A LexA-based yeast two-hybrid system for studying light-switchable interactions of phytochromes with their interacting partners

Li, Hong; Qin, Xinyan; Song, Pengyu; Run, Han; Li, Jigang

doi:10.1007/s42994-021-00034-5

Cited by 10 publications

(10 citation statements)

References 57 publications

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“…We thus asked whether phyB might attenuate the interaction between SPA1 and HY5 through direct competition with HY5 for SPA1 binding. In yeast‐two‐hybrid systems based on LexA and GAL4 assays (Lu et al, 2015; Li et al, 2021), both phyB and HY5 interacted with both full‐length SPA1 and a C‐terminal truncated protein, but not an N‐terminal truncation, whereas phyA interacted with the full‐length protein and N‐terminal truncated protein, but not the C‐terminal truncation (Figure S7A, B). Compared with dark conditions, exposure to B light enhanced the interaction between phyA or phyB and SPA1 (Figure S7C).…”

Section: Resultsmentioning

confidence: 99%

Arabidopsis phytochromes A and B synergistically repress SPA1 under blue light

Jia

Song

Wang

et al. 2023

JIPB

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In Arabidopsis, although studies have demonstrated that phytochrome A (phyA) and phyB are involved in blue light signaling, how blue lightactivated phytochromes modulate the activity of the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)-SUPPRESSOR OF PHYA-105 (SPA1) E3 complex remains largely unknown. Here, we show that phyA responds to early and weak blue light, whereas phyB responds to sustainable and strong blue light. Activation of both phyA and phyB by blue light inhibits SPA1 activity. Specifically, blue light irradiation promoted the nuclear import of both phytochromes to stimulate their binding to SPA1, abolishing SPA1's interaction with LONG HYPOCOTYL 5 (HY5) to release HY5, which promoted seedling photomorphogenesis.

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Section: Resultsmentioning

confidence: 99%

Arabidopsis phytochromes A and B synergistically repress SPA1 under blue light

Jia

Song

Wang

et al. 2023

JIPB

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“…We first performed Y2H assays to investigate whether 14‐3‐3λ/κ could potentially interact with phyB. Because the full‐length phyB fused with the LexA DNA‐binding domain (phyB‐LexA) displayed strong self‐activation activity in yeast cells (Li et al ., 2021), we expressed phyB‐N‐LexA (the N‐terminal 621‐amino acid domain of phyB fused with LexA) and AD‐14‐3‐3λ/κ fusion proteins in yeast cells. Notably, both 14‐3‐3λ and κ interacted with phyB‐N in yeast cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The phyB‐N‐LexA (amino acid (aa) 1–621 region of the phyB sequence) and the full‐length and various truncated forms of PIF3 fused to the AD domain constructs used in LexA‐based yeast‐two‐hybrid (Y2H) assays were described previously (Li et al ., 2010, 2021; Qi et al ., 2020). The details of the generation of the prey vectors expressing the various 14‐3‐3 proteins fused to the AD domain and bait vectors expressing the LexA‐fused various fragments of PIF3 are described in Methods S2.…”

Section: Methodsmentioning

confidence: 99%

14‐3‐3 proteins regulate photomorphogenesis by facilitating light‐induced degradation of PIF3

et al. 2022

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14-3-3s are highly conserved phosphopeptide-binding proteins that play important roles in various developmental and signaling pathways in plants. However, although protein phosphorylation has been proven to be a key mechanism for regulating many pivotal components of the light signaling pathway, the role of 14-3-3 proteins in photomorphogenesis remains largely obscure. PHYTOCHROME-INTERACTING FACTOR3 (PIF3) is an extensively studied transcription factor repressing photomorphogenesis, and it is well-established that upon red (R) light exposure, photo-activated phytochrome B (phyB) interacts with PIF3 and induces its rapid phosphorylation and degradation. PHOTOREGULATORY PROTEIN KINASES (PPKs), a family of nuclear protein kinases, interact with phyB and PIF3 in R light and mediate multisite phosphorylation of PIF3 in vivo.Here, we report that two members of the 14-3-3 protein family, 14-3-3k and j, bind to a serine residue in the bHLH domain of PIF3 that can be phosphorylated by PPKs, and act as key positive regulators of R light-induced photomorphogenesis.Moreover, 14-3-3k and j preferentially interact with photo-activated phyB and promote the phyB-PIF3-PPK complex formation, thereby facilitating phyB-induced phosphorylation and degradation of PIF3 upon R light exposure.Together, our data demonstrate that 14-3-3k and j work in close concert with the phyB-PIF3 module to regulate light signaling in Arabidopsis.

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“…For LexA‐based yeast two‐hybrid assays, the pEG202 vector expressing SWC6 fused to LexA DNA‐binding domain and pJG4‐5 vectors expressing the full‐length phyB, phyB‐NTD, phyB‐CTD, phyA‐NTD, and phyA‐CTD fused to B42AD domain were co‐transformed into the EGY48 strain. For the phyA‐SWC6 interaction, the pJG4‐5 vector expressing AD‐SWC6 and the pLexA‐JL vector expressing phyA‐LexA (Li et al, 2021) were used. Six independent clones were grown on Synthetic Defined (SD) medium (−Trp, −His, −Ura) supplemented with galactose, raffinose, 1× BU salt, 80 mg/L X‐gal (TaKaRa) and 20 μmol/L phycocyanobilin (PCB) under red (3 μmol/m 2 /s) or far‐red (3 μmol/m 2 /s) light for the interaction test.…”

Section: Methodsmentioning

confidence: 99%

Phytochrome B interacts with SWC6 and ARP6 to regulate H2A.Z deposition and photomorphogensis in Arabidopsis

Wei

Wang

et al. 2021

JIPB

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Light serves as a crucial environmental cue which modulates plant growth and development, and which is controlled by multiple photoreceptors including the primary red light photoreceptor, phytochrome B (phyB). The signaling mechanism of phyB involves direct interactions with a group of basic helix‐loop‐helix (bHLH) transcription factors, PHYTOCHROME‐INTERACTING FACTORS (PIFs), and the negative regulators of photomorphogenesis, COP1 and SPAs. H2A.Z is an evolutionarily conserved H2A variant which plays essential roles in transcriptional regulation. The replacement of H2A with H2A.Z is catalyzed by the SWR1 complex. Here, we show that the Pfr form of phyB physically interacts with the SWR1 complex subunits SWC6 and ARP6. phyB and ARP6 co‐regulate numerous genes in the same direction, some of which are associated with auxin biosynthesis and response including YUC9, which encodes a rate‐limiting enzyme in the tryptophan‐dependent auxin biosynthesis pathway. Moreover, phyB and HY5/HYH act to inhibit hypocotyl elongation partially through repression of auxin biosynthesis. Based on our findings and previous studies, we propose that phyB promotes H2A.Z deposition at YUC9 to inhibit its expression through direct phyB‐SWC6/ARP6 interactions, leading to repression of auxin biosynthesis, and thus inhibition of hypocotyl elongation in red light.

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A LexA-based yeast two-hybrid system for studying light-switchable interactions of phytochromes with their interacting partners

Cited by 10 publications

References 57 publications

Arabidopsis phytochromes A and B synergistically repress SPA1 under blue light

Arabidopsis phytochromes A and B synergistically repress SPA1 under blue light

14‐3‐3 proteins regulate photomorphogenesis by facilitating light‐induced degradation of PIF3

Phytochrome B interacts with SWC6 and ARP6 to regulate H2A.Z deposition and photomorphogensis in Arabidopsis

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