Fig. 3.

AtRAP interacts with LSU2. (a) Interaction between AtRAP and LSU2 in yeast two-hybrid assay. Full-length LSU2 was fused downstream of GAL4-AD in pACT2. Full-length AtRAP was fused downstream of GAL4-BD of pAS2-1. Yeast cells co-transformed with pACT2 and pAS2-1 fusion derivatives were selected on Synthetic Defined (SD) lacking leucine, tryptophan and histidine (SD/-LWH) and SD laking leucine, tryptophan, histidine and adenine (SD/-LWHA) agar media. (b) Co-immunoprecipitation assay in Nicotiana benthamiana leaves. AtRAP and LSU2, tagged with YFP and FLAG, respectively, were coexpressed in N. benthamiana leaves by agro-infiltration and immunoprecipitated with anti-YFP antibody-conjugated agarose beads. Expression of AtRAP-YFP and LSU2-FLAG in total protein extracts was confirmed by Western blotting using anti-YFP and anti-FLAG antibodies, respectively. Total protein extract from leaves infiltrated with the infiltration buffer was used as a negative control (mock). (c, d) AtRAP is co-localized with LSU2 in chloroplasts. AtRAP and LSU2, which were respectively tagged with YFP and CFP, were coexpressed in N. benthamiana leaves by agro-infiltration. The fluorescence signals of YFP and CFP are shown in the yellow and cyan channels, respectively. Both the (c) infiltrated leaves and (d) protoplasts of N. benthamiana were subjected to confocal microscopy. Chloroplasts emit red fluorescence. (e) AtRAP-YFP localizes in chloroplasts from transgenic plants expressing AtRAP-YFP under the 35S promoter. The fluorescence signal of YFP is shown in the yellow channel. Chloroplasts emit red fluorescence. Bars: (c, e) 10 µm; (d) 25 µm.