Trans t1 competent cells

The cDNA was synthesized using the PrimeScriptTMRT reagent Kit with gDNA Eraser (Perfect Real Time) (TAKARA, Toyoko, Japan). Four differentially expressed genes (SfurDES2, SfurDES5, SfurFAR2, SfurFAR3) were randomly selected to amplify the full-length ORF sequence of these genes by using TransStart FastPfu Fly PCR Supermix (TransGen Biotech, Beijing, China). PCR conditions were: 5 min at 94 °C, followed by 40 cycles of 94 °C for 20 s, 20 s at 52 °C, and 45 s at 72 °C, followed by incubation at 72 °C for 10 min, carried out in a Bio-Rad thermocycler (Bio-Rad DNA Engine Peltier Thermal Cycler, Bio-Rad, Hercules, CA, USA). The primers were listed in Table S2, designed by Primer 5.0 software. The products were gel-purified and ligated into a pEASY-blunt vector (TransGen Biotech, Beijing, China). The ligation products were transformed into Trans T1 competent cells (TransGen Biotech, Beijing, China). All sequencing was performed by Tsingke Biotechnology Co., Ltd. (Beijing, China).

To search for laccase proteins in A. areolatum, the protein sequences corresponding to laccase in C. cinerea and P. ostreatus were used as queries using BLASTp [51 ,54 (link),66 ]. In addition, we also searched for three HMM profiles (PF00394, PF07731, and PF07732) in the genome of A. areolatum using HMMER 3.0 software to identify laccase genes. Subsequently, gene-specific primers of the identified laccase genes were designed with Primer Premier 5.0 software (Table S4) [67 ]. Target laccase sequences were then amplified using PrimeSTAR HS DNA polymerase (Takara, Dalian, China) under the following conditions: 98 °C for 1 min; then 30 cycles of 98 °C for 10 s, 58 °C for 15 s, and 72 °C for 1 min; and then 72 °C for 10 min for a final extension. The PCR products were recovered using EasyPure Quick Gel Extraction Kit (TransGen, Beijing, China), and the products were ligated into the pEASY-Blunt cloning vector (TransGen, Beijing, China), according to the manufacturers’ instructions. Plasmids were transformed into Trans-T1 competent cells (TransGen, Beijing, China) and sequenced by Ruibiotech (Beijing, China). The sequences of these laccases have been deposited into the GeneBnak database with the accession numbers MT648837-MT648850.

Contigs and gaps were confirmed and filled by RT-PCR using specific primer pairs 1, 2, 3, 4, 1-2, 2-3, and 3-4 F/R (Table S1) designed according to the consensus sequences of the assembled viral contigs with more than 70-nt overlapping. The sugarcane yellow leaf virus (ScYLV, AF157029.1) genome sequence was used for the alignment and positioning of the contigs. The terminal sequences of the viral genome were obtained using 5′ and 3′ rapid amplification of cDNA ends (RACE) kits according to the manufacture (Invitrogen). The PCR products were collected using Wizard SV Gel and PCR Clean-Up System (Promega). The purified PCR products were then cloned into the pEASY-T5 vector (TransGen Biotech, China) and used to transformed Trans-T1 competent cells (TransGen Biotech) following the manufacturers' instructions. The clones harboring the transformed vector were identified by PCR and Sanger sequencing (Sangon Biotech (Shanghai) Co., China). The results of sequencing were assembled using DNAMAN (version 6) program (Lynnon Biosoft, San Ramon, CA, USA) with more than 70-nt overlapping regions to form the full-length viral genome.