VdERG2 was involved in ergosterol biosynthesis, nutritional differentiation and virulence of Verticillium dahliae

VdERG2 was involved in ergosterol biosynthesis, nutritional differentiation and virulence of Verticillium dahliae | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article VdERG2 was involved in ergosterol biosynthesis, nutritional differentiation and virulence of Verticillium dahliae Junyuan Lv, Shichao Liu, Xiaojian Zhang, Lihong Zhao, Tao Zhang, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2131818/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract The ergosterol biosynthesis pathway plays an important role in model pathogenic bacteria Saccharomyces cerevisiae , but little is known about the biosynthesis of ergosterol in pathogenic fungus Verticillium dahliae . In this study, we identified the VdERG2 gene encoding sterol C-8 isomerase from V. dahliae and investigated its function in virulence by generating gene deletion mutants (ΔVdEGR2) and complemented mutants (C-ΔVdEGR2). Deletion of VdERG2 reduced ergosterol content. The conidial germination rate and conidial yield of ΔVdERG2 decreased significantly, and abnormal conidia were produced. In spite of VdERG2 did not affect the utilization of carbon sources by V. dahliae , but ΔVdERG2 observed a decrease in melanin production when cellulose and pectin were used as sole carbon sources, respectively. The ability of mutants ΔVdERG2 to produce microsclerotia and melanin decreased and the knockout of VdERG2 led to a significant decrease in the expression of microsclerotia and melanin-related genes VaflM , Vayg1, VDH1, VdLAC , VdSCD and VT4HR . In addition, mutants ΔVdEGR2-1 and ΔVdEGR2-2 were very sensitive to congo red (CR), sodium dodecyl sulfate (SDS) and hydrogen peroxide (H 2 O 2 ) stresses, indicating that VdEGR2 was involved in cell wall and oxidative stress response. The absence of VdERG2 weakened the penetration ability of mycelium on cellophane and affected the growth of mycelium on cellophane. Although ΔVdERG2 could infect cotton, its pathogenicity was significantly impaired. These phenotypic defects in ΔVdERG2 could be complemented by reintroduction of a full-length VdERG2 gene. In summary, as a single conservative secretory protein, VdERG2 played a crucial role in ergosterol biosynthesis, nutritional differentiation and virulence in V. dahliae . Verticillium dahliae Ergosterol biosynthesis Nutritional differentiation Cell wall stress Oxidative stress Virulence Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction As a soil-borne fungus, V. dahliae can cause Verticillium wilt in more than 200 host plants, including important economic crop (Johnson and Dung 2010, Song, et al. 2020). The characteristic of Verticillium wilt is that fungal hyphae fills woody vascular tissues of plants, resulting in plant leaves wilting and dying (Prieto, et al. 2009, Subbarao 2009). For example, cotton production in many parts of the world is seriously threatened by this disease. At present, once plants are infected, there is no efficient fungicide to cure them. The pathogen has strong variability and co-evolution ability with the host, and its pathogenic mechanism is very complex (Depotter, et al. 2018, ZHANG, et al. 2022b). The interaction mechanism between pathogens and hosts still unclear (Depotter, et al. 2018). Therefore, studying the molecular mechanism of V. dahliae pathogenicity will help to control Verticillium wilt of cotton. Ergosterol is the main sterol component in fungal plasma membrane and a fungal specific sterol (Jordá and Puig 2020, Munn, et al. 1999, Zinser, et al. 1993). Ergosterol can maintain the integrity and fluidity of cell membrane, regulate cell membrane permeability and membrane binding protein activity (Abe and Hiraki 2009, Wang, et al. 2020). In fungi, ergosterol is synthesized by acetyl coenzyme A through a complex process (Hu, et al. 2017, Zhang, et al. 2020). The biosynthesis of ergosterol has been well described in S. cerevisiae and the ergosterol pathway includes the following nine non-essential genes: HMG1 , HMG2 , ERG2 , ERG3 , ERG4 , ERG5 , ERG6 , ERG24 , and ERG28 (Bhattacharya, et al. 2018) . Deletion strains for each of the 9 nonessential genes were viable but showed disruption of ergosterol biosynthesis and accumulation of aberrant sterols leading to susceptibility to stress agents and osmotic stress (Gupta, et al. 2003, Kodedová and Sychrová 2015, Valachovic, et al. 2006). But ERG1 , ERG18 and ERG26 were essential for fungal survival in S. cerevisiae and Candida albicans (Liu, et al. 2013). So far, there is little information about ergosterol pathway in V. dahliae . In previous studies, sterol C-24 reductase encoded by FgERG4 was involved in ergosterol biosynthesis, affecting differentiation and reducing pathogenicity in Fusarium graminearum (Liu, et al. 2013). The sterol C-14 reductase encoded by FgERG24B enhanced the intrinsic resistance of F. graminearum to amine fungicides(Liu, et al. 2011). ERG24 and ERG2 were the main targets of amine fungicides (Hernández, et al. 2016, Liu, et al. 2011, Vlainić, et al. 2021). The C-8 isomerase encoded by ERG2 catalyzed the biosynthesis of ergosterol, resulting in the formation of episterol by C-7 unsaturated in sterol B ring (Lees, et al. 2020). High expression of ERG2 gene in S. cerevisiae inhibits the defect of ergosterol synthesis and indicated that ERG2 played a role in ergosterol synthesis (Bhattacharya, et al. 2018, Johnston, et al. 2020, Sanglard, et al. 2003). Although ERG2 has been well documented in S. cerevisiae , ERG2 has not been reported in V. dahliae . Therefore, the main purpose of this study is to investigate the role of VdERG2 in the physiology and virulence of V. dahliae . In this study, we found that VdERG2 not only played a role in the biosynthesis of ergosterol, but also participated in the regulation of nutritional differentiation and virulence of V. dahliae . Materials And Methods 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 × 10 7 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 (10 7 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 (H 2 O 2 ) 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 10 7 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 × 10 7 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. Results Identification of Vd ERG 2 in dahliae Using S. cerevisiae sterol C-8 isomerase Erg2 as query, we identified VdERG2 (VDAG_01363) from the V. dahliae strain VdLs. 17 (https://www.ncbi.nlm.nih.gov/genome/832). The nucleotide sequence of VdERG2 is 678 bp, encoding 225 amino acids. The protein homology analysis using DNAMAN showed that the amino acid sequence of VdERG2 was relatively conservative at the C-terminal with that of S. cerevisiae and other filamentous fungi, but mutated greatly at the N-terminal. Their homology was 73.82% (Figure 1A). SignalP V5.0 predicted that VdERG2 may contain a signal peptide at the N-terminal. YTK12-pSUC2-VdERG2sp was normally grown on CMD-W and YPRAA mediums according to the yeast signal peptide trap recovery test (Figure 1B). TTC staining showed that yeast strain YTK12 carrying pSUC2-VdERG2sp secreted sucrase to hydrolyze sucrose into monosaccharides, which reacted with TTC to produce triphenyltetrazole chloride red insoluble in water (Figure 1B). The above results indicated that VdERG2 was a highly conserved secretory protein in V. dahliae . The knockout and complemented mutants of VdERG2 in dahliae In order to analyze the function of VdERG2 in detail, this study used the method of homologous recombination (Figure 2A), according to the gene sequence of VdERG2 in the genome of V. dahliae VdLs. 17, the upstream and downstream 0.9 Kb fragments were selected, and the upstream of the target gene, hygromycin resistance gene fragment ( HPH ) and the downstream fragment of the target gene were fused to the knockout vector B303 by homologous cloning method to construct the knockout vector B303-VdERG2. VdERG2 was knockout by Agrobacterium mediated fungal transformation using wild type Vd080 conidia. Two mutants ΔVdERG2-1 and ΔVdERG2-2 were determined by PCR and southern blotting (Figure 2C and D). In the genome of V. dahliae , the 1.2 Kb fragment of the upstream promoter region of the target gene VdERG2 and the target gene VdERG2 fragment were selected, and the complemented vector pCAMBIA1302-Neo-VdERG2 was constructed by homologous recombination further genetic transformation to knockout mutants. Two complemented mutants C-ΔVdERG2-1 and C-ΔVdERG2-2 were identified by PCR (Figure 2B). Decrease of ergosterol biosynthesis in the VdERG2 knockout mutant High-performance liquid chromatography (HPLC) analysis ofergosterol extracted from mycelia of wild type, ΔVdERG2-1, ΔVdERG2-2, C-ΔVdERG2-1 and C-ΔVdERG2-2 revealed that there was an ergosterol-specific absorption peak at the retention time of 16.5 min