1. Growth conditions of strains and plants
Vd080, a virulent strain of V. dahliae preserved in our laboratory, grew in darkness on Potato Dextrose Agar (PDA) at 25 °C. Susceptible variety Jimian 11 was preserved in our laboratory for pathogen infection experiment. Cotton grows at 28 °C in a 16 h light/8 h dark cycle greenhouse. YTK12 yeast strain was preserved in our laboratory.
2. Vector and bioinformatics analysis
The gene knockout vector B303-Hyg and gene complemented vector pCAMBIA1302-neo were provided preserved in our laboratory. The pSUC2 vector was preserved in our laboratory. DNAMAN was used for multi-sequence alignment of each protein. Potential signal peptides are predicted by the signal peptide prediction server SignalP V5.0 prediction (http://www.cbs.dtu.dk/services/SignalP/index.php) (Almagro Armenteros, et al. 2019).
3. Yeast signal peptide capture test
The function of signal peptides (SP) was verified by yeast secretion system. The SP sequence of VdERG2 was cloned into pSUC2 vector with specific primers, and the obtained plasmid was transformed into YTK12 yeast strain. According to the previous method, positive colonies were screened on CMD-W medium, and the utilization of raffinose by yeast was observed on YPRAA medium to verify the function of signal peptide (Liu, et al. 2021b). The activity of sucrose transferase was detected using 2% TTC solution to verify the function of signal peptide (Meng, et al. 2022).
4. Knockout and complemented of VdERG2 Gene
According to the previous method, both VdERG2 knockout mutants and complemented mutants were obtained through Agrobacterium-mediated transformation (ATMT) (Li, et al. 2012, Paz, et al. 2011, Wang, et al. 2016a). The upstream and downstream 0.9 Kb sequence of VdERG2 gene was selected. The target fragment was amplified from wild type genomic DNA by specific primers (B303-VdERG2-Up-F/R, B303-VdERG2-Down-F/R). A hygromycin resistant fragment (HPH) was amplified from B303 vector by specific primers (VdERG2-HPH-F/R). Three fragments were ligated with linearized B303 vector by homologous recombination ligase (ClonExpress Ultra One Step Cloning Kit, Vazyme, Nanjing, China). After the obtained plasmid (B303-Up-HPH-Down) was transformed into the susceptible state of GV3101, the ATMT method was used to screen the positive transformants on PDA medium containing hygromycin resistance, and the specific primers (VdERG2-F/R, HPH-F/R) were used for PCR verification. Using the same method, the 1.2 Kb upstream fragment of VdERG2 gene, VdERG2 fragment and linearized pCAM-BIA1302 vector were connected and transformed to obtain the recombinant plasmid (pCAM-BIA1302-Up-VdERG2). Based on the knock-out mutant strain, the positive transformants were screened according to the vector resistance, and the specific primers (VdERG2-F/R, HPH-F/R) were used for PCR verification. The primers used in this assay were listed in Table S1.
5. Southern blotting
According to the previous method, DIGHigh prime DNA marker and detection kit II (Roche, Germany) were used for southern blotting. The fragment of HPH was amplified with specific primers (HPH-F/R) as probe. Genomic DNA was extracted from knockout mutant and wild type by CTAB method. In the experiment, HindIII was used for enzyme digestion of genomic DNA (Glenn and Andreou 2013). The primers used in this assay were listed in Table S1.
6. Determination of ergosterol biosynthesis
To extract ergosterol, a 5 mL spore suspension (1 × 107 CFU/mL) of each strain was inoculated in 50mL Potato Dextrose Broth (PDB) medium, and incubated at 25 °C for 3 days. Mycelia were harvested by filtration and washed three times with sterile water. The resulting mycelia were dried at 60 °C for 3 h, and the dried mycelia were ground into a powder.
Specific Extraction of Ergosterol Refer to previously published method for extraction (Liu, et al. 2013). The instrument used in this test was Waters 2695 high performance liquid chromatograph equipped with Waters 2996 ultraviolet detector (Ping and Rong 2006). Ergosterol was separated at 30 °C on a Waters C18 Column (5 µm, 4.6 mm × 250 mm) analytical column using 100% methanol (chromatography pure) as mobile phase. The detection wavelength was 282 nm, and the standard was purchased from Shang hai yuan ye Bio-Technology Co, Ltd. The experiment was repeated three times (Chiocchio and Matković 2011, Gessner 2020, Nahar, et al. 2020).
