**3. Results**

### *3.1. The Orthologs of WC-1 and WC-2 in F. asiaticum*

Since *F. asiaticum* belongs to a sub-lineage of the *Fusarium graminearum* species complex, the orthologous genes of *wc1* and *wc2* in *F. asiaticum*, namely *Fawc1* and *Fawc2*, were identified as follows: BLASTP search of the *F. gaminearum* genome in Ensembl Fungi database, using the amino acid sequences of *Neurospora crassa* WC-1 (NCU02356) and WC-2 (NCU00902) proteins [34] as queries, resulted in the corresponding orthologs namely FgWC1 (FGSG\_07941) and FgWC2 (FGSG\_00710), respectively. The corresponding genes and their flanking sequences were used as references to design specific primers to amplify the open reading frame (ORF) sequences of their orthologs, *Fawc1* and *Fawc2*, in *F. asiaticum* strain EXAP-08 via the *Pfu* DNA polymerase. Sequencing of the cloned PCR products indicated that *Fawc1* (KX905081.1) and *Fawc2* (MT019868) of *F. asiaticum* showed high similarity to *Fgwc1* (99.05%) and *Fgwc2* (99.39%) of *F. graminearum*. The deduced amino acid sequences of FaWC1 and FaWC2 were analyzed via the SMART online tool, and the results indicated that FaWC1 possessed a Zn-finger DNA-binding domain as well as three PAS domains, of which the N-terminal most PAS domain should be the special subclass called the LOV domain (for light, oxygen, and voltage) being responsible for binding the chromophore molecule of flavin adenine dinucleotide (FAD), while the FaWC2 just essentially contained a single PAS domain and a Zn-finger DNA binding domain. In general, FaWC1 and FaWC2 show high similarity with their orthologs in *F. graminearum* and *N. crassa* (Figure 1A). Gene expression analysis showed that both *Fawc1* and *Fawc2* were induced to peak levels by 15 min light exposure, while the long period (12 h) of illumination caused less induction of the transcription levels of these two genes (Figure 1B).

**Figure 1.** Identification of two photoreceptor genes, *Fawc1* and *Fawc2*, in *F. asiaticum*. (**A**). Schematic demonstration of the domains of WC1 and WC2 orthologs from *N. crassa*, *F. graminearum*, and *F. asiaticum*. Accessions of the amino acid sequences are as follows: WC1 (NCU02356); WC2 (NCU00902); FgWC1 (FGSG\_07941); FgWC2 (FGSG\_00710); FaWC1 (KX905081.1); FaWC2 (MT019868). Domains of these photoreceptor proteins were analyzed via the SMART online tool (http://smart.embl-heidelberg.de/). PAS: Per-period circadian protein; Arnt: Ah receptor nuclear translocator protein; Sim: Single-minded protein, NLS: Nuclear Location Singal, Zn: Zinc finger binding to DNA consensus sequence. (**B**). Transcript levels of *Fawc1* and *Fawc2* are regulated by light. The horizontal axis indicates light treatment time. The bars present mean values ± SD of three replicate samples.

### *3.2. Generation and Characterization of the* Δ*Fawc1 and* Δ*Fawc2 Mutants*

To reveal the functions of *Fawc1* and *Fawc2*, homologous recombination cassettes used for gene knockout purposes were created as shown in Figure 2. After protoplast transformation, the transformants with hygromycin resistance successfully grew up on the selection medium. To characterize the knockout mutants of Δ*Fawc1* and Δ*Fawc2* in which the hygromycin resistance cassette (*hph*) had correctly replaced each target gene, PCR assays with the specific primer pairs were performed with the genomic DNA, and the correct transformants for each gene were selected for further analysis. To verify that the *Fawc1* or *Fawc2* was completely knocked out, reverse transcription (RT)-PCR was applied, revealing that the transcripts of *Fawc1* and *Fawc2* were present in the EXAP-08 wild-type strain but absent in the mutants of Δ*Fawc1* and Δ*Fawc2*, respectively. To confirm the functions of these two genes, the wild-type *Fawc1* and *Fawc2* connected with their native promoters, and terminators were transformed into Δ*Fawc1* and Δ*Fawc2* to obtain the complementation strains. As FaWC1 possesses both signal input and output domains, the LOV and zinc finger (ZnF) domains, respectively, the truncated versions of FaWC1, lacking either LOV or ZnF domains (Figure 2), were expressed in the Δ*Fawc1*

mutant to explore the functions of these domains in light signaling and other life aspects of *F. asiaticum*. All the fungal strains used in this study and their genotypes are listed in Table 1.