in all strains (Figure 3A). However, the ergosterol content of the ΔVdERG2-1 and ΔVdERG2-2 mutants were significantly lower than that of the wild type and complemented mutants (Figure 3B). It indicated that VdERG2 played an important role in ergosterol synthesis pathway in V. dahliae . VdERG2 was involved in the regulation of conidia in dahliae After the strains were cultured in liquid medium for 5 days, the number of spores were observed under the fluorescence microscope and counted through the blood cell counting plate. It was found that the conidia produced in ΔVdERG2-1 and ΔVdERG2-2 were significantly less than those in the wild type and the complemented mutant (Figure S1 and Figure 4A). The same concentration spore suspensions with the same concentration of spores of each strain were coated on PDA for 6 h, and the spore germination was observed. It was found that the germination rates of conidia in ΔVdERG2-1 and ΔVdERG2-2 were lower than those in the wild type and the complemented mutant (Figure S2 and Figure 4B). The conidia were made into frozen sections, and the spore morphology was observed under scanning electron microscope. It was found that ΔVdERG2-1 and ΔVdERG2-2 could lead to abnormal spore morphology (Figure 4C). The above results indicated that the deletion of VdERG2 could lead to decrease of spores production and spores germination rate, with influencing on the normal morphology of spores. VdERG2 did not affect the utilization of carbon source, but reduced microsclerotia formation and melanin production In order to further explore the function of VdERG2 in the utilization of carbon sources, sucrose, cellulose, skim milk, pectin and starch were selected as carbon sources. The results showed that VdEGR2 did not affect the utilization of carbon sources, but when cellulose and pectin were used as carbon sources, the melanin and microsclerotia of the mutant showed a decreasing trend (Figure S3). In order to further investigate whether VdERG2 affects the production of melanin and microsclerotia. The results showed that ΔVdERG2-1 and ΔVdERG2-2 significantly reduced the production of melanin and microsclerotia compared with wild type and complement mutant strains (Figure 5A). Analysis using RT-qPCR to detect in the expression of melanin and microsclerotia-related genes VT4HR, VaflM , Vayg1, VdLAC , VdSCD and VDH1 . The results showed that their expression levels decreased significantly (Figure 5B). VdERG2 responded to CR, SDS and H 2 O 2 stresses As a soil-borne fungus, there are various stresses in the soil, affecting the growth of pathogen V. dahliae . In this study, 1 mol/L KCl, 1 mol/L NaCl, 1 mol/L Sorbitol, 0.004% (mass/mass) SDS and 0.02% (mass/mass) CR were added to PDA medium to simulate abiotic stress. Normal PDA culture was used as the control. The results showed that the growth of ΔVdERG2 in KCl, NaCl, Sorbitol and normal PDA medium were not different from those of wild type and complemented mutant (Figure S4). Under CR stress, the colony diameters of ΔVdERG2-1 and ΔVdERG2-2 were 37.39 mm and 38.33 mm, respectively, which were lower than those in normal PDA (63.5 mm and 63.75 mm). After gene complemented, the colony diameter increased, which was consistent with the wild type (Figure 6A). Under SDS stress, the colony diameters of ΔVdERG2-1 and ΔVdERG2-2 were 31.61 mm and 32.03 mm, respectively, which were lower than those in normal PDA (63.5 mm and 63.75 mm). After gene complemented, the colony diameter increased, which was consistent with that of wild type (Figure 6A). The above results showed that VdERG2 was involved in the response of V. dahliae to CR and SDS-induced cell wall stress, the cell wall of mutant ΔVdERG2 was defective. In order to study the role of VdERG2 in response to oxidative stress, we examined the growth status of these strains under H 2 O 2 stress. The sensitivity of each strain to H 2 O 2 was examined by measuring the diameter of inhibition zone. Three gradients (7.5%, 15% and 30%) of H 2 O 2 concentration were designed in the experiment. The results showed that the inhibition zones of ΔVdERG2-1 and ΔVdERG2-2 were larger than those of wild type and complemented strains, and the phenotypes became more and more obvious with the increase of H 2 O 2 concentration (Figure 6B). VdERG2 weakened hyphal penetration and affected hyphal growth on cellophane In order to detect the penetration ability on cellophane, conidia of each strain were placed on PDA medium containing glass paper for 3 days at 25 °C. The results showed that all strains could grow normally on cellophane (Figure 7A). By observing the growth of each strain on the cellophane, it was found that the mycelium growth of the mutant was sparse and chaotic, while the mycelium growth of the wild type and complemented strains were uniform and normal (Figure 7C). By observing the back of the cellophane, it was found that all strains had conidia (Figure 7B). When removing the cellophane, it was observed that the colony diameters of ΔVdERG2-1 and ΔVdERG2-2 were significantly smaller than those of the wild type and the complemented mutant (Figure 7A). The above results showed that VdERG2 could weaken the penetration of mycelium and affect the growth of mycelium on cellophane. VdERG2 positively regulated the virulence of dahliae To further study the function of VdERG2 in virulence of V. dahliae , the pathogenicity of wild type Vd080, ΔVdERG2-1, ΔVdERG2-2, C-ΔVdERG2-1 and C-ΔVdERG2-2 strains were determined using cotton root dipping method. The results showed that compared with the control inoculated with water, the leaves of cotton inoculated with knockout strains turned yellow and wilted at 21 days after inoculation, and only a few cotyledons fell off. After inoculation with mutant strains, the leaves wilted and necrotic, and the true leaves fell off, which was consistent with the incidence of wild type strains. The results showed that the pathogenicity of V. dahliae to cotton decreased after knocking out VdERG2 (Figure 8A). The stems of cotton were cut to observe the browning. The results showed that the browning of stems inoculated with knock-out mutant strains was significantly lighter than that inoculated with wild type and complemented mutant strains (Figure 8B). Fungal recovery test showed that the colonies of diseased cotton stems inoculated with knockout mutants were less than those of wild type and complemented mutants (Figure 8C). At the 14, 18 and 21 days after inoculation, the disease index of cotton was investigated. The results showed that the disease index increased over time, 21 days post inoculation, the disease index of cotton plants infected by deletion mutants (ΔVdERG2-1 and ΔVdERG2-2) were 34.56 and 34.22, respectively. While that of cotton plants infected by wild type, C-ΔVdERG2-1 and C-ΔVdERG2-2 were 71.11, 72.07 and 71.94, respectively (Figure 8D). When the fungal biomass in roots was evaluated by RT-qPCR to assess the role of VdERG2 in systemic infection, the fungal biomass of C-ΔVdERG2-1 and C-ΔVdERG2-2 strains did not differ significantly, whereas the fungal biomass of ΔVdERG2-1 and ΔVdERG2-2 strains decreased by 4.73 times and 4.54 times, respectively, compared with the wild type strain (Figure 8E). The above results show that VdERG2 positively regulated the pathogenicity of V. dahliae . Discussion The main sterol components in fungal plasma membrane are involved in a variety of cell functions. Because of the important biochemical characteristics of ergosterol, many enzymes involved in its biosynthetic pathway have become the attract sites for antifungal drugs (Liu, et al. 2011, Liu, et al. 2013). ERG2 is a non-essential enzyme in S. cerevisiae , but high expression inhibits ergosterol biosynthesis (Hu, et al. 2017). In this study, we used ERG2 in S. cerevisiae as the query object and identified VdERG2 in the genome of V. dahliae VdLs. 17. VdERG2 was identified as a single conserved secretory protein in V. dahliae (Figure 1). Compared with the wild type, we found that the biosynthesis of ergosterol in the knockout mutant was decreased, indicating that VdERG2 was played a role in the biosynthesis of ergosterol in V. dahliae (Figure 3). At the same time, it also indicated that the biosynthesis pathway of ergosterol in V. dahliae might be slightly different from that reported in S. cerevisiae due to the possibility of gene redundancy exists. Verticillium wilt caused by V. dahliae is called cancer (Cai, et al. 2009, Gong, et al. 2017). Once the plant is infected, it is difficult for fungicides to effectively treat it, which may cause serious economic losses. Therefore, it is particularly important to study the molecular mechanism of V. dahliae and host plants. In previous studies, knockout of FgERG4 in F. graminearum led to a decline in conidial production and malformation of conidia (Liu, et al. 2013), knockout of VdHP1 in