Because of its effectiveness in previous studies, we used heterologous expression to assess gene function in the current study [56,57]. To generate expression constructs of the cytochrome P450 genes TRI13 and F1155_1930, the coding region (with introns) of each gene was fused to the Aureobasidium pullulans TEF1 promoter sequence (AbTEF1pro), cloned into the plasmid pRF-GU as described [58], and then transformed into F. verticillioides strain FRC M-3125. In this fungus, AbTEF1pro was expected to drive constitutive expression of TRI13 and F1155_1930 [59]. PCR primers used to generate the constructs are listed in Supplementary Table S1. AbTEF1pro was amplified from plasmid pTEFEGFP [59] using PCR primers TEF1-3 and TEF1-5-up, which included the sequence GGACTTAAU at its 5′ end to facilitate USER cloning (New England Biolabs, Ipswich, USA). The TRI13 coding region was amplified from 66739 genomic DNA with PCR primers TRI13-5 and TRI13-3-down, and the F1155_1930 coding region was amplified with primers CPM1-5 and CPM1-3-down. To facilitate fusion of AbTEF1pro and coding regions, the 5′ primer for each coding region (i.e., TRI13-5 and CPM1-5) included a 15-base overlap with the 3′ end of AbTEF1pro. In addition, the 3′ primer for each coding region (i.e., TRI13-3-down and CPM1-3-down) included the sequence GGGTTTAAU to facilitate USER cloning. To generate fusion constructs, the AbTEF1pro and individual coding-region amplicons were employed as a DNA template in a second round of PCR, using primers TEF-5-up and Tri13-3-down to amplify the AbTEF1pro-TRI13 fusion construct, and primers TEF-5-up and CPM1-3-down to amplify the AbTEF1pro-F1155_1930 fusion construct. The resulting fusion constructs were cloned separately into plasmid pRF-GU using the USER protocol as previously described (Hao e al. 2019). Briefly, each amplified fusion construct was ligated separately with pRF-GU that had been digested with restriction enzymes PacI and Nt.BbcCI (New England Biolabs). A 2-μL aliquot of each ligation reaction was transformed separately into Escherichia coli TOP10 competent cells (Invitrogen, Waltham, USA). The resulting colonies were screened by PCR with primers TEF-5-up and TRI13-3-down (AbTEF1pro-TRI13) and primers TEF-5-up and CPM1-3-down (AbTEF1pro-F1155_1930) to confirm the presence of the fusion constructs, and selected plasmids (AbTEF1pro-TRI13 and AbTEF1pro-F1155_1930 expression vectors) were subjected to Sanger sequencing using TEF1-664F, TRI13-399R or CPM1-437R to confirm that fusion construct sequences were correct.Each expression vector was transformed into Agrobacterium tumefaciens strain AGL1 as previously describe [58]. Briefly, the A. tumefaciens transformants were grown overnight in Luria Broth [60] supplemented with spectinomycin to an OD600 of 0.4. A 200 μL aliquot of the A. tumefaciens cell suspension was then mixed with 200 μL of F. verticillioides conidia (1 × 107 conidia per mL) harvested from a 7-day-old V8 Juice agar culture. Two hundred μL of the resulting cell-conidia mixture were spread onto a 0.45-μm pore nitrocellulose filter (Whatman-Cytiva, Marlborough, USA) on co-culture medium [61] amended with 200 μM acetosyringone. After incubation at 25 °C for two days, the filters were transferred to V8 Juice agar medium containing 150 μg/mL hygromycin B and 200 μM cefotaxime (Sigma Aldrich Chemical Co., St. Louis, Missouri, USA). Hygromycin B-resistant colonies of F. verticillioides were transferred to fresh V8 Juice agar containing 150 μg/mL hygromycin and 200 µg/mL cefotaxime. Genomic DNA was isolated from the resulting cultures and screened by PCR to confirm the presence of the expression constructs. Primers TEF-5-up & Tri13-3-down and TEF-5-up and CPM1-3-down were used to confirm the presence of the AbTEF1pro-TRI13 and AbTEF1pro-F1155_1930 constructs, respectively. Three independently acquired F. verticillioides transformants for each expression construct were selected for further analysis.