 10.1093/hr/uhaf119
Figure Lengend Snippet: RhCASPL1D1 interacts with RhPIP2s. A) Split-ubiquitin yeast two-hybrid assay. RhCASPL1D1 was fused to the N terminus of ubiquitin (RhCASPL1D1-Cub), and RhPIP2;1, RhPIP2;2 as well as RhPIP2;7 were fused to the C terminus of mutated ubiquitin (NubG-RhPIP2;1/RhPIP2;2/RhPIP2;7). The TSU2-APP/NubG-Fe65 and RhCASPL1D1-Cub/NubG combinations were used as controls. The transformed yeasts were plated on Trp-Leu-His-Ade-lacking synthetic defined medium to test protein interactions. B) Interaction of RhCASPL1D1 and RhPIP2s in bimolecular fluorescence complementation (BiFC) assay. RhCASPL1D1-cYFP and RhPIP2;1/RhPIP2;2/RhPIP2;7-nYFP constructs were co-infiltrated in N. benthamiana leaves. PM-mCherry was used as a plasmalemma marker. The cYFP/nYFP combination was used as a negative control. YFP fluorescence was visualized with confocal microscopy after 3-day infiltration. Scale bars, 50 μm. C) RhCASPL1D1-RhPIP2;7 interaction detected by co-IP. The combinations of RhCASPL1D1-FLAG and RhPIP2;7-MYC constructions were co-transformed into N. benthamiana leaves with negative control, GFP-FLAG/RhPIP2;7-MYC combination. Immunoprecipitation was conducted by incubation of leaf total proteins and FLAG beads. The input and co-immunoprecipitated proteins were analyzed using anti-FLAG and anti-MYC antibodies. D) RhCASPL1D1 co-localizes with RhPIP2;7 in N. benthamiana epidermal cells. RhCASPL1D1-GFP and RhPIP2;7-mCherry were co-expressed in N. benthamiana leaves. GFP + RhPIP2;7-mCherry, RhCASPL1D1-GFP + mCherry were regarded as negative control, respectively. Fluorescence was visualized via confocal microscopy 3 days after infiltration. The fluorescence intensity profiles along the white arrows (in the merge micrographs) were analyzed by ImageJ. Pearson correlation coefficients (Rp) for co-localization were calculated from the fluorescence signal in the white dashed boxes using ImageJ. Scale bars, 50 μm.
Article Snippet: Two days after infiltration, the co-localization of GFP and mCherry was observed with a laser confocal microscopy (Zeiss LSM 800).
Techniques: Ubiquitin Proteomics, Y2H Assay, Transformation Assay, Bimolecular Fluorescence Complementation Assay, Construct, Marker, Negative Control, Fluorescence, Confocal Microscopy, Co-Immunoprecipitation Assay, Immunoprecipitation, Incubation
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