V. dahliae reduces conidial smoothness (Zhang, et al. 2022a). In this study, it was found that the knockout of VdERG2 led to the decrease of conidial yield and conidial germination rate, and abnormal conidial morphology (Figure 2). As a soil-borne fungus, environmental conditions in the soil can cause various abiotic stresses and changes in nutritional conditions, thus affecting the normal growth of V. dahliae . Previous studies have shown that VdHog1 , VdPbs2 , VdSsk1 , Vdssk2 and VdCrz1 played important roles in responding to osmotic stress, CR stress and SDS stress in V. dahliae (Tian, et al. 2016, Wang, et al. 2016b, Xiong, et al. 2015, Yu, et al. 2019, Zheng, et al. 2019). In the presence of CR, knockout of VdSsk1 or VdSsk2 caused high expression of genes related to cell wall biosynthesis (Yu, et al. 2019, Zheng, et al. 2019). Knockout of VdCrz1 resulted in high sensitivity to SDS stress (Xiong, et al. 2015). In this study, it was found that ΔVdERG2 was not sensitive to osmotic stress, but highly sensitive to CR and SDS stress (Figure 6). Overcoming the natural barrier of cell wall is a key step for fungal pathogens to infect plant hosts (Ferreira, et al. 2006, Underwood 2012). When VdOGDH was knockout to supplement different carbon sources, the vegetative growth of V. dahliae was significantly inhibited (Li, et al. 2020). After knockout VdSNF1 , the growth of the mutant was significantly inhibited only when pectin or galactose was used as carbon source (Tzima, et al. 2011). In this study, when sucrose, cellolose, skim milk, pectin and starch as carbon sources, the grouth of ΔVdERG2 was similar to those of the wild type and the complemented mutant, interestingly, the melanin and microsclerotia production of the knockout mutant was weakened when cellulose and pectin were used as carbon sources (Figure S3). Previous studies have shown that melanin and microsclerotia are essential for survival and diffusion in V. dahliae life cycle (Tzima, et al. 2011). The virulence of V. dahliae seems to be increasingly related to the development of microsclerotia. The ΔVdSsk2 strain showed severe delayed microsclerotia formation and melanization. Although the microsclerotia development of ΔVdste11 was similar to that observed in the wild type, it was obviously lack of melanin biosynthesis, indicating that both VdSsk2 and VdSte11 were involved in the microsclerotia of micronucleus (Zheng, et al. 2019). In this study, it was found that VdERG2 could inhibit the formation of micronucleus and melanin, thereby affecting virulence (Figure 5). Therefore, VdERG2 not only participated in the response to CR and SDS abiotic stresses but inhibited the formation of microsclerotia, thereby affecting virulence. The enzymes in ergosterol biosynthesis pathway are also involved in the regulation of pathogenicity. Previous studies have shown that FgERG4 knockout mutant in F. graminearum can successfully invade wheat and tomato, but the virulence of the mutant decreased significantly (Liu, et al. 2013). Knockout of Fghyd2 and Fghyd3 in F. graminearum reduced the symptom spikelets in wheat (Quarantin, et al. 2019). H 2 O 2 , one of the earliest cellular responses to infection, is produced when plant host cells are challenged by pathogens or inducers of pathogenic origin (Fassler and West 2011). VdSkn7 mutant showed severe growth defect H 2 O 2 under heat shock and cell wall disturbance, and its toxicity was significantly lower than that of wild type (Tang, et al. 2020). In this study, we found that ΔVdERG2 was very sensitive to H2O2 (Figure 6). Foc_M35-1 reduced the pathogenicity to host plants without affecting the mycelium penetration ability of F . oxysporum (Zhang, et al. 2021). In this study, we found that the deletion of VdERG2 reduced the penetration ability of V. dahliae mycelium and led to the chaotic growth of mycelium (Figure 7). After inoculation with conidial suspension of ΔVdERG2 strain, the cotton cotyledons turned yellow and few deciduous leaves appeared, compared with wild type and complement mutant strains, ΔVdERG2 disease index decreased significantly (Figure 8). It is concluded that VdERG2 may be a positive regulator with multi-faceted functions in pathogenicity of V. dahliae. In conclusion, sterol C-8 isomerase VdERG2 of V. dahliae was a single conservative secretory protein and involved in the ergosterol biosynthesis, the regulation of conidia and inhibited the formation of melanin and microsclerotia. ΔVdERG2 was more sensitive to abiotic stresses (CR and SDS), and weakened the penetration ability of hyphae, thereby reducing the pathogenicity to cotton. This study showed that sterol C-8 isomerase was of great significance for the development, adaptability and pathogenicity of V. dahliae , which may provide a new perspective for us to further understand the molecular mechanism of ergosterol biosynthesis pathway in virulence of V. dahliae . Declarations Supplementary Materials : Supplemental file 1, PDF file Author Contributions: Conceptualization, Hongjie Feng and Heqin Zhu; Data curation, Junyuan Lv; Formal analysis, Lihong Zhao and Ruiyuan Zhao; Investigation, Shichao Liu and Xiaojian Zhang; Methodology, Junyuan Lv, Feng Wei, Jinglong Zhou and Hongjie Feng; Supervision, Tao Zhang, Zhigang Zhang and Zili Feng; Writing–original draft, Junyuan Lv; Writing–review & editing, Hongjie Feng, Caihong Li and Yalin Zhang. Funding : This work was supported by the Natural Science Foundation of Hunan Province, China (No.2021JJ40285), National Natural Science Foundation of China (Grant No. 32201752), the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences, and Hunan Technology Professionals Project (2020TJ-Q17). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. 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Frontiers in microbiology 10: 606 doi: Zinser E, Paltauf F, Daum G (1993) Sterol composition of yeast organelle membranes and subcellular distribution of enzymes involved in sterol metabolism. Journal of bacteriology 175: 2853-2858 doi: Additional Declarations No competing interests reported. Supplementary Files supplementalmaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 18 Oct, 2022 Reviews received at journal 17 Oct, 2022 Reviewers agreed at journal 12 Oct, 2022 Reviewers invited by journal 06 Oct, 2022 Editor assigned by journal 06 Oct, 2022 Submission checks completed at journal 05 Oct, 2022 First submitted to journal 04 Oct, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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(A) Homology analysis of VdERG2 with ERG2 in Saccharomyces cerevisiae and other filamentous fungi. Protein sequences from top to bottom were derived from \u003cem\u003eFusarium verticillioide\u003c/em\u003e, \u003cem\u003eFusarium oxysporum\u003c/em\u003e, \u003cem\u003eFusarium graminearume\u003c/em\u003e, \u003cem\u003eBotrytis cinerea\u003c/em\u003e, \u003cem\u003eV. dahliae\u003c/em\u003e and \u003cem\u003eS. cerevisiae\u003c/em\u003e. (B) Signal peptide capture test to verify the signal peptide functions of VdERG2.\u003c/p\u003e","description":"","filename":"F1.png","url":"https://assets-eu.researchsquare.com/files/rs-2131818/v1/77db39f02f820a6ac3df3972.png"},{"id":27571307,"identity":"13d65501-83de-49fe-887a-8b7cb1b27d8e","added_by":"auto","created_at":"2022-10-10 17:43:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89029,"visible":true,"origin":"","legend":"\u003cp\u003eThe knockout and complemented mutants of VdERG2 in \u003cem\u003eV. dahliae\u003c/em\u003e. (A) The homologous recombination of \u003cem\u003eVdERG2\u003c/em\u003ein \u003cem\u003eV. dahliae. \u003c/em\u003e(B) Identification of \u003cem\u003eVdERG2\u003c/em\u003e gene knockout transformations by PCR. (C) Identification of \u003cem\u003eVdERG2\u003c/em\u003e gene complementary transformations by PCR. (D) Identification of \u003cem\u003eVdERG2\u003c/em\u003e gene knockout transformations by southern blotting.