PROJECT SUMMARY Almost all cellular organisms employ an array of photoreceptors to detect their light environment. Arguably the most influential are the phytochromes (Phys), a diverse group essential for plant development and the behavior of many bacterial, fungal, and algal species. By reversible photointerconversion of their bilin (or open-chain tetrapyrrole) chromophores between red light-absorbing Pr and far-red light-absorbing Pfr states, Phys act as photoswitches in various signaling cascades responsive to light intensity, duration, direction, and spectral quality. Moreover, through the thermal reversion of Pfr back to Pr, some Phys sense temperature through enthalpic effects on this reaction, and perceive photoperiod through the nighttime depletion of Pfr. The cumulative effects of this Pr/Pfr interconversion impact numerous physiological processes important to agriculture and the biology of harmful plant and human pathogens. In addition, their unique photochemistries provide invaluable optogenetic tools, including novel fluorophores for tissue imaging, and engineered photoswitches for regulating cellular events with remarkable temporal and spatial precision. Recently, we made great strides in understanding how Phys signal, with emerging structures suggesting that microbial and plant Phys use two distinct output modalities. Both start with light-triggered isomerization of the bilin, which drives a - stranded to -helical rearrangement of a hairpin loop that links the signature PHY and GAF domains. While photoactivated microbial Phys then connect torsional strain generated within the dimer to regulate an appended output domain (typically with histidine kinase activity), plant Phys have rearranged their domain organization to create a photosensitive dimeric platform that likely enables reversible binding and eventual degradation of the family of PIF transcriptional repressors. While current models helped illuminate gross changes required for endstate conversion, the intermediates of photoexcitation and ensuing structural changes necessary for creating a signaling-competent Pfr state remain uncertain. The objectives of this proposal are to complete these pictures through continued X-ray crystallographic and cryo-electron microscopic approaches followed by informed biochemical analyses of representatives in their inactive and active states. Specific aims are to: (1) exploit time-resolved serial X-ray crystallography to structurally define the intermediates generated by Phys after photon absorption; (2) generate more comprehensive structures of bacterial Phys, including models of full- length dimeric photoreceptors with their signal output modules; (3) develop a model for how plant Phys signal through structural studies on Pfr; (4) apply steady-state and time-resolved protein surface mapping to support the Phy photoconversion pathway(s) seen structurally; (5) develop models of Phys interacting with their downstream effectors, and (6) appreciat...