**4. Discussion**

Light is a strong environment cue orchestrating numerous biological processes in various organisms. Understanding the photobiology mechanisms in fungi can be useful in designing feasible strategies to decrease the detrimental effects of fungi while enhancing those qualities that are beneficial. Most known fungal responses to light are mediated by the well-conserved fungal blue light receptor white collar complex (WCC) [41,42]. In this paper, we functionally investigated the WCC orthologs, *Fawc1* and *Fawc2*, in the plant pathogenic species *F. asiaticum*.

The FaWC1 and FaWC2 characterized in *F. asiaticum* strain EXAP-08 showed high similarity to their counterparts in *F. graminearum* sensu stricto, although they are distinct from each other in geographic, metabolic, and pathogenic phenotypes [9,16,43,44]. The FaWC1 contains a LOV domain for binding the flavin chromophore, a nuclear localization domain (NLS), a Zinc finger domain (ZnF) for DNA binding and transcription factor functions, and a PAS domain for protein–protein interaction. The FaWC2 also contains a PAS domain, NLS domain, and ZnF domain. All these protein domain characteristics of FaWC1 and FaWC2 are highly similar to WC-1 and WC-2 of *N. crassa*, respectively. Light generally increases cellular metabolism and at the same time causes significant oxidative stress to the organism, and to deal with this stress, protective pigments, e.g., carotenoids, are made [45]. Stimulated carotenogenesis as influenced with light treatment is a common photoresponsive phenotype among filamentous fungi [25], including several *Fusarium* species. Experiments in this study showed a clear dependence on WCC for light-induced carotenoid biosynthesis in *F. asiaticum*.

Another common reason for fungi to sense light should be to survive through the environmental stress posed by sunlight, especially the radiation of ultraviolet (UV), which can cause DNA damage or induce the accumulation of toxic reactive oxygen species (ROS) [45]. The ability to sense less harmful wavelengths of visible light can help the organisms to anticipate the emergence of, and promote tolerance to, the harmful UV. This premise has been shown among diverse fungal species through functional study with the orthologs of blue light receptor WCC [46]. It is the photoinduction of DNA repair enzymes, photolyases, and UV endonucleases, by the conserved blue light signaling pathway (WCC) that should be mainly responsible for the light-induced UV resistance in varied fungi [40,47–50]. In the present study, this reliable discipline has been confirmed in *F. asiaticum* in which the loss of *Fawc1* or *Fawc2* resulted in a deficiency of light-induced expression of photolyase gene (*Faphr1*) and consequently hypersensitivity to UV. Taken together, the two marker responses to light signals, including the expression of pigment synthesis and DNA repair enzymes to protect the fungal cells from the damage caused by UV during sunlight exposure, are indeed mediated cooperatively by FaWC1 and FaWC2 in *F. asiaticum*.

Fungal development can also be regulated by light signals. According to varied reports, light signals should have specific roles in certain species including *Aspergillus nidulans* [51], *Cercospora zeae-maydis* [27], *Botrytis cinerea* [29], and *Alternaria alternate* [52]. A key factor required in the regulation of fungal reproductive development events is the velvet complex composed of VeA, VelB, and LaeA [53], and the interactions between photoreceptors and velvet proteins have been demonstrated in *A. nidulans* [54]. As for the FGSC, the development of perithecia and production of ascospores are recognized to be important for the success of these pathogens in their disease cycle [55]. As found in the *F. graminearum* strain Z-3639, WCC should be required for the normal maturity of perithecia during sexual development [40]. However, another group later reported that WCC could negatively regulate sexual development in another strain of *F. graminearum*; the Z-3643 [30]. In the present study, the perithecial maturation and ascospore formation of *F. asiaticum* are dependent on the presence of the WCC. Consequently, the role of the conserved light receptors in regulating fungal reproductive development could be varied case by case.

Besides fungal development and metabolism, the involvement of light receptors in fungal pathogenesis is increasingly attracting research attention [20]. The orthologs of WCC have been recognized in several pathogenic species. However, functional di fferences have also been reported among them [27–30,40,52]. For example, in *Magnaporthe oryzae*, constant light suppresses disease development, which is mediated via MGWC-1 [28]. In the maize leaf pathogen *C. zeae-maydis*, the WC-1 ortholog Crp-1 is required for stomata tropism and for appressorium and lesion formation, implying that Crp-1 positively contributed to its virulence [27]. While in the necrotrophic pathogen *B. cinerea*, the WCC and its direct target BcLTF1, a light-responsive transcription factor homologous to the SUB1 in *N. crassa*, are required to cope with the oxidative stress that is caused by either excessive exposure to light or arising during host invasion, and thus required for achieving full virulence under excessive light [29].The present study with *F. asiaticum* shows that the deletion of *Fawc1* resulted in decreased virulence. This is in contrast with the reports of *F. graminearum*, in which the WCC seems to be not involved in regulating virulence [30,40]. Even in those fungal species in which WCC are involved in determining virulence expression, the ways are quite di fferent from each other; for example, WCC orthologs are involved in a ffecting the infection activities of *B. cinerea*, *C. zeae-maydis*, and *M. oryzae* in a light-dependent manner [27–29]. While in contrast, WCC are required for the full virulence of *C. neoformans* and *F. oxysporum* in mammals in a light-independent way [20,47,49]. Since the WCC proteins possess domains for both signal input (LOV domain) and output (Zn-finger domain), it occurs in several fungi that the WCC orthologs have both light-dependent and independent regulatory roles; for example, in *Trichoderma reesei*, the WCC photoreceptors, BLR1 and BLR2, play a role in the alteration of carbohydrate metabolic functions and the transport of compounds with distinct, both positive and negative targets in darkness [25].

To address the issue that whether the light signal and light-perceiving ability by fungi can a ffect pathogenicity, we have further revealed that the LOV domain of FaWC1 is required, while the ZnF domain is dispensable for inducing carotenoid synthesis and UV tolerance. These findings imply that the WCC-mediated light signaling in *F. asiaticum* does rely on the LOV domain for perceiving light signals, while the ZnF domain of FaWC1 seems to be not involved in regulating the downstream light responses. This phenomenon can be interpreted according to what has been found in several other fungi that indicates that the WC-2 ZnF domain is indeed responsible for the light signal output to regulate the expression of downstream genes [56]. More importantly, the ZnF domain of FaWC1 should have its downstream targets, which are not for light signaling but most likely for pathogenicity regulation. These findings have raised two open questions for future study: (1) to identify the light-independent targets of FaWC1 that are involved in pathogenicity expression; and (2) to resolve the upstream mechanisms that can orchestrate the photoreceptor to well balance between sensing light and regulating light-independent pathogenicity behaviors.