\u003c/p\u003e","description":"","filename":"F2.png","url":"https://assets-eu.researchsquare.com/files/rs-2131818/v1/b07de0f0ca6d8557d3f88739.png"},{"id":27571825,"identity":"cad09e16-3211-45f3-8b3b-dbd7c0910c86","added_by":"auto","created_at":"2022-10-10 17:53:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":116811,"visible":true,"origin":"","legend":"\u003cp\u003eDecrease of ergosterol biosynthesis in the \u003cem\u003eVdERG2\u003c/em\u003e knockout mutant. (A) The ergosterol content in wild type, ΔVdERG2 and C-ΔVdERG2 strains were determined by high performance liquid chromatography (HPLC). Commercial standard of ergosterol was used as the control. (B) The ergosterol content of the wild type, ΔVdERG2 and C-ΔVdERG2 strains. Values represent means ± standard deviation of three replicates. The asterisks represent statistical differences performed by a t-test in comparison with the wild type strains (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"F3.png","url":"https://assets-eu.researchsquare.com/files/rs-2131818/v1/13b35aa96cfed76f2201ddd0.png"},{"id":27571314,"identity":"a44cfbb4-9723-4f3f-9077-fcf747bb292c","added_by":"auto","created_at":"2022-10-10 17:43:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":219154,"visible":true,"origin":"","legend":"\u003cp\u003eVdERG2 involved in the regulation of conidia in \u003cem\u003eV. dahlia. \u003c/em\u003e(A) The spore concentration of the wild type, ΔVdERG2 and C-ΔVdERG2 strains grown in liquid Czapek Dox medium for 5 days. (B) Spore germination rate of wild type, ΔVdERG2 and C-ΔVdERG2 strains cultured in solid PDA medium for 6 h. (C) The morphology of conidia was observed by scanning electron microscope. Scale bar = 20 μm. Values represent means ± standard deviation of three replicates. The asterisks represent statistical differences performed by a t-test in comparison with the wild type strains (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"F4.png","url":"https://assets-eu.researchsquare.com/files/rs-2131818/v1/6109ee4d2feeb0aea5220502.png"},{"id":27571310,"identity":"78dfa0c6-bae1-4ef3-985e-abcc0a6e9694","added_by":"auto","created_at":"2022-10-10 17:43:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":210655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eVdERG2\u003c/em\u003e inhibited the production of microsclerotia. (A) Growth of microsclerotia of wild type, ΔVdERG2 and C-ΔVdERG2 strains cultured on BMM solid medium containing nitrocellulose membrane for 30 days. (B) Analysis using quantitative RT-qPCR to detect the expression of melanin-related genes \u003cem\u003eVaflM\u003c/em\u003e, \u003cem\u003eVayg1, VDH1, VdLAC\u003c/em\u003e, \u003cem\u003eVdSCD\u003c/em\u003e and \u003cem\u003eVT4HR\u003c/em\u003e. Values represent means ± standard deviation of three replicates. The asterisks represent statistical differences performed by a t-test in comparison with the wild type strains (*p\u0026lt;0.05, **p\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"F5.png","url":"https://assets-eu.researchsquare.com/files/rs-2131818/v1/17248026b1f15c96852dcf9f.png"},{"id":27571311,"identity":"5a1751d3-fd3c-474d-afb7-8112d3a3184a","added_by":"auto","created_at":"2022-10-10 17:43:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":253526,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eVdERG2\u003c/em\u003e responded to CR ,SDS and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stresses in \u003cem\u003eV. dahliae. \u003c/em\u003e(A) Phenotype analysis and colony diameter determination of the wild type, ΔVdERG2 and C-ΔVdERG2 strains grown on PDA or PDA supplemented with 0.004% SDS and 0.02% CR for 14 days. (B) Similar numbers of spores (10\u003csup\u003e8\u003c/sup\u003e CFU/mL) of the wild type, ΔVdERG2 and C-ΔVdERG2 strains of \u003cem\u003eV. dahliae\u003c/em\u003e were added to PDA plates. Five millimetre diameter sterile filter paper disks were placed in the centre of the plates, to which 5 μl of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (7.5%, 15% and 30%) was added. The plates were incubated at 25°C for 2 days and the inhibition zones were recorded in millimetres. The suppression zone of the above plates after 2 days' incubation. Values represent means ± standard deviation of three replicates. The asterisks represent statistical differences performed by a t-test in comparison with the wild type strains (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"F6.png","url":"https://assets-eu.researchsquare.com/files/rs-2131818/v1/9b8dcc2f42e79846123125e6.png"},{"id":27571670,"identity":"81532e2a-a36e-4bc2-95c3-2abc25d4d429","added_by":"auto","created_at":"2022-10-10 17:48:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":333069,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eVdERG2\u003c/em\u003e weakened hyphal penetration and affected hyphal growth on cellophane of \u003cem\u003eV. dahliae\u003c/em\u003e. (A) The cellophane membrane penetration assay. Penetration symptoms of the wild type, ΔVdERG2 and C-ΔVdERG2 strains grown on PDA medium overlaid with a cellophane layer (above) for 3days and removal of the cellophane membrane for 5 days. (B) The existence of spores on the back of cellophane membrane were observed by scanning electron microscope. Scale bar = 50 μm. (C) The morphology of hyphae on cellophane membrane were observed by scanning electron microscope. Scale bar = 50 μm.\u003c/p\u003e","description":"","filename":"F7.png","url":"https://assets-eu.researchsquare.com/files/rs-2131818/v1/6c4c720b974369d41127ea9e.png"},{"id":27571312,"identity":"8f491850-93fc-4f28-983f-36a3bd7fe23d","added_by":"auto","created_at":"2022-10-10 17:43:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":295680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eVdERG2\u003c/em\u003e positively regulated the pathogenicity of \u003cem\u003eV. dahliae\u003c/em\u003e in cotton (A) Disease symptoms of cotton after the wild type, ΔVdERG2 and C-ΔVdERG2 strains infection. Photographs were taken at 21 days after fungal inoculation. (B) Vascular discoloration of cotton stem tissue. (C) Reisolation of V. dahliae strains from the stem of inoculated cotton plants at 25 °C for 5 days. (D) Disease index of cotton plants at 14, 18, and 21 days after the wild type, ΔVdERG2 and C-ΔVdERG2 strains infection. (E) Fungal biomass in stems of cotton after the wild type, ΔVdERG2 and C-ΔVdERG2 strains infection at 21 days. \u003cem\u003eVdβt\u003c/em\u003e was used as the detection gene, and \u003cem\u003eGhUBQ7\u003c/em\u003e of upland cotton was used as the endogenous control gene. Values represent means ± standard deviation of three replicates. The asterisks represent statistical differences performed by a t-test in comparison with the wild type strains (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"F8.png","url":"https://assets-eu.researchsquare.com/files/rs-2131818/v1/0984c9421ffdd26212340d7e.png"},{"id":27571826,"identity":"351b349e-6522-4db4-b7f8-1ce706f84f39","added_by":"auto","created_at":"2022-10-10 17:53:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2250254,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2131818/v1/d4b08702-e26d-443e-9dde-5866efdf4fd2.pdf"},{"id":27571315,"identity":"878937c2-a556-490a-94fd-0c291d3d5299","added_by":"auto","created_at":"2022-10-10 17:43:01","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":6008051,"visible":true,"origin":"","legend":"","description":"","filename":"supplementalmaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-2131818/v1/c8a654619e25c2faffa1c461.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"VdERG2 was involved in ergosterol biosynthesis, nutritional differentiation and virulence of Verticillium dahliae","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs\u0026nbsp;a soil-borne fungus, \u003cem\u003eV. dahliae\u003c/em\u003e can cause Verticillium wilt in more than 200 host plants, including important economic crop\u0026nbsp;(Johnson and Dung 2010, Song, et al. 2020). The characteristic of Verticillium wilt is that fungal hyphae fills woody vascular tissues of plants, resulting in plant leaves wilting and dying\u0026nbsp;(Prieto, et al. 2009, Subbarao 2009). For example, cotton production in many parts of the world is seriously threatened by this disease. At present, once plants are infected, there is no efficient fungicide to cure them. The pathogen has strong variability and co-evolution ability with the host, and its pathogenic mechanism is very complex\u0026nbsp;(Depotter, et al. 2018, ZHANG, et al. 2022b). The interaction mechanism between pathogens and hosts still unclear\u0026nbsp;(Depotter, et al. 2018). Therefore, studying the molecular mechanism of \u003cem\u003eV. dahliae\u003c/em\u003e pathogenicity will help to control Verticillium wilt of cotton.\u003c/p\u003e\n\u003cp\u003eErgosterol is the main sterol component in fungal plasma membrane and a fungal specific sterol\u0026nbsp;(Jord\u0026aacute; and Puig 2020, Munn, et al. 1999, Zinser, et al. 1993). Ergosterol can maintain the integrity and fluidity of cell membrane, regulate cell membrane permeability and membrane binding protein activity\u0026nbsp;(Abe and Hiraki 2009, Wang, et al. 2020). In fungi, ergosterol is synthesized by acetyl coenzyme A through a complex process\u0026nbsp;(Hu, et al. 2017, Zhang, et al. 2020). The biosynthesis of ergosterol has been well described in \u003cem\u003eS. cerevisiae\u003c/em\u003e and the ergosterol pathway includes the following nine non-essential genes: \u003cem\u003eHMG1\u003c/em\u003e, \u003cem\u003eHMG2\u003c/em\u003e, \u003cem\u003eERG2\u003c/em\u003e, \u003cem\u003eERG3\u003c/em\u003e, \u003cem\u003eERG4\u003c/em\u003e, \u003cem\u003eERG5\u003c/em\u003e, \u003cem\u003eERG6\u003c/em\u003e, \u003cem\u003eERG24\u003c/em\u003e, and \u003cem\u003eERG28\u0026nbsp;\u003c/em\u003e(Bhattacharya, et al. 2018)\u003cem\u003e.