7. Oxidative stress
Oxidative stress was detected using the method of previous studies (Rehman, et al. 2018, Tang, et al. 2020). To test oxidative stresses, a 100 μL conidial suspension (107 spores/ml) of each strain was spread onto PDA plates, and filter paper discs containing 5 μL of 7.5%, 15% and 30% hydrogen peroxide (H2O2) were placed onto the centre of each plate. The zone of growth inhibition was measured after 3 days.
8. Expression analysis of related genes
To study the impact of VdERG2 knockout on the regulation of other genes related to microsclerotia and melanin, some genes related to melanin and microsclerotia formation were as follows: class II hydrophobin gene (VDH1) (Klimes, et al. 2008, Klimes and Dobinson 2006), pigment biosynthesis protein Ayg1 (Vayg1) (Fan, et al. 2017), scytalone dehydratase (VdSCD) (Duressa, et al. 2013), laccase (VdLAC) (Li, et al. 2020), tetrahydroxynaphthalene reductase (VT4HR) (Wang, et al. 2018), and versicolorin reductase (VaflM) (Wang, et al. 2016b).
Conidia were harvested from a 6-day-old culture of wild type, ΔVdERG2, and C-ΔVdERG2 strains and the concentration was adjusted to 107 CFU/mL. 1 μL of the respective suspensions was inoculated in 200 mL of PDB and incubated on the shaker (180 rpm) at 25 °C. After 5 days, the culture was filtered through four layers of clean gauze to collect hyphae. Total RNA was extracted from the respective hyphae using the RNA Extraction Kit (YPHBio, Tianjin, China). First strand cDNA was synthesized with HiScript II QRT SuperMix for qPCR (+g DNA wiper) (Vazyme) according to the instructions. QRT-PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme). The primers used in this assay were listed in Table S1.
9. Stress response and carbon utilization detection
For the determination of abiotic stress, 1 mol/L KCl, 1 mol/L NaCl, 1 mol/L Sorbitol, 0.004% SDS and 0.02% CR were added to PDA medium (Liu, et al. 2021a, Zhang, et al. 2022a). Normal PDA medium was used as a control. The spore suspension was cultured on various types of PDA and at 25 °C. In order to study the role of VdERG2 in the growth of V. dahliae during the absorption or utilization of specific carbon sources, sucrose (30 g/L), cellolose (5 g/L), skim milk (18 g/L), pectin (10 g/L) and starch (17 g/L) were added to Czapek-Dox medium without sucrose, respectively. The colony diameter of all strains were measured after 14 days of culture (Guo, et al. 2022, Liu, et al. 2021c, Su, et al. 2018, Zhang, et al. 2022a, Zhang, et al. 2015). All experiments were repeated three times.
10. Mycelium penetration test
Preparation of spore suspension according to previous methods (Fraczek, et al. 2019). 5 μL spore suspension of V. dahliae strains were added to the PDA plate with a layer of cellophane. After 3 days of culture, the growth was observed and photographed. Three days later, in the ultra-clean worktable, PDA plates were removed the cellophane with mycelium, reclosed the petri dish, after three days of culture, photographed the colony morphology.
The mycelia above the removed cellophane were made into frozen sections, and the growth of mycelia was observed under scanning electron microscope. The back of the cellophane was placed on the metal table and sprayed with gold under vacuum. The spores or hyphae were observed under a scanning electron microscope.
11. Pathogenicity test
The spore suspensions of various strains were prepared and the concentration was adjusted to 1 × 107 CFU/mL. The cotton seedlings with the same growth and two true leaves were gently pulled out of vermiculite. The roots were washed with water, and the seedlings without damage to the roots were selected. The cotton seedlings were soaked in the spore suspension for 10 min by dipping the roots. Subsequently, they were retransplanted into nutrient soil, and placed in the cotton greenhouse for cultivation.
The disease index (DI) was investigated at three time points 14, 18 and 21 days after inoculation. The disease index was based on the previous method (Gong, et al. 2017). According to symptoms on cotyledons and true leaves, which were divided into five grades (0, 1, 2, 3 and 4) (Wang, et al. 2004, Zhang, et al. 2012).
The stems of cotton plants on the 21 day after inoculation were selected for fungal recovery test. Fungal recovery test was based on the method described before (Zhang, et al. 2016, Zhang, et al. 2019).
Moreover, the stems of cotton were longitudinally cut with a scalpel, and the browning degree of the stem was observed under the stereomicroscope (Leica, M165 FC, Germany), and photographed. At 21 days after inoculation, the total DNA of cotton plant was extracted using the plant genome extraction kit (Vazyme), and calculated by 2−ΔΔCT method. Vdβt was the target sequence for V. dahliae detection, and GhUBQ7 was the reference gene.