**2. Materials and Methods**

### *2.1. Fungal Strains and Culture Conditions*

The wild-type strain EXAP-08 was previously isolated from postharvest asparagus spears with serious rot symptoms and characterized as *F. asiaticum*, and it is used as the recipient strain for genetic modifications in this study. The wild-type strain and the resulting mutants are listed in Table 1, and all the fungal strains were purified by single spore isolation and were stored as spore suspension in 20% glycerol at −80 ◦C. The fungal cultures were grown on complete medium (CM) (10 g glucose, 2 g peptone, 1 g yeas<sup>t</sup> extract, 1 g casamino acids, nitrate salts, trace elements, 0.01% of vitamins, 10 g agar and 1 L water, pH 6.5) for mycelial growth, carboxymethyl cellulose (CMC) medium [31] for conidiation, and carrot agar medium [32] for sexual development. For light-responsive phenotypic analyses, plates were incubated for 3–7 days at 25 ◦C as indicated, either under illumination (20 W m<sup>−</sup><sup>2</sup> white light obtained with fluorescent bulbs) or under dark conditions.



### *2.2. Sequence Analysis of Fawc1 and Fawc2 of F. Asiaticum*

The amino acid sequences of WC-1and WC-2 of *Neurospora crassa* were used to blast against the *Fusarium graminearum* genome at the Ensembl Fungi database (http://fungi.ensembl.org/index.html). The obtained target sequences were used as references to design primers to amplify the corresponding orthologs in *F. asiaticum* wild-type strain EXAP-08. The amplified products were subcloned to T-vector and subsequently sequenced. The obtained sequences of *Fawc1* and *Fawc2* were further aligned with their orthologs from other fungal species retrieved from publically available databases by DNAMAN version 5.2.2 (LynnonBiosoft Company, Pointe-Claire, QC, Canada).

### *2.3. Generation of Mutants and Complementation Strains in F. asiaticum*

The flanks 5 and 3 of *Fawc1* and *Fawc2* were amplified from EXAP-08 genomic DNA, and the *hph* was amplified from plasmid *p22*. Overlap PCR was performed to obtain the 5 -*Fawc1*-*hph*-*Fawc1*-3 gene knockout cassettes by mixing an equimolar ratio of 5 -*Fawc1*, *hph*, and *Fawc1*-3 as templates, and the 5 -*Fawc2*-*hph*-*Fawc2*-3 cassette was prepared in a similar way. The resulted products were separated by gel electrophoresis and then recovered by a gel extraction kit. The purified gene knockout cassettes (2 μg for each cassette) were subjected to protoplast transformation of the wild-type strain EXAP-08 according to the reported method [33]. To screen for the correct mutants of Δ*Fawc1* and Δ*Fawc2*, the transformants grown on selective media containing 75 μg/mL hygromycin B were purified and subjected to genomic DNA extraction via the standard CTAB protocol. Site specific primers were used to carry out PCR assay to screen for the knockout mutants.

To generate the complementation strains, wild-type *Fawc1* and *Fawc2* connected with their 1.5 kb 5- and 1 kb 3-flanking sequences were amplified and subsequently cloned into the flu6 plasmid that contains the geneticin (G418) resistance gene, resulting in *flu6*-*Fawc1*-com and *flu6*-*Fawc2*-com expression vectors. Then, the complementation vectors were transformed into the protoplasts of the corresponding gene deletion mutants. CM containing 100 μg/mL of G418 was used to select the successful transformants. To generate mutant strains carrying truncated FaWC1 that lacked either LOV or ZnF domains, the *Fawc1*ΔLOV and *Fawc1*ΔZnF fragments with deletion of the coding regions for LOV and ZnF domains, respectively, were generated by overlap PCR, and the resulted products were gel-purified and cloned into the flu6 plasmid, leading to the expression vectors *flu6*-*Fawc1*ΔLOV and *flu6*-*Fawc1*<sup>Δ</sup>ZnF, which were delivered into Δ*Fawc1* mutant to generate the Δ*Fawc1-C*ΔLOV and Δ*Fawc1-C*ΔZnF strains. All the primers used in the mutant generation and diagnosis PCR reactions are listed in supplementary Table S1.

### *2.4. Extraction of RNA and Quantitative RT-PCR Analysis*

For gene expression analysis, samples were prepared as follows: aliquots of 200 μL conidia suspension (10<sup>6</sup>/mL) were inoculated on solid CM medium with cellophane overlays and incubated at 23 ◦C. The mycelium samples were harvested after either 48 h culture in total darkness, or by ending the 48 h culture with light illumination of indicated time duration. Total RNA was extracted using QIAGEN Reagent (Germany), and 1 μg of each RNA sample was used for reverse transcription with the Prime Script™ RT reagen<sup>t</sup> Kit (Perfect Real Time) (TakaRa Biotechnology, Co., Dalian, China). The real-time PCR amplifications were conducted in a CFX96TM Real-Time System (BIO-BAD, Inc., USA) using TakaRa SYBR Premix Ex Taq (TakaRa Biotechnology, Co., Dalian, China). For each sample, the expression of the β-tubulin gene was used as an internal reference. There were three replicates for each sample. The experiment was repeated three times. The gene expression levels were calculated using the 2−ΔΔCt method.