\u003c/em\u003e Deletion strains for each of the 9 nonessential genes were viable but showed disruption of ergosterol biosynthesis and accumulation of aberrant sterols leading to susceptibility to stress agents and osmotic stress\u0026nbsp;(Gupta, et al. 2003, Kodedov\u0026aacute; and Sychrov\u0026aacute; 2015, Valachovic, et al. 2006). But \u003cem\u003eERG1\u003c/em\u003e, \u003cem\u003eERG18\u003c/em\u003e and \u003cem\u003eERG26\u003c/em\u003e were essential for fungal survival in \u003cem\u003eS. cerevisiae\u003c/em\u003e and \u003cem\u003eCandida albicans\u003c/em\u003e (Liu, et al. 2013).\u003c/p\u003e\n\u003cp\u003eSo far, there is little information about ergosterol pathway in \u003cem\u003eV. dahliae\u003c/em\u003e. In previous studies, sterol C-24 reductase encoded by \u003cem\u003eFgERG4\u003c/em\u003e was involved in ergosterol biosynthesis, affecting differentiation and reducing pathogenicity in \u003cem\u003eFusarium graminearum\u003c/em\u003e (Liu, et al. 2013). The sterol C-14 reductase encoded by\u003cem\u003e\u0026nbsp;FgERG24B\u003c/em\u003e enhanced the intrinsic resistance of \u003cem\u003eF. graminearum\u003c/em\u003e to amine fungicides(Liu, et al. 2011). \u003cem\u003eERG24\u003c/em\u003e and \u003cem\u003eERG2\u003c/em\u003e were the main targets of amine fungicides\u0026nbsp;(Hern\u0026aacute;ndez, et al. 2016, Liu, et al. 2011, Vlainić, et al. 2021). The C-8 isomerase encoded by ERG2 catalyzed the biosynthesis of ergosterol, resulting in the formation of episterol by C-7 unsaturated in sterol B ring\u0026nbsp;(Lees, et al. 2020).\u0026nbsp;High expression of \u003cem\u003eERG2\u003c/em\u003e gene in \u003cem\u003eS. cerevisiae\u003c/em\u003e inhibits the defect of ergosterol synthesis and indicated that \u003cem\u003eERG2\u003c/em\u003e played a role in ergosterol synthesis\u0026nbsp;(Bhattacharya, et al. 2018, Johnston, et al. 2020, Sanglard, et al. 2003). Although \u003cem\u003eERG2\u003c/em\u003e has been well documented in \u003cem\u003eS. cerevisiae\u003c/em\u003e, \u003cem\u003eERG2\u003c/em\u003e has not been reported in \u003cem\u003eV. dahliae\u003c/em\u003e. Therefore, the main purpose of this study is to investigate the role of \u003cem\u003eVdERG2\u003c/em\u003e in the physiology and virulence of \u003cem\u003eV. dahliae\u003c/em\u003e. In this study, we found that \u003cem\u003eVdERG2\u003c/em\u003e not only played a role in the biosynthesis of ergosterol, but also participated in the regulation of nutritional differentiation and virulence of \u003cem\u003eV. dahliae\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e1. Growth conditions of strains and plants\u003c/p\u003e\n\u003cp\u003eVd080, a virulent strain of \u003cem\u003eV. dahliae\u003c/em\u003e preserved in our laboratory, grew in darkness on Potato Dextrose Agar (PDA) at 25 \u0026deg;C. Susceptible variety Jimian 11 was preserved in our laboratory for pathogen infection experiment. Cotton grows at 28 \u0026deg;C in a 16 h light/8 h dark cycle greenhouse. YTK12 yeast strain was preserved in our laboratory.\u003c/p\u003e\n\u003cp\u003e2. Vector and bioinformatics analysis\u003c/p\u003e\n\u003cp\u003eThe 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).\u003c/p\u003e\n\u003cp\u003e3. Yeast signal peptide capture test\u003c/p\u003e\n\u003cp\u003eThe 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\u0026nbsp;(Liu, et al. 2021b). The activity of sucrose transferase was detected using 2% TTC solution to verify the function of signal peptide\u0026nbsp;(Meng, et al. 2022).\u003c/p\u003e\n\u003cp\u003e4. Knockout and complemented of \u003cem\u003eVdERG2\u003c/em\u003e Gene\u003c/p\u003e\n\u003cp\u003eAccording to the previous method, both \u003cem\u003eVdERG2\u003c/em\u003e knockout mutants and complemented mutants were obtained through \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation (ATMT) (Li, et al. 2012, Paz, et al. 2011, Wang, et al. 2016a). The upstream and downstream 0.9 Kb sequence of \u003cem\u003eVdERG2\u003c/em\u003e 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 (\u003cem\u003eHPH\u003c/em\u003e) 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 \u003cem\u003eVdERG2\u003c/em\u003e gene, \u003cem\u003eVdERG2\u003c/em\u003e 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.\u003c/p\u003e\n\u003cp\u003e5. Southern blotting\u003c/p\u003e\n\u003cp\u003eAccording to the previous method, DIGHigh prime DNA marker and detection kit II (Roche, Germany) were used for southern blotting\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eThe fragment of \u003cem\u003eHPH\u003c/em\u003e 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, \u003cem\u003eHindIII\u003c/em\u003e was used for enzyme digestion of genomic DNA (Glenn and Andreou 2013). The primers used in this assay were listed in Table S1.\u003c/p\u003e\n\u003cp\u003e6. Determination of ergosterol biosynthesis\u003c/p\u003e\n\u003cp\u003eTo extract ergosterol, a 5 mL spore suspension (1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU/mL) of each strain was inoculated in 50mL Potato Dextrose Broth (PDB) medium, and incubated at 25 \u0026deg;C for 3 days. Mycelia were harvested by filtration and washed three times with sterile water. The resulting mycelia were dried at 60 \u0026deg;C for 3 h, and the dried mycelia were ground into a powder.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSpecific Extraction of Ergosterol Refer to previously published method for extraction\u0026nbsp;(Liu, et al. 2013). The instrument used in this test was Waters 2695 high performance liquid chromatograph equipped with Waters 2996 ultraviolet detector\u0026nbsp;(Ping and Rong 2006). Ergosterol was separated at 30 \u0026deg;C on a Waters C18 Column (5 \u0026micro;m, 4.6 mm\u0026nbsp;\u0026times;\u0026nbsp;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\u0026nbsp;(Chiocchio and Matković 2011, Gessner 2020, Nahar, et al. 2020).\u003c/p\u003e\n\u003cp\u003e7. Oxidative stress\u003c/p\u003e\n\u003cp\u003eOxidative stress was detected using the method of previous studies\u0026nbsp;(Rehman, et al. 2018, Tang, et al. 2020). To test oxidative stresses, a 100 \u0026mu;L conidial suspension (10\u003csup\u003e7\u003c/sup\u003e spores/ml) of each strain was spread onto PDA plates, and filter paper discs containing 5 \u0026mu;L of 7.5%, 15% and 30% hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) were placed onto the centre of each plate. The zone of growth inhibition was measured after 3 days.\u003c/p\u003e\n\u003cp\u003e8. Expression analysis of related genes\u003c/p\u003e\n\u003cp\u003eTo study the impact of \u003cem\u003eVdERG2\u003c/em\u003e 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 (\u003cem\u003eVDH1\u003c/em\u003e)\u0026nbsp;(Klimes, et al. 2008, Klimes and Dobinson 2006), pigment biosynthesis protein Ayg1 (\u003cem\u003eVayg1\u003c/em\u003e)\u0026nbsp;(Fan, et al. 2017), scytalone dehydratase (\u003cem\u003eVdSCD\u003c/em\u003e)\u0026nbsp;(Duressa, et al. 2013), laccase (\u003cem\u003eVdLAC\u003c/em\u003e)\u0026nbsp;(Li, et al. 2020), tetrahydroxynaphthalene reductase (\u003cem\u003eVT4HR\u003c/em\u003e)\u0026nbsp;(Wang, et al. 2018), and versicolorin reductase (\u003cem\u003eVaflM\u003c/em\u003e)\u0026nbsp;(Wang, et al. 2016b).\u003c/p\u003e\n\u003cp\u003eConidia were harvested from a 6-day-old culture of wild type,\u0026nbsp;\u0026Delta;VdERG2, and C-\u0026Delta;VdERG2 strains and the concentration was adjusted to 10\u003csup\u003e7\u003c/sup\u003e CFU/mL. 1 \u0026mu;L of the respective suspensions was inoculated in 200 mL of PDB and incubated on the shaker (180 rpm) at 25 \u0026deg;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.\u003c/p\u003e\n\u003cp\u003e9. Stress response and carbon utilization detection\u003c/p\u003e\n\u003cp\u003eFor 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\u0026nbsp;(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 \u0026deg;C. In order to study the role of VdERG2 in the growth of V\u003cem\u003e. dahliae\u003c/em\u003e 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.\u003c/p\u003e\n\u003cp\u003e10. Mycelium penetration test\u003c/p\u003e\n\u003cp\u003ePreparation of spore suspension according to previous methods (Fraczek, et al. 2019). 5 \u0026mu;L spore suspension of \u003cem\u003eV. dahliae\u003c/em\u003e 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.\u003c/p\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003cp\u003e11. Pathogenicity test\u003c/p\u003e\n\u003cp\u003eThe spore suspensions of various strains were prepared and the concentration was adjusted to 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e 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.\u003c/p\u003e\n\u003cp\u003eThe 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\u0026nbsp;(Gong, et al. 2017). According to symptoms on cotyledons and true leaves, which were divided into five grades (0, 1, 2, 3 and 4)\u0026nbsp;(Wang, et al. 2004, Zhang, et al. 2012).