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. 2016 Feb 3;5:e13292. doi: 10.7554/eLife.13292

Figure 4. PCH1 directly interacts with phyB in a light-dependent manner.

(A) and (B) yeast two-hybrid between PCH1 (fused to GAL4 DNA binding domain, DBD) and preys (ELF3, ELF4, N-/C- termini (Nt or Ct) and full length (FL) LUX, COP1, TZP and the Ct of phyA, B, C, D, and E fused to GAL4 activating domain, AD). –LW select (minus Leu and Trp) for presence of both DBD and AD constructs and–LWH+3AT plates (minus Leu, Trp and His, with 2 mM 3AT added) tested interactions. (C) Transient tobacco co-immunoprecipitation (IP) assay with PCH1-His6-FLAG3 and phyB-GFP or GFP. IPs were done against FLAG followed by westerns using either anti-FLAG, phyB or GFP antibodies. (D) The in-vivo PCH1-phyB interaction is light-sensitive. A schematic of the light treatment is above western. PCH1ox3 seedlings entrained in 12L:12D white light (WL) were either exposed to WL for 12 hr (WL ZT12, lane 1 to 3), subjected to extended dark (WL to DD) for 24 or 48 hr (lane 4 and 5), red light for 12 hr (WL to Rc ZT12, lane 6), or an end-of-day far-red pulse for 10 min after 12 hr of WL (WL EOD-FRp ZT12, lane 7). WT and PCH1ox3 in phyB-9 plants are western controls. IPs were done against FLAG followed by westerns using either anti-FLAG or phyB antibodies. Anti-RPT5 was used as a loading control. The asterisk at the FLAG-IP / anti-phyB notes an unspecific band that migrates faster than phyB that is present in every lane. (E) PCH1 preferentially binds the Pfr form of phyB in in vitro. Recombinant His6-PCH1-His6-FLAG3 or His6-YPet-His6-FLAG3 was incubated with phyB-HA transcribed and translated by rabbit-reticulate lysate. PΦB absent (apo) phyB precipitations were incubated in the dark, while red (Pfr) or far red light (Pr) were incubated with 20 μM PΦB. His-affinity capture was followed by immunoblotting for anti-HA or anti-FLAG.

DOI: http://dx.doi.org/10.7554/eLife.13292.018

Figure 4.

Figure 4—figure supplement 1. Interaction map of PCH1-associated proteins.

Figure 4—figure supplement 1.

An updated interaction map from previous described (Huang et al., 2015), which integrate our AP-MS and protein-protein interaction data to illustrate both directly and indirectly interacting proteins that co-precipitate with PCH1. Solid lines indicate direct interactions determined in this study or previously defined, while dotted lines indicate association.
DOI: http://dx.doi.org/10.7554/eLife.13292.019