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Literature Sharing | Both phytochrome A and phyB interact with PHYTOCHROME-INTERACTING FACTORs through an evolutionary conserved phyOPM-APA interaction

Release time:

2025-05-20

This study reveals that both phytochrome A (phyA) and phytochrome B (phyB) interact with PHYTOCHROME-INTERACTING FACTORs (PIFs) through the APA (active phyA-binding) motif via the C-terminal output module (phyOPM), rather than the previously assumed N-terminal photosensory module (phyPSM). While phyB also binds the APB (active phyB-binding) motif via its N-terminal phyBPSM, the APA motif interaction is common to both phyA and phyB through phyOPM.

To enable light-dependent interaction assays in yeast without adding external chromophore, researchers engineered the AH109 strain to produce phycocyanobilin (PCB) by expressing four biosynthetic genes, creating the AH109C strain. This strain successfully formed spectrally active phytochrome, as confirmed by red fluorescence and characteristic absorption spectra. Using AH109C, yeast two-hybrid assays detected red light-dependent interactions between phyB (including phyBPSM and full-length phyB) and known partners such as PIF3, SPA1, SPA2, ELF3, and TZP, while no interaction was seen in the dark, demonstrating that AH109C enables efficient light-dependent interaction assays without exogenous PCB.

Fig. 1 Yeast two-hybrid assay (Y2H) demonstrates light-dependent interaction between phytochrome and interacting proteins in AH109C

Using the yeast two-hybrid assay, researchers found that phyA interacts with PIF3 through the APA motif, not the APB motif. Full-length phyA bound to wild-type PIF3 and APB-mutant PIF3 under red light, but not to APA-mutant or double-mutant PIF3. The phyA N-terminal photosensory module (phyAPSM) did not interact with any PIF3 variant, while the C-terminal output module (phyAOPM) interacted with wild-type and APB-mutant PIF3, but not with APA-mutant forms. Similar results were observed with PIF1. These findings show that phyA interacts with PIF1 and PIF3 specifically through the phyAOPM-APA interaction.

Fig. 2 PhyA interacts with PIF3 through the phyAOPM-APA interaction

Yeast two-hybrid assays showed that phyB interacts with PIF1 and PIF3 through two distinct interfaces: phyBPSM binds the APB motif, while phyBOPM binds the APA motif. Full-length phyB interacted with PIF3 and its single mutants (PIF3mAPA, PIF3mAPB) under red light but not with the double mutant (PIF3mAPA/mAPB), indicating both motifs contribute to binding. Domain analysis confirmed phyBPSM interacts via APB and phyBOPM via APA. Similar results were observed with PIF1, demonstrating that phyB uses both its N-terminal and C-terminal domains to bind PIFs through APB and APA motifs, respectively.

Fig. 3 PhyB interacts with PIF3 through the phyPSM-APB or the phyBOPM-APA Interaction

Experiments showed that the phyBOPM-APA interaction promotes PIF3 degradation under red light, similar to the phyBPSM-APB interaction. Using transgenic lines in a phyA mutant background, PIF3, PIF3mAPA, and PIF3mAPB were degraded under red light, but the double mutant PIF3mAPA/mAPB was not, confirming that both APA and APB contribute to degradation. Further tests revealed that only the APB-dependent interaction triggers mutual degradation of both phyB and PIF3, while the APA-dependent interaction leads to PIF3 degradation alone. Thus, phyBOPM-APA promotes PIF3 degradation without phyB destruction, unlike the phyBPSM-APB interaction.

Fig. 4 The phyOPM-APA interaction promotes the PIF3 degradation but not the mutual phyB destruction.

Yeast two-hybrid assays showed that Marchantia phytochrome (Mp-phy) interacts with Mp-PIF through the Mp-phyOPM-APA interaction under red light, but not with an APA-mutated Mp-PIF. The Mp-phyPSM domain showed no interaction, indicating that, like Arabidopsis phyA, Mp-phy uses its OPM domain to bind PIF via the conserved APA motif. This suggests that the phyOPM-APA interaction evolved before the divergence of bryophyte and angiosperm phytochromes.

Fig. 5: Marchantia Mp-phy interacts with its PIF also through the Mp-phyOPM-APA interaction.

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