Interactions between Core Elements of the Botrytis cinerea Circadian Clock Are Modulated by Light and Different Protein Domains

Abstract

Botrytis cinerea possesses a complex light-sensing system composed of eleven photoreceptors. In B. cinerea, bcwcl1 encodes for the BcWCL1 protein, the orthologue of the blue-light photoreceptor WC-1 from Neurospora crassa. The functional partner of BcWCL1 is the BcWCL2 protein, both interacting in the nucleus and forming the B. cinerea white collar complex (BcWCC). This complex is required for photomorphogenesis and circadian regulation. However, no molecular evidence shows a light-dependent interaction between the BcWCC components or light-sensing capabilities in BcWCL1. In this work, by employing a yeast two-hybrid system that allows for the in vivo analysis of protein–protein interactions, we confirm that BcWCL1 and BcWCL2 interact in the absence of light as well as upon blue-light stimulation, primarily through their PAS (Per-Arnt-Sim) domains. Deletion of the PAS domains present in BcWCL1 (BcWCL1PAS∆) or BcWCL2 (BcWCL2PAS∆) severely impairs the interaction between these proteins. Interestingly, the BcWCL1PAS∆ protein shows a blue-light response and interacts with BcWCL2 or BcWCL2PAS∆ upon light stimulation. Finally, we demonstrate that BcWCL1 and BcWCL1PAS∆ respond to blue light by introducing a point mutation in the photoactive cysteine, confirming that both proteins are capable of light sensing. Altogether, the results revealed the complexity of protein–protein interactions occurring between the core elements of the B. cinerea circadian clock.

Expanding the molecular versatility of an optogenetic switch in yeast

“…This plate was incubated at 25°C in a Synergy H1 microplate reader (Biotek, United States ) for simultaneous acquisition of Optical Density at 600 nm (OD 600 ) and Luminescence (Lum) from each well every 10 min for 24 h ( Romero et al, 2021 ). The Luc experiments were performed in three conditions: constant darkness (DD), constant blue-light illumination (BL), and a single blue-light pulse (BLP) of 2 h (h) duration ( Romero et al, 2021 ; Rojas et al, 2022 ). In the DD assays, the 96-well plate was incubated inside the plate reader (dark) at 25°C with data acquisition of Lum and OD 600 every 10 min.…”

Domains Required for FRQ-WCC Interaction within the Core Circadian Clock ofNeurospora

“…In Neurospora , WCC acts as a responsive hub coordinating light signaling and the circadian clock that is obligate in constant darkness (18). In consonance with its dual role as a light sensor and a transcription factor for frq in the clock, WC-1 bears a light-, oxygen-, and voltage-sensing (LOV) domain, a transactivation domain, two motifs required for DNA binding—the zinc-finger (ZnF) and its nearby DBD (defective in DNA binding) motif, and two Per-Arnt-Sim (PAS) domains for WC-2 interaction (19); WC-2, an accessory of WC-1, contains a PAS and a ZnF DNA-binding domain (19).…”

Domains required for the interaction of the central negative element FRQ with its transcriptional activator WCC within the core circadian clock of Neurospora

“…In Neurospora , WCC acts as a responsive hub coordinating the circadian clock that is obligate in constant darkness and light signaling ( 19 ). In consonance with its dual role as a light sensor and a transcription factor for frq in the clock, WC-1 bears a light-, oxygen-, and voltage-sensing domain, a transactivation domain, two motifs required for DNA binding—the zinc finger (ZnF) and its nearby DBD (defective in DNA binding) motif, and two Per-Arnt-Sim (PAS) domains for WC-2 interaction ( 20 , 21 ); WC-2, an accessory of WC-1, contains a PAS and a ZnF DNA-binding domain ( 20 ).…”