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Circadian clocks control gene expression to provide an internal representation of local time. We report reconstitution of a complete cyanobacterial circadian clock in vitro, including the central oscillator, signal transduction pathways, downstream transcription factor, and promoter DNA. The entire system oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of each component simultaneously without user intervention. We identified the molecular basis for loss of cycling in an arrhythmic mutant and explored fundamental mechanisms of timekeeping in the cyanobacterial clock. We find that SasA, a circadian sensor histidine kinase associated with clock output, engages directly with KaiB on the KaiC hexamer to regulate period and amplitude of the central oscillator. SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex and enhance rhythmicity of the oscillator, particularly under limiting concentrations of KaiB. Thus, the expanded in vitro clock reveals previously unknown mechanisms by which the circadian system of cyanobacteria maintains the pace and rhythmicity under variable protein concentrations.
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This content will become publicly available on November 8, 2025
The circadian clock regulates receptor-mediated immune responses to an herbivore-associated molecular pattern
Summary Plants activate induced defenses through the recognition of molecular patterns. Like pathogen-associated molecular patterns (PAMPs), herbivore-associated molecular patterns (HAMPs) can be recognized by cell surface pattern recognition receptors leading to defensive transcriptional changes in host plants. Herbivore-induced defensive outputs are regulated by the circadian clock, but the underlying molecular mechanisms remain unknown. To investigate how the plant circadian clock regulates transcriptional reprogramming of a specific HAMP-induced pathway, we characterized the daytime and nighttime transcriptional response to caterpillar-derived In11 peptide, in the legume crop cowpea (Vigna unguiculata). Using diurnal and free-running conditions, we found that daytime In11 elicitation resulted in stronger late-induced gene expression than nighttime. Plants with a conditional arrhythmic phenotype in constant light (LL) conditions lost time-of-day dependent responses to In11 treatment, and this was associated with arrhythmic expression of circadian clock core transcription factorLate Elongated Hypocotyl VuLHY1andVuLHY2. Reporter assays with VuLHY homologs indicated that they interact with the promoter of daytime In11-inducedKunitz Trypsin Inhibitor(VuKTI) via a canonical and a polymorphic CCA1/LHY Binding Site (CBS), consistent with a mechanism of direct regulation by circadian clock transcription factors. This study improves our understanding of the time-dependent mechanisms that regulate herbivore-induced gene expression.
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- Award ID(s):
- 2139986
- PAR ID:
- 10618399
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Institution:
- bioRxiv
- Sponsoring Org:
- National Science Foundation
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