\u003c/p\u003e\n\u003cp\u003eThe 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\u0026nbsp;(Zhang, et al. 2016, Zhang, et al. 2019).\u003c/p\u003e\n\u003cp\u003eMoreover, 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\u0026nbsp;(Vazyme), and calculated by 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;CT\u003c/sup\u003e method.\u0026nbsp;\u003cem\u003eVd\u0026beta;t\u003c/em\u003e was the target sequence for \u003cem\u003eV. dahliae\u003c/em\u003e detection, and \u003cem\u003eGhUBQ7\u003c/em\u003e was the reference gene.\u003c/p\u003e"},{"header":"Results","content":"\u003col\u003e\n\u003cli\u003eIdentification of \u003cem\u003eVd\u003c/em\u003e\u003cem\u003eERG\u003c/em\u003e\u003cem\u003e2\u003c/em\u003e in \u003cem\u003e dahliae\u003c/em\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eUsing \u003cem\u003eS. cerevisiae\u003c/em\u003e sterol C-8 isomerase Erg2 as query, we identified \u003cem\u003eVdERG2\u003c/em\u003e (VDAG_01363) from the \u003cem\u003eV. dahliae\u003c/em\u003e strain VdLs. 17 (https://www.ncbi.nlm.nih.gov/genome/832). The nucleotide sequence of \u003cem\u003eVdERG2\u003c/em\u003e is 678 bp, encoding 225 amino acids. The protein homology analysis using DNAMAN showed that the amino acid sequence of VdERG2 was relatively conservative at the C-terminal with that of \u003cem\u003eS. cerevisiae\u003c/em\u003e and other filamentous fungi, but mutated greatly at the N-terminal. Their homology was 73.82% (Figure 1A). SignalP V5.0 predicted that VdERG2 may contain a signal peptide at the N-terminal. YTK12-pSUC2-VdERG2sp was normally grown on CMD-W and YPRAA mediums according to the yeast signal peptide trap recovery test (Figure 1B). TTC staining showed that yeast strain YTK12 carrying pSUC2-VdERG2sp secreted sucrase to hydrolyze sucrose into monosaccharides, which reacted with TTC to produce triphenyltetrazole chloride red insoluble in water (Figure 1B). The above results indicated that VdERG2 was a highly conserved secretory protein in \u003cem\u003eV. dahliae\u003c/em\u003e.\u003c/p\u003e\n\u003col start=\"2\"\u003e\n\u003cli\u003eThe knockout and complemented mutants of \u003cem\u003eVdERG2\u003c/em\u003e in \u003cem\u003e dahliae\u003c/em\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eIn order to analyze the function of VdERG2 in detail, this study used the method of homologous recombination (Figure 2A), according to the gene sequence of \u003cem\u003eVdERG2\u003c/em\u003e in the genome of \u003cem\u003eV. dahliae\u003c/em\u003e VdLs. 17, the upstream and downstream 0.9 Kb fragments were selected, and the upstream of the target gene, hygromycin resistance gene fragment (\u003cem\u003eHPH\u003c/em\u003e) and the downstream fragment of the target gene were fused to the knockout vector B303 by homologous cloning method to construct the knockout vector B303-VdERG2. \u003cem\u003eVdERG2\u003c/em\u003e was knockout by \u003cem\u003eAgrobacterium \u003c/em\u003emediated fungal transformation using wild type Vd080 conidia. Two mutants \u0026Delta;VdERG2-1 and \u0026Delta;VdERG2-2 were determined by PCR and southern blotting (Figure 2C and D). In the genome of \u003cem\u003eV. dahliae\u003c/em\u003e, the 1.2 Kb fragment of the upstream promoter region of the target gene \u003cem\u003eVdERG2\u003c/em\u003e and the target gene \u003cem\u003eVdERG2\u003c/em\u003e fragment were selected, and the complemented vector pCAMBIA1302-Neo-VdERG2 was constructed by homologous recombination further genetic transformation to knockout mutants. Two complemented mutants C-\u0026Delta;VdERG2-1 and C-\u0026Delta;VdERG2-2 were identified by PCR (Figure 2B).\u003c/p\u003e\n\u003col start=\"3\"\u003e\n\u003cli\u003eDecrease of ergosterol biosynthesis in the \u003cem\u003eVdERG2\u003c/em\u003e knockout mutant\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eHigh-performance liquid chromatography (HPLC) analysis ofergosterol extracted from mycelia of wild type, \u0026Delta;VdERG2-1, \u0026Delta;VdERG2-2, C-\u0026Delta;VdERG2-1 and C-\u0026Delta;VdERG2-2 revealed that there was an ergosterol-specific absorption peak at the retention time of 16.5 min in all strains (Figure 3A). However, the ergosterol content of the \u0026Delta;VdERG2-1 and \u0026Delta;VdERG2-2 mutants were significantly lower than that of the wild type and complemented mutants (Figure 3B). It indicated that \u003cem\u003eVdERG2\u003c/em\u003e played an important role in ergosterol synthesis pathway in \u003cem\u003eV. dahliae\u003c/em\u003e.\u003c/p\u003e\n\u003col start=\"4\"\u003e\n\u003cli\u003e\u003cem\u003eVdERG2\u003c/em\u003e was involved in the regulation of conidia in \u003cem\u003e dahliae\u003c/em\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eAfter the strains were cultured in liquid medium for 5 days, the number of spores were observed under the fluorescence microscope and counted through the blood cell counting plate. It was found that the conidia produced in \u0026Delta;VdERG2-1 and \u0026Delta;VdERG2-2 were significantly less than those in the wild type and the complemented mutant (Figure S1 and Figure 4A). The same concentration spore suspensions with the same concentration of spores of each strain were coated on PDA for 6 h, and the spore germination was observed. It was found that the germination rates of conidia in \u0026Delta;VdERG2-1 and \u0026Delta;VdERG2-2 were lower than those in the wild type and the complemented mutant (Figure S2 and Figure 4B). The conidia were made into frozen sections, and the spore morphology was observed under scanning electron microscope. It was found that \u0026Delta;VdERG2-1 and \u0026Delta;VdERG2-2 could lead to abnormal spore morphology (Figure 4C). The above results indicated that the deletion of \u003cem\u003eVdERG2\u003c/em\u003e could lead to decrease of spores production and spores germination rate, with influencing on the normal morphology of spores.\u003c/p\u003e\n\u003col start=\"5\"\u003e\n\u003cli\u003e\u003cem\u003eVdERG2\u003c/em\u003e did not affect the utilization of carbon source, but reduced microsclerotia formation and melanin production\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eIn order to further explore the function of VdERG2 in the utilization of carbon sources, sucrose, cellulose, skim milk, pectin and starch were selected as carbon sources. The results showed that \u003cem\u003eVdEGR2\u003c/em\u003e did not affect the utilization of carbon sources, but when cellulose and pectin were used as carbon sources, the melanin and microsclerotia of the mutant showed a decreasing trend (Figure S3). In order to further investigate whether \u003cem\u003eVdERG2\u003c/em\u003e affects the production of melanin and microsclerotia. The results showed that \u0026Delta;VdERG2-1 and \u0026Delta;VdERG2-2 significantly reduced the production of melanin and microsclerotia compared with wild type and complement mutant strains (Figure 5A). Analysis using RT-qPCR to detect in the expression of melanin and microsclerotia-related genes \u003cem\u003eVT4HR, VaflM\u003c/em\u003e, \u003cem\u003eVayg1, VdLAC\u003c/em\u003e, \u003cem\u003eVdSCD\u003c/em\u003e and \u003cem\u003eVDH1\u003c/em\u003e. The results showed that their expression levels decreased significantly (Figure 5B).\u003c/p\u003e\n\u003col start=\"6\"\u003e\n\u003cli\u003e\u003cem\u003eVdERG2\u003c/em\u003e responded to CR, SDS and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stresses\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eAs a soil-borne fungus, there are various stresses in the soil, affecting the growth of pathogen \u003cem\u003eV. dahliae\u003c/em\u003e. In this study, 1 mol/L KCl, 1 mol/L NaCl, 1 mol/L Sorbitol, 0.004% (mass/mass) SDS and 0.02% (mass/mass) CR were added to PDA medium to simulate abiotic stress. Normal PDA culture was used as the control. The results showed that the growth of \u0026Delta;VdERG2 in KCl, NaCl, Sorbitol and normal PDA medium were not different from those of wild type and complemented mutant (Figure S4). Under CR stress, the colony diameters of \u0026Delta;VdERG2-1 and \u0026Delta;VdERG2-2 were 37.39 mm and 38.33 mm, respectively, which were lower than those in normal PDA (63.5 mm and 63.75 mm). After gene complemented, the colony diameter increased, which was consistent with the wild type (Figure 6A). Under SDS stress, the colony diameters of \u0026Delta;VdERG2-1 and \u0026Delta;VdERG2-2 were 31.61 mm and 32.03 mm, respectively, which were lower than those in normal PDA (63.5 mm and 63.75 mm). After gene complemented, the colony diameter increased, which was consistent with that of wild type (Figure 6A). The above results showed that \u003cem\u003eVdERG2\u003c/em\u003e was involved in the response of \u003cem\u003eV. dahliae\u003c/em\u003e to CR and SDS-induced cell wall stress, the cell wall of mutant \u0026Delta;VdERG2 was defective.