Fungal LOV proteins facilitate photoadaptation via blue light regulation of dimer formation. Despite considerable homology of these proteins in closely related fungi, deviations in signaling exist. Here we report the crystal structure of ENVOY (ENV1), a homolog of N. crassa VVD in the fungus T. reesei, a model organism for plant cell wall degradation. Structural studies contradict a model of reversible competitive dimerization. Rather, evolutionary pressures have facilitated a two-residue shift in the position of a key Cys residue (Cys96) that enables the integration of environmental stress and light responses. A Cys96Thr variant abolishes adaptive responses to light and oxidative stress in a carbon source-dependent manner in vivo. Phylogenetic analysis verifies an evolutionary relevance of the Cys residue shift in different orders within Sordariomycetes. In this manner, we identified a widespread oxidative stress signaling mechanism that couples metabolic sensing and blue light responses not previously identified in LOV proteins.

Light-oxygen-voltage (LOV)3 domain-containing photoreceptors are widely distributed in nature, where they couple blue light absorption to regulation of a diverse array of signal transduction pathways (1). In general, LOV proteins can be divided into two subclasses: 1) short LOV proteins (sLOV) that exist as the isolated LOV domain with short ancillary N-or C-terminal caps (2-4) and 2) modular LOV proteins that couple blue light activation to allosteric regulation of effector domains (1). Although the modular LOV proteins are present in plants, bacteria, and all fungi, the sLOV variety are predominantly found in bacteria and only some fungi (4). Currently, LOV proteins have received widespread attention because of their important roles in regulation of circadian function (3, 5, 6), growth and development (7,8), stress responses (9 -11), and adaptation to and regulation of pathogenicity (12). In addition, their wide ranging utility has led to the development of optogenetic tools that harness their modular design (13,14). Despite substantial research, key questions involving LOV photocycles remain.LOV domain chemistry is characterized by blue light-induced formation of a covalent adduct between a bound flavin cofactor (FMN, FAD, or riboflavin) and a conserved Cys residue in a GXNCRFLQ motif. Concomitant with adduct formation is protonation of the N5 position of the isoalloxazine ring. Current models indicate that signal transduction is coupled to N5 protonation via allosteric regulation of N-or C-terminal effector elements remote from the flavin active site (3,15,16). The covalent adduct is defined by a broad UV-visible absorption band centered at ϳ390 nm (LOV 390 ). Upon return to the dark, the adduct state spontaneously decays to an oxidized flavin (LOV 450 ) on a timescale of seconds to days (17,18). Currently the biological role of the wide range in photocycle lifetimes is unknown; however, several studies have suggested that the range facilitates adaptation to changing levels of light intensity (17,19,20). For these reasons, chemical tuning of the LOV photocycle lifetime through understanding of the adduct decay mechanism has been attempted in several systems (2,17,18,(21)(22)(23)(24).Several lines of reasoning have led to a general mechanism of adduct decay. First, solvent isotope effect experiments indicate that a single proton abstraction event is rate-limiting (17, 18). Second, adduct decay can be catalyzed by the presence of small molecule bases such as imidazole (25). Third, residue substitutions at regions that regulate solvent access to the flavin active site have a substantial effect on LOV photocycle lifetimes (18,26). Combined, these experiments implicate N5 deprotonation as the rate-determining step in adduct decay. Consistent with such a model, mutation of residues that regulate accessibility of small molecules to the N5 position or that tune hydrogen bonding characteristics affect kinetics of LOV proteins (17,18,21,22,24,26,27). Importantly, the natural base responsible for N5 deprotonation remai...

Blue light-signaling pathways regulated by members of the light-oxygen-voltage (LOV) domain family integrate stress responses, circadian rhythms and pathogenesis in fungi. The canonical signaling mechanism involves two LOV-containing proteins that maintain homology to Neurospora crassa Vivid (NcVVD) and White Collar 1 (NcWC1). These proteins engage in homo- and heterodimerization events that modulate gene transcription in response to light. Here, we clone and characterize the VVD homolog in Botrytis cinerea (BcVVD). BcVVD retains divergent photocycle kinetics and is incapable of LOV mediated homodimerization, indicating modification of the classical hetero/homodimerization mechanism of photoadaptation in fungi.