**Figure 2.** Generation of the transgenic mutants of *F. asiaticum.* Schematic diagrams of homologous recombination occurred between the replacement vector carrying the hygromycin resistance marker (*hph*) and the target gene (*Fawc1*) of *F. asiaticum* strain EXAP-08, resulting in the knockout mutant (Δ*Fawc1*). Meanwhile, the wild-type *Fawc1* or its truncated versions, *Fawc1*ΔLOV and *Fawc1*<sup>Δ</sup>Zn, were amplified from genomic DNA of EXAP-08 and cloned into the flu6 plasmid, and then transformed into the Δ*Fawc1* mutant, resulting in the complementation strain Δ*Fawc1*-*C*, and Δ*Fawc1-C*ΔLOV and Δ*Fawc1-C*ΔZnF mutant strains. The knockout mutant Δ*Fawc2* and its complementation strain Δ*Fawc1*-*C* were generated via a similar strategy.

### *3.3. The Marker Responses to Light Signal Are Mediated by WCC and Dependent on LOV but not ZnF Domain of FaWC1 in F. asiaticum*

As fungi commonly sense light to indicate the presence of deleterious ultraviolet (UV) radiation, the light-induced survivability through UV damage is recognized as a marker response to light signal in fungi [35,36]. In the present study, the survival of serially diluted spores of all the tested strains was similarly poor if the fungal cultures were kept in the dark after UV-C treatment. However, when white light illumination was applied, the wild type (WT) could survive much better from UV-C irradiation than those cultures in the dark, but no recovery of survival spores was observed with Δ*Fawc1* and Δ*Fawc2*. The complementation strains, Δ*Fawc1-C* and Δ*Fawc2-C*, demonstrated similar photoreactivation levels as the WT, indicating that the defect in the light-induced UV-C resistance in the Δ*Fawc1* and Δ*Fawc2* mutants is indeed caused by the loss of these two genes (Figure 3A). Interestingly, the Δ*Fawc1-C*ΔLOV showed similar UV-C susceptibility as the Δ*Fawc1* and Δ*Fawc2* mutants, while in contrast, the Δ*Fawc1-C*ΔZnF restored UV-C tolerance to the WT level. Gene expression analysis showed that the transcript level of the photolyase gene Faphr1 was significantly induced by light in WT, Δ*Fawc1-C*, Δ*Fawc2-C*, and Δ*Fawc1-C*ΔZnF strains; however, no remarkable change of *Faphr1* expression as influenced with light has been observed in the Δ*Fawc1*, Δ*Fawc2*, and Δ*Fawc1-C*ΔLOV strains (Figure 3B).

**Figure 3.** Effect of light on UV-C resistance. (**A**). Serial dilutions of all strains were point-inoculated onto complete agar medium (CM). After the UV irradiation of indicated dosages, the plates were incubated for one day in light (left) or darkness (right). (**B**). The relative expression level of the deduced photolyase gene *Faphr1* in wild-type and mutant strains as influenced by light. DD, samples cultured for 48 h in darkness; LL, samples experienced 47 h culture in darkness followed by one hour of light illumination. The bars present mean values ± SD of three replicate samples.

Another marker response to the light signal in fungi is pigment production [18,37]. The WT strain could produce significantly more orange-colored carotenoid pigment in constant light compared to dark condition after growth in liquid complete medium (CM) for three days (Figure 4A). In contrast, there was no observable orange pigment accumulated by Δ*Fawc1* and Δ*Fawc2* in light and dark conditions (Figure 4A). Similarly, the Δ*Fawc1-C*ΔLOV mutant, which lacks the LOV domain of FaWC1, showed similar pigmentation phenotypes as the Δ*Fawc1* and Δ*Fawc2* mutants. However, the Δ*Fawc1-C*ΔZnF with truncating the ZnF domain of FaWC1 demonstrated enhanced carotenogenesis in response to light, which was similar to the WT strain. Quantitative measurement assay also showed that in darkness, all strains produced basic carotenoid levels, and light treatment caused a significant increment of carotenoid accumulation in WT, Δ*Fawc1-C*, Δ*Fawc2-C*, and Δ*Fawc1-C*ΔZnF strains, but light failed to alter the pigmentation behavior in Δ*Fawc1*, Δ*Fawc2-C*, and Δ*Fawc1-C*ΔLOV strains (Figure 4B). Gene expression analysis showed that the transcript levels of the carotenoid biosynthetic genes *CarRA* and *CarB* [37,38] were up-regulated in light versus dark condition in the WT, ΔFawc1-C, ΔFawc2-C, and Δ*Fawc1-C*ΔZnF strains. Contrarily, the expression of *CarRA* and *CarB* could not be induced by light in the Δ*Fawc1*, Δ*Fawc2*, and Δ*Fawc1-C*ΔLOV strains (Figure 4C,D).

**Figure 4.** Effect of light on carotenogenesis. (**A**). Carotenoid pigment accumulation of the tested strains cultured in liquid CM for four days under constant light (LL) or darkness (DD). (**B**). Measurement of carotenoid contents in the mycelium of each strain harvested from the liquid shaking culture in (**A**). (**C**) and (**D**). Relative expression levels of deduced carotenoid biosynthesis genes *CarRA* and *CarB* in wild-type and mutant strains under light (LL) or darkness (DD). The bars in **B**, **C**, and **D** present mean values ± SD of three replicate samples.

