Additional file 1 of Functional analysis of cutinase transcription factors in Fusarium verticillioides
Creators: name: Peng, Minghui
name: Wang, Jiajia
name: Lu, Xiange
name: Wang, Meiduo
name: Wen, Gaolong
name: Wu, Congxian
name: Lu, Guodong
name: Wang, Zonghua
name: Shim, Won Bo
name: Yu, Wenying
Publisher: name: figshare
Year: 2024
Subjects: subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Plant Biology

description: Additional file 1: Figure S1. Sequence structure of FvCtf1α, FvFarA, FvFarB, and FvCut, and phylogenetic tree analysis of CTF transcription factors. a Schematic of FvCtf1α, FvFarA, and FvFarB of F. verticillioides showed a GAL4-like Zn(II)2Cys6 (purple) and a fungal specific transcription factor (blue). Sequence structure of CTF transcription factors identified by SMART software and their schematic were drawn using IBS 1.0 software, respectively. b The phylogenetic tree of Ctf1 was constructed based on the amino acid sequence from eight selected fungi including M. oryzae (Mo), N. crassa (Nc), and F. verticillioides (Fv), F. oxysporum (Fo), Fusarium graminearum (Fg), Botrytis cinerea (Bc), C.albicans (Ca), as well the orthologs from the fungus F. solani (Fs). Using the Clustal W method of the Megalign program, the tree was constructed using MEGA 6.0 software by Maximum Likelihood with 1000 bootstrap reapplication. Bootstrap support values greater than 50% are indicated at the relevant nodes and Bayesian posterior probabilities are ≥ 95%. The decimal under the branch indicates the degree of genetic variation of the gene. Numbers around nodes indicated the bootstrap value. The bar marker showed the genetic distance. c Schematic of four FvCut showed a SP (dark blue) and a cutinase catalytic domain (pink) in F. verticillioides. Sequence structure of FvCut was identified by SMART software and schematic were drawn using IBS 1.0 software. SP: signal peptide. Figure S2. Gene deletion and mutant complementation of cutinases transcription factor. a Diagram showing that the target gene coding region was replaced by the HPH cassette. HPH: Hygromycin. The upstream fragment is a probe used for hybridization. b PCR verification of knockout mutants ΔFvctf1α. The ORF of the target gene FvCTF1α from the candidate transformant was amplified by PCR with no band appeared, and the connection product (UA) of the upstream fragment of the target gene and the HPH fragment was obtained by PCR. c–e Confirmation of the mutants by Southern blot. The genomic DNA is digested by different enzymes that of WT (Fv7600) and ΔFvctf1α digested by EcoRI, that of WT and ΔFvfarA digested by BamHI, that of WT and ΔFvfarB were digested by HindIII, and then separated by agarose gel, respectively. Anticipated band sizes were obtained. f–h Confirmation of mutants and mutant complementation by qRT-PCR. qRT-PCR was used to quantify transcript level of FvCTF1α, FvFARA, and FvFARB genes by comparison with the reference gene β-tubulin2 using the 2−ΔΔCt method. Error bars represent the standard deviation. ND means that the value is not detected. qRT-PCR was conducted at least twice with three independent biological replicates. Figure S3. Gene deletion of cutinases genes. a, b PCR verification of knockout mutants ΔFvcut3 and ΔFvcut4. The ORF of the target gene FvCUT3 and FvCUT4 from the candidate transformant was amplified by PCR with no band appeared, and the connection product (UA) of the upstream fragment of the target gene and the HPH fragment was obtain by PCR, respectively. c qRT-PCR was used to confirm mutants. qRT-PCR was used to quantify transcript level of FvCUT3 and FvCUT4 genes by comparison with the reference gene β-tubulin2 using the 2−ΔΔCt method. Error bars represent the standard deviation. ND means that the value is not detected. qRT-PCR was conducted at least twice with three independent biological replicates. Figure S4. FvCut3 and FvCut4 were not contributed to hype growth and pathogenicity on sugarcane. a The vegetative growths of ΔFvcut3, ΔFvcut4, and WT were monitored on CMII, MM medium, respectively. b Sugarcane (Badila) stem were split longitudinally to visually inspect rot symptoms 7 days after inoculation with wide type and ΔFvcut3. c Sugarcane stem were split longitudinally to visually inspect rot symptoms 7 days after inoculation with wide-type and ΔFvcut4. Figure S5. FvFarA and FvFarB were not contributed to hype growth and pathogenicity on sugarcane. a The vegetative growths of ΔFvfarA, ΔFvfarB, and WT were monitored on CMII, MM medium, respectively. b The colony diameters of the cultures were measured and analyzed by t-test. c Sugarcane were split longitudinally to visually inspect rot symptoms 7 days after inoculation. Sugarcanes were inoculated with immersed ΔFvfarA, ΔFvfarB, and WT conidia toothtips at the internodal region, respectively, and incubated for 7 days. Control were inoculated with sterile toothtip. Three independent biological repetitions were performed. Figure S6. The RNA-Seq analysis of the ∆Fvctf1α mutant. a Valcanic maps for Differential gene expression (DEGs) identified by 1.2-fold |log2_fold change| in FKPM values. DEGs analysis was conducted using cuffdiff v2.1.1 with parameters: -FDR(False Discovery Rate) = 0.05 -library-norm-method classic-fpkm -u/-multi-read-correct -b/-frag-bias-correct. A gene that was considered to be differentially expressed must have at least 1.2-fold expression changes between WT and Fvctf1α mutant. Red dots, significantly upregulated genes. Green dots, significantly downregulated genes. Blue dots, nondifferentially expressed genes. b, c The enrich genes of DEGs by GO analysis, the x-axis displays the number of genes and the right y-axis shows GO terms, with the down-regulated DEGs enriching to 17 GO terms and the up-regulated DEGs enriching to 19 GO terms. d, e KEEG analysis of DEGs enrich pathway, the x-axis displays the number of genes and the right y-axis shows KEEG terms, with the down-regulated DEGs enriching to 20 pathways and the up-regulated DEGs enriched to 5 pathways.