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. 2022 Feb 8;34(5):1724–1744. doi: 10.1093/plcell/koac037

Figure 4.

Figure 4

CPR5 interacts and colocalizes with PRL1 and FIP1. A, Subcellular localization of CPR5 protein. Agrobacteria carrying the construct of 35S:CFP-CPR5 (the CFP–CPR5 fusion gene is driven by the 35S promoter) was infiltrated into N. benthamiana. The magenta box in the left indicates the nucleus which is enlarged on the right. N, nucleus . B, The interaction between CPR5-N (N-terminus, 1–339 aa) and FIP1 was tested using yeast two-hybrid system. AD, yeast GAL4 activation domain; DB, yeast GAL4 DNA-binding domain. –L/T, SD base with leucine/tryptophan dropout supplement; –L/T/H/A+3-amino-1,2,4-triazole (3-AT), SD base with leucine/tryptophan/histidine/adenine dropout supplement and Figure 4: (continued) 10 mM 3-AT. C, Co-IP was carried out by transiently co-expressing the Myc empty vector (Myc), Myc-tagged PRL1 (Myc-PRL1), or Myc-tagged FIP1 (Myc-FIP1) with HA-tagged CPR5 (HA-CPR5) in N. benthamiana. Protein extracts were IPed with α-Myc and resolved by SDS–PAGE. Input and IPed proteins were detected with both α-Myc and anti-HA antibody (α-HA). D, Subcellular localization of PRL1 and FIP1 proteins. Agrobacteria carrying the constructs of 35S:VENUS-PRL1 (VENUS fused to the N-terminus of PRL1) and 35S:VENUS-FIP1 as well as 35S:mCherry-NLS (mCherry-NLS, the nuclear localization signal of SV40 protein fused to the C-terminus of mCherry) were co-infiltrated into N. benthamiana. mCherry-NLS was used as a marker of nucleus. E, BiFC assay was performed by transiently co-expressing 35S:mCherry-NLS, 35S:cYFP-CPR5 (the C-terminus of yellow fluorescent protein fused to the N-terminus of CPR5) or 35S:cYFP-PRL1 with 35S:SR34-nYFP (the N-terminus of YFP fused to the C-terminus of SR34) or 35S:PRL1-nYFP or 35S:FIP1-nYFP in N. benthamiana. mCherry-NLS and SR34 were used as markers of nucleus and NS, respectively. The empty vectors of 35S:nYFP and 35S:cYFP were used as negative controls. “/” is used to separate each pair of split-YFPs (cYFP alone or fusion protein/nYFP alone or fusion protein). For instance, PRL1/FIP1 stands for cYFP-PRL1/FIP1-nYFP. F, Top: Schematic diagram of the FRET-FLIM assay for detecting the CPR5/PRL1/FIP1 ternary complex. The transfer of excitation energy from CFP to a functional YFP formed from the split-YFPs (N, nYFP; C, cYFP) to generate FRET. The CFP lifetime is detected by FLIM. Bottom: The fluorescence lifetime heat maps. This assay was carried out by transiently expressing 35S:CFP-CPR5 alone (CFP-CPR5) or co-expressing 35S:CFP-CPR5 with either 35S:YFP-NLS (the nuclear localization signal of SV40 protein fused to the C-terminus of YFP) (CFP-CPR5/YFP) or 35S:cYFP-PRL1/35S:FIP1-nYFP (CFP-CPR5/BiFC-YFP) in N. benthamiana. Both CFP-CPR5 and CFP-CPR5/YFP are used as negative controls. Scale bar, CFP lifetime (ns, nanosecond). G, Box plot represents quantification of the CFP lifetime in Figure 4F. Experiments were conducted 3 times with similar results (n = 30–80). The letter above the bar indicates a statistically significant difference between groups at P < 0.01.