Kai proteins globally regulate circadian gene expression of cyanobacteria. The KaiC phosphorylation cycle, which persists even without transcription or translation, is assumed to be a basic timing process of the circadian clock. We have reconstituted the self-sustainable oscillation of KaiC phosphorylation in vitro by incubating KaiC with KaiA, KaiB, and adenosine triphosphate. The period of the in vitro oscillation was stable despite temperature change (temperature compensation), and the circadian periods observed in vivo in KaiC mutant strains were consistent with those measured in vitro. The enigma of the circadian clock can now be studied in vitro by examining the interactions between three Kai proteins.

OsCEBiP, a chitin-binding protein, and OsCERK1, a receptor-like kinase, are plasma membrane (PM) proteins that form a receptor complex essential for fungal chitin-driven immune responses in rice. The signaling events immediately following chitin perception are unclear. Investigating the spatiotemporal regulation of the rice small GTPase OsRac1, we find that chitin induces rapid activation of OsRac1 at the PM. Searching for OsRac1 interactors, we identified OsRacGEF1 as a guanine nucleotide exchange factor for OsRac1. OsRacGEF1 interacts with OsCERK1 and is activated when its C-terminal S549 is phosphorylated by the cytoplasmic domain of OsCERK1 in response to chitin. Activated OsRacGEF1 is required for chitin-driven immune responses and resistance to rice blast fungus infection. Further, a protein complex including OsCERK1 and OsRacGEF1 is transported from the endoplasmic reticulum to the PM. Collectively, our results suggest that OsCEBiP, OsCERK1, OsRacGEF1, and OsRac1 function as key components of a "defensome" critically engaged early during chitin-induced immunity.

No abstract

The circadian clock in cyanobacteria persists even without the transcription/translation feedbacks proposed for eukaryotic systems. The period of the cyanobacterial clock is tuned to the circadian range by the ATPase activity of a clock protein known as KaiC. Here, we provide structural evidence on how KaiC ticks away 24 h while coupling the ATPase activity in its N-terminal ring to the phosphorylation state in its C-terminal ring. During the phosphorylation cycle, the C-terminal domains of KaiC are repositioned in a stepwise manner to affect global expansion and contraction motions of the C-terminal ring. Arg393 of KaiC has a critical function in expanding the C-terminal ring and its replacement with Cys affects the temperature compensation of the period-a fundamental property of circadian clocks. The conformational ticking of KaiC observed here in solution serves as a timing cue for assembly/disassembly of other clock proteins (KaiA and KaiB), and is interlocked with its auto-inhibitory ATPase underlying circadian periodicity of cyanobacteria.