Additional file 1 of Chitin synthases containing myosin motor-like domain are required for cell wall integrity and virulence of vascular wilt pathogen Verticillium dahliae
Creators: name: Chen, Bin
name: Tian, Juan
name: Feng, Zhidi
name: Wang, Huan
name: Sun, Jie
name: Kong, Zhaosheng
Publisher: name: figshare
Year: 2024
Subjects: subject: Plant Biology
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. Domain structures of class I-VIII chitin synthases in Verticillium dahliae. Class V and class VII CHSs have an N-terminal myosin motor-like domainand a C-terminal Chitin_synth_2 domain. Meanwhile, other CHSs contain at least a Chitin_synth_1, Chitin_synth_2, or Chitin_synth_1N domain. All chitin synthases have multiple transmembranedomains in V. dahliae. The boxes in different colors show the myosin motor-like domains, chitin_synth_1 domain, chitin_synth_1N domain, chitin_synth_2 domain, cytochrome b5-like Heme/Steroid binding domain, and the DEK_C terminal domainin Pfam database. The transmembrane helicesdomain was indicated by black box. Figure S2. Identification of ΔVdChs7, ΔVdChs5, and ΔVdChs5Chs7 knockout mutants. a Strategy for gene knockout and primers used for testing mutants. HPH represents hygromycin phosphotransferase encoding gene. b PCR confirmation of the knockout mutants and the complemented strains with the primers Test-Hyg-F/R. Lanes 1, 2, 3, 4, 5, 6, 7, and 8 indicate V592, ΔVdChs7, ΔVdChs7/VdChs7-GFP, ΔVdChs5, ΔVdChs5/VdChs5-GFP, ΔVdChs5Chs7, ΔVdChs5Chs7/VdChs7-GFP, and ΔVdChs5Chs7/VdChs5-GFP strains, respectively. c PCR confirmation of the knockout mutants and the complemented strains with the primers Test-VdChs7-F/R. d PCR confirmation of the knockout mutants and the complemented strains with the primers Test-VdChs5-F/R. e PCR confirmation of the ΔVdChs5Chs7 knockout mutant and the wild-type strain V592 with the primers Test-1F/R. f PCR confirmation of the ΔVdChs5Chs7 knockout mutant and the wild-type strain V592 with the primers Test-2F/R. Lanes 1 and 2 indicate wild-type strain V592 and ΔVdChs5Chs7, respectively. Figure S3. RT-qPCR analyses of the VdChs5and VdChs7expression during Verticillium dahliae infection into cotton. The samples were collected from taproot of the infected cotton at 12 h, 24 h, 48 h, 3 d, 5 d, and 8 d post inoculation with conidial suspension of wild-type strain V592. Conidial suspension for infectionand the mycelia 12 h, 24 h, and 48 h post inoculation with conidia on Czapek-Dox mediumwere set as control. The V. dahliae β-tubulinwas used as an endogenous control for gene expression analysis. The error bars represent the standard deviation. ** indicates the statistical significancecompared with 0 h by Student’s t-test. Figure S4. The chitin content of the V592, ΔVdChs7, ΔVdChs5, and ΔVdChs5Chs7 knockout mutants and the complemented strains. Conidia were inoculated into 100 mL liquid CM at a concentration of 106 conidia/mL and incubated at 26 °C with shakingfor 3 days. Three aliquots of 10 mg lyophilized mycelia were used as independent samples for cell wall analysis and the experiment was repeated three times from different biological samples. The valuesshown are micrograms of cell wall component per 10 mg dry mycelia. ** and *** indicate significant differences at P &lt; 0.01 and P &lt; 0.001, respectively, according to Student’s t-test. Figure S5. HIGS silencing of VdChs5Chs7 had no impact on the cotton growth. a Growth phenotype of different HIGS-treated cotton plants without inoculating V592. The seedlings were photographed at 30 days post inoculation. b The transverse section of stems of different HIGS-treated cotton plants without inoculating V592 at 30 dpi. Scale bars, 0.5 mm. c The longitudinal section of stems of different HIGS-treated cotton plants without inoculating V592 at 30 dpi. Scale bars, 0.5 mm.