Collectively, the above data sugges<sup>t</sup> that both FaWC1 and FaWC2 are responsible for light signaling to induce the marker responses, including carotenoid accumulation and UV damage tolerance. Moreover, the LOV and ZnF domains of FaWC1 are required and dispensable, respectively, for mediating the light responses in *F. asiaticum*.

### *3.4. Perithecia Maturation and Ascospore Development of F. asiaticum Are Regulated by WCC Photoreceptor*

Sexual reproduction is usually vital for the dissemination of fungal pathogens in their lifecycles, and the near-UV light is known to induce the perithecia maturation and ascospore formation in FGSC [39]. However, two independent studies reported inconsistent e ffects of the WCC photoreceptor on the sexual development of *F. graminearum*, which was probably due to the di fference of the wild-type background strains used in each study [30,40]. The present work with *F. asiaticum* showed that the WT strain was able to form mature perithecia, in which the sexual ascospores could be found apparently. In contrast, the Δ*Fawc1* and Δ*Fawc2* mutants could produce a comparable amount of perithecia as WT, but these mutants' perithecia failed to develop into the black-pigmented mature stage, being deficient in ascospore formation (Figure 5). Re-introducing the wild-type *Fawc1* and *Fawc2* into the corresponding gene deletion mutants had fully recovered their perithecia maturation and ascospore formation abilities, suggesting that these WCC photo receptor components, FaWC1 and FaWC2, are indeed required for sexual reproduction development in this fungus. Additionally, the Δ*Fawc1-C*ΔZnF and Δ*Fawc1-C*ΔLOV demonstrated phenotypes in sexual development as WT and Δ*Fawc1*, respectively, indicating that it should be the LOV domain, but not the ZnF domain, that is required for mediating the light signal to regulate the sexual reproduction of *F. asiaticum*.

**Figure 5.** Regulation of *Fawc1* and *Fawc2* on the sexual reproduction development processes of *F. asiaticum*. The perithecium formed by each strain on carrot agar medium was observed via stereomicroscope. Via picking up the perithecia and pressing them on glass slides for microscopic analysis, mature ascospores were observed from the perithecia of EXAP-08, Δ*Fawc1-C*, Δ*Fawc1-C*<sup>Δ</sup>ZnF, and Δ*Fawc2-C* strains, while in contrast, only mycelium biomass could be found in the immature perithecium of Δ*Fawc1*, Δ*Fawc2*, and Δ*Fawc1-C*ΔLOV mutant strains.

### *3.5. FaWC1 and FaWC2 Play Di*ff*erent Roles in Regulating Virulence Expression*

In the infection assay with wheat coleoptiles, inoculation with the WT caused apparent brown rot symptom in the host plant materials (Figure 6). In contrast, the Δ*Fawc1* mutant showed more than 80% reduction in pathogenicity in comparison with WT. Meanwhile, the complementation strain Δ*Fawc1-C* had a recovered pathogenicity level similar to that of the WT strain, suggesting that FaWC1 is involved in regulating the pathogenicity of *F. asiaticum*. However, the mutant with the deletion of *Fawc2* caused equivalent disease severity to the WT. These data suggested that FaWC1 and FaWC2 played independently different roles in regulating the pathogenicity of this fungus.

**Figure 6.** Virulence assay on wheat confirms that *Fawc1* regulate the virulence of *F. asiaticum* in a light-independent manner. (**A**). Wheat coleoptiles were inoculated with 5 μL conidial suspensions and were kept humid inside a plastic box. Fungal strains for test include the wild-type EXAP-08, Δ*Fawc1*, Δ*Fawc1*-*C*, Δ*Fawc1-C*<sup>Δ</sup>LOV, Δ*Fawc1-C*<sup>Δ</sup>ZnF, Δ*Fawc2*, and Δ*Fawc2*-*C*. Photographs were taken at four days post inoculation. (**B**). Statistical analysis of lesion sizes caused by each fungal genotype. Different letters represent a significant difference at *p* < 0.05. The bars present mean values ± SD (*n* = 20).

In order to assess whether and how light signaling via the WCC pathway could be involved in fungal pathogenicity expression, the mutant strains lacking the LOV and ZnF domains of FaWC1 were further analyzed in the host infection assay. Unexpectedly, the Δ*Fawc1-C*ΔLOV mutant showed similar

pathogenicity as the WT and complementation strains. While in contrast, the pathogenicity of the Δ*Fawc1-C*ΔZnF mutant was similar to the Δ*Fawc1* mutant, being significantly reduced compared to WT (Figure 6). Consequently, it can be concluded that the FaWC1 LOV domain, which is required for sensing light signals, exerts no influence on pathogenicity; on the other hand, the ZnF domain of FaWC1 is involved in regulating pathogenicity, although this domain is dispensable for mediating light signals in *F. asiaticum*.