\u003c/p\u003e\n\u003cp\u003eIn order to study the role of \u003cem\u003eVdERG2\u003c/em\u003e in response to oxidative stress, we examined the growth status of these strains under H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stress. The sensitivity of each strain to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was examined by measuring the diameter of inhibition zone. Three gradients (7.5%, 15% and 30%) of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration were designed in the experiment. The results showed that the inhibition zones of \u0026Delta;VdERG2-1 and \u0026Delta;VdERG2-2 were larger than those of wild type and complemented strains, and the phenotypes became more and more obvious with the increase of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration (Figure 6B).\u003c/p\u003e\n\u003col start=\"7\"\u003e\n\u003cli\u003e\u003cem\u003eVdERG2\u003c/em\u003e weakened hyphal penetration and affected hyphal growth on cellophane\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eIn order to detect the penetration ability on cellophane, conidia of each strain were placed on PDA medium containing glass paper for 3 days at 25 \u0026deg;C. The results showed that all strains could grow normally on cellophane (Figure 7A). By observing the growth of each strain on the cellophane, it was found that the mycelium growth of the mutant was sparse and chaotic, while the mycelium growth of the wild type and complemented strains were uniform and normal (Figure 7C). By observing the back of the cellophane, it was found that all strains had conidia (Figure 7B). When removing the cellophane, it was observed that the colony diameters of \u0026Delta;VdERG2-1 and \u0026Delta;VdERG2-2 were significantly smaller than those of the wild type and the complemented mutant (Figure 7A). The above results showed that \u003cem\u003eVdERG2\u003c/em\u003e could weaken the penetration of mycelium and affect the growth of mycelium on cellophane.\u003c/p\u003e\n\u003col start=\"8\"\u003e\n\u003cli\u003e\u003cem\u003eVdERG2\u003c/em\u003e positively regulated the virulence of \u003cem\u003e dahliae\u003c/em\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eTo further study the function of \u003cem\u003eVdERG2\u003c/em\u003e in virulence of \u003cem\u003eV. dahliae\u003c/em\u003e, the pathogenicity of wild type Vd080, \u0026Delta;VdERG2-1, \u0026Delta;VdERG2-2, C-\u0026Delta;VdERG2-1 and C-\u0026Delta;VdERG2-2 strains were determined using cotton root dipping method. The results showed that compared with the control inoculated with water, the leaves of cotton inoculated with knockout strains turned yellow and wilted at 21 days after inoculation, and only a few cotyledons fell off. After inoculation with mutant strains, the leaves wilted and necrotic, and the true leaves fell off, which was consistent with the incidence of wild type strains. The results showed that the pathogenicity of\u003cem\u003e V. dahliae\u003c/em\u003e to cotton decreased after knocking out \u003cem\u003eVdERG2\u003c/em\u003e (Figure 8A). The stems of cotton were cut to observe the browning. The results showed that the browning of stems inoculated with knock-out mutant strains was significantly lighter than that inoculated with wild type and complemented mutant strains (Figure 8B). Fungal recovery test showed that the colonies of diseased cotton stems inoculated with knockout mutants were less than those of wild type and complemented mutants (Figure 8C). At the 14, 18 and 21 days after inoculation, the disease index of cotton was investigated. The results showed that the disease index increased over time, 21 days post inoculation, the disease index of cotton plants infected by deletion mutants (\u0026Delta;VdERG2-1 and \u0026Delta;VdERG2-2) were 34.56 and 34.22, respectively. While that of cotton plants infected by wild type, C-\u0026Delta;VdERG2-1 and C-\u0026Delta;VdERG2-2 were 71.11, 72.07 and 71.94, respectively (Figure 8D). When the fungal biomass in roots was evaluated by RT-qPCR to assess the role of VdERG2 in systemic infection, the fungal biomass of C-\u0026Delta;VdERG2-1 and C-\u0026Delta;VdERG2-2 strains did not differ significantly, whereas the fungal biomass of \u0026Delta;VdERG2-1 and \u0026Delta;VdERG2-2 strains decreased by 4.73 times and 4.54 times, respectively, compared with the wild type strain (Figure 8E). The above results show that \u003cem\u003eVdERG2\u003c/em\u003e positively regulated the pathogenicity of \u003cem\u003eV. dahliae\u003c/em\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe main sterol components in fungal plasma membrane are involved in a variety of cell functions. Because of the important biochemical characteristics of ergosterol, many enzymes involved in its biosynthetic pathway have become the attract sites for antifungal drugs\u0026nbsp;(Liu, et al. 2011, Liu, et al. 2013). ERG2 is a non-essential enzyme in \u003cem\u003eS. cerevisiae\u003c/em\u003e, but high expression inhibits ergosterol biosynthesis\u0026nbsp;(Hu, et al. 2017). In this study, we used \u003cem\u003eERG2\u003c/em\u003e in\u003cem\u003e\u0026nbsp;S. cerevisiae\u003c/em\u003e as the query object and identified \u003cem\u003eVdERG2\u003c/em\u003e in the genome of \u003cem\u003eV. dahliae\u003c/em\u003e VdLs. 17. VdERG2 was identified as a single conserved secretory protein in \u003cem\u003eV. dahliae\u003c/em\u003e (Figure 1). Compared with the wild type, we found that the biosynthesis of ergosterol in the knockout mutant was decreased, indicating that \u003cem\u003eVdERG2\u003c/em\u003e was played a role in the biosynthesis of ergosterol in \u003cem\u003eV. dahliae\u003c/em\u003e (Figure 3). At the same time, it also indicated that the biosynthesis pathway of ergosterol in \u003cem\u003eV. dahliae\u003c/em\u003e might be slightly different from that reported in \u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003edue to the possibility of gene redundancy exists.\u003c/p\u003e\n\u003cp\u003eVerticillium wilt caused by \u003cem\u003eV. dahliae\u003c/em\u003e is called cancer\u0026nbsp;(Cai, et al. 2009, Gong, et al. 2017). Once the plant is infected, it is difficult for fungicides to effectively treat it, which may cause serious economic losses. Therefore, it is particularly important to study the molecular mechanism of \u003cem\u003eV. dahliae\u003c/em\u003e and host plants. In previous studies, knockout of \u003cem\u003eFgERG4\u003c/em\u003e in \u003cem\u003eF. graminearum\u003c/em\u003e led to a decline in conidial production and malformation of conidia\u0026nbsp;(Liu, et al. 2013), knockout of \u003cem\u003eVdHP1\u003c/em\u003e in \u003cem\u003eV. dahliae\u003c/em\u003e reduces conidial smoothness\u0026nbsp;(Zhang, et al. 2022a). In this study, it was found that the knockout of \u003cem\u003eVdERG2\u003c/em\u003e led to the decrease of conidial yield and conidial germination rate, and abnormal conidial morphology (Figure 2).\u003c/p\u003e\n\u003cp\u003eAs\u0026nbsp;a soil-borne fungus, environmental conditions in the soil can cause various abiotic stresses and changes in nutritional conditions, thus affecting the normal growth of\u003cem\u003e\u0026nbsp;V. dahliae\u003c/em\u003e. Previous studies have shown that \u003cem\u003eVdHog1\u003c/em\u003e, \u003cem\u003eVdPbs2\u003c/em\u003e, \u003cem\u003eVdSsk1\u003c/em\u003e, \u003cem\u003eVdssk2\u003c/em\u003e and \u003cem\u003eVdCrz1\u003c/em\u003e played important roles in responding to osmotic stress, CR stress and SDS stress in \u003cem\u003eV. dahliae\u003c/em\u003e (Tian, et al. 2016, Wang, et al. 2016b, Xiong, et al. 2015, Yu, et al. 2019, Zheng, et al. 2019). In the presence of CR, knockout of \u003cem\u003eVdSsk1\u003c/em\u003e or \u003cem\u003eVdSsk2\u003c/em\u003e caused high expression of genes related to cell wall biosynthesis\u0026nbsp;(Yu, et al. 2019, Zheng, et al. 2019). Knockout of \u003cem\u003eVdCrz1\u003c/em\u003e resulted in high sensitivity to SDS stress\u0026nbsp;(Xiong, et al. 2015). In this study, it was found that \u0026Delta;VdERG2 was not sensitive to osmotic stress, but highly sensitive to CR and SDS stress (Figure 6). Overcoming the natural barrier of cell wall is a key step for fungal pathogens to infect plant hosts\u0026nbsp;(Ferreira, et al. 2006, Underwood 2012). When \u003cem\u003eVdOGDH\u003c/em\u003e was knockout to supplement different carbon sources, the vegetative growth of \u003cem\u003eV. dahliae\u003c/em\u003e was significantly inhibited\u0026nbsp;(Li, et al. 2020). After knockout \u003cem\u003eVdSNF1\u003c/em\u003e, the growth of the mutant was significantly inhibited only when pectin or galactose was used as carbon source\u0026nbsp;(Tzima, et al. 2011). In this study, when sucrose, cellolose, skim milk, pectin and starch as carbon sources, the grouth of \u0026Delta;VdERG2 was similar to those of the wild type and the complemented mutant,\u0026nbsp;interestingly, the melanin and microsclerotia production of the knockout mutant was weakened when cellulose and pectin were used as carbon sources (Figure S3). Previous studies have shown that melanin and microsclerotia are essential for survival and diffusion in \u003cem\u003eV. dahliae\u003c/em\u003e life cycle\u0026nbsp;(Tzima, et al. 2011). The virulence of\u003cem\u003e\u0026nbsp;V. dahliae\u003c/em\u003e seems to be increasingly related to the development of microsclerotia. The \u0026Delta;VdSsk2 strain showed severe delayed microsclerotia formation and melanization. Although the microsclerotia development of \u0026Delta;Vdste11 was similar to that observed in the wild type, it was obviously lack of melanin biosynthesis, indicating that both \u003cem\u003eVdSsk2\u003c/em\u003e and \u003cem\u003eVdSte11\u003c/em\u003e were involved in the microsclerotia of micronucleus\u0026nbsp;(Zheng, et al. 2019). In this study, it was found that \u003cem\u003eVdERG2\u003c/em\u003e could inhibit the formation of micronucleus and melanin, thereby affecting virulence (Figure 5). Therefore, \u003cem\u003eVdERG2\u003c/em\u003e not only participated in the response to CR and SDS abiotic stresses but inhibited the formation of microsclerotia, thereby affecting virulence.\u003c/p\u003e\n\u003cp\u003eThe enzymes in ergosterol biosynthesis pathway are also involved in the regulation of pathogenicity. Previous studies have shown that \u003cem\u003eFgERG4\u003c/em\u003e knockout mutant in \u003cem\u003eF. graminearum\u003c/em\u003e can successfully invade wheat and tomato, but the virulence of the mutant decreased significantly (Liu, et al. 2013). Knockout of \u003cem\u003eFghyd2\u003c/em\u003e and \u003cem\u003eFghyd3\u003c/em\u003e in \u003cem\u003eF. graminearum\u003c/em\u003e reduced the symptom spikelets in wheat (Quarantin, et al. 2019). H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, one of the earliest cellular responses to infection, is produced when plant host cells are challenged by pathogens or inducers of pathogenic origin (Fassler and West 2011). \u003cem\u003eVdSkn7\u003c/em\u003e mutant showed severe growth defect H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e under heat shock and cell wall disturbance, and its toxicity was significantly lower than that of wild type (Tang, et al. 2020). In this study, we found that \u0026Delta;VdERG2 was very sensitive to H2O2 (Figure 6). Foc_M35-1 reduced the pathogenicity to host plants without affecting the mycelium penetration ability of \u003cem\u003eF\u003c/em\u003e.\u003cem\u003e\u0026nbsp;oxysporum\u003c/em\u003e (Zhang, et al. 2021). In this study, we found that the deletion of VdERG2 reduced the penetration ability of \u003cem\u003eV. dahliae\u003c/em\u003e mycelium and led to the chaotic growth of mycelium (Figure 7). After inoculation with conidial suspension of \u0026Delta;VdERG2 strain, the cotton cotyledons turned yellow and few deciduous leaves appeared, compared with wild type and complement mutant strains, \u0026Delta;VdERG2 disease index decreased significantly (Figure 8). It is concluded that \u003cem\u003eVdERG2\u003c/em\u003e may be a positive regulator with multi-faceted functions in pathogenicity of \u003cem\u003eV. dahliae.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn conclusion, sterol C-8 isomerase \u003cem\u003eVdERG2\u003c/em\u003e of \u003cem\u003eV. dahliae\u003c/em\u003e was a single conservative secretory protein and involved in the ergosterol biosynthesis, the regulation of conidia and inhibited the formation of melanin and microsclerotia. \u0026Delta;VdERG2 was more sensitive to abiotic stresses (CR and SDS), and weakened the penetration ability of hyphae, thereby reducing the pathogenicity to cotton. This study showed that sterol C-8 isomerase was of great significance for the development, adaptability and pathogenicity of \u003cem\u003eV. dahliae\u003c/em\u003e, which may provide a new perspective for us to further understand the molecular mechanism of ergosterol biosynthesis pathway in virulence of \u003cem\u003eV. dahliae\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials\u003c/strong\u003e: Supplemental file 1, PDF file\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, Hongjie Feng and Heqin Zhu; Data curation, Junyuan Lv; Formal analysis, Lihong Zhao and Ruiyuan Zhao; Investigation, Shichao Liu and Xiaojian Zhang; Methodology, Junyuan Lv, Feng Wei, Jinglong Zhou and Hongjie Feng; Supervision, Tao Zhang, Zhigang Zhang and Zili Feng; Writing\u0026ndash;original draft, Junyuan Lv; Writing\u0026ndash;review \u0026amp; editing, Hongjie Feng, Caihong Li and Yalin Zhang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This work was supported by the Natural Science Foundation of Hunan Province, China (No.2021JJ40285), National Natural Science Foundation of China (Grant No. 32201752), the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences, and Hunan Technology Professionals Project (2020TJ-Q17).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflflict of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbe F, Hiraki T (2009) Mechanistic role of ergosterol in membrane rigidity and cycloheximide resistance in Saccharomyces cerevisiae. 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Journal of bacteriology 175: 2853-2858 doi:\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-genetics-and-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cuge","sideBox":"Learn more about [Current Genetics](https://www.springer.com/journal/294)","snPcode":"294","submissionUrl":"https://submission.nature.com/new-submission/294/3","title":"Discover Genetics and Evolution","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Verticillium dahliae, Ergosterol biosynthesis, Nutritional differentiation, Cell wall stress, Oxidative stress, Virulence","lastPublishedDoi":"10.21203/rs.3.rs-2131818/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2131818/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe ergosterol biosynthesis pathway plays an important role in model pathogenic bacteria\u003cem\u003e Saccharomyces cerevisiae\u003c/em\u003e, but little is known about the biosynthesis of ergosterol in pathogenic fungus \u003cem\u003eVerticillium dahliae\u003c/em\u003e.\u003cstrong\u003e \u003c/strong\u003eIn this study, we identified the \u003cem\u003eVdERG2\u003c/em\u003e gene encoding sterol C-8 isomerase from \u003cem\u003eV. dahliae\u003c/em\u003e and investigated its function in virulence by generating gene deletion mutants (ΔVdEGR2) and complemented mutants (C-ΔVdEGR2). Deletion of \u003cem\u003eVdERG2\u003c/em\u003e reduced ergosterol content. The conidial germination rate and conidial yield of ΔVdERG2 decreased significantly, and abnormal conidia were produced. In spite of \u003cem\u003eVdERG2\u003c/em\u003e did not affect the utilization of carbon sources by \u003cem\u003eV. dahliae\u003c/em\u003e, but ΔVdERG2 observed a decrease in melanin production when cellulose and pectin were used as sole carbon sources, respectively. The ability of mutants ΔVdERG2 to produce microsclerotia and melanin decreased and the knockout of \u003cem\u003eVdERG2\u003c/em\u003e led to a significant decrease in the expression of microsclerotia and melanin-related genes \u003cem\u003eVaflM\u003c/em\u003e, \u003cem\u003eVayg1, VDH1, VdLAC\u003c/em\u003e, \u003cem\u003eVdSCD\u003c/em\u003e and \u003cem\u003eVT4HR\u003c/em\u003e. In addition, mutants ΔVdEGR2-1 and ΔVdEGR2-2 were very sensitive to congo red (CR), sodium dodecyl sulfate (SDS) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) stresses, indicating that VdEGR2 was involved in cell wall and oxidative stress response. The absence of \u003cem\u003eVdERG2\u003c/em\u003e weakened the penetration ability of mycelium on cellophane and affected the growth of mycelium on cellophane. Although ΔVdERG2 could infect cotton, its pathogenicity was significantly impaired. These phenotypic defects in ΔVdERG2 could be complemented by reintroduction of a full-length \u003cem\u003eVdERG2\u003c/em\u003e gene. In summary, as a single conservative secretory protein,\u003cem\u003e VdERG2\u003c/em\u003e played a crucial role in ergosterol biosynthesis, nutritional differentiation and virulence in \u003cem\u003eV. dahliae\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"VdERG2 was involved in ergosterol biosynthesis, nutritional differentiation and virulence of Verticillium dahliae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-10 17:42:58","doi":"10.21203/rs.3.rs-2131818/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-10-18T08:44:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-10-17T18:21:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"f1572339-baf9-479c-a1f1-e617520e13d8","date":"2022-10-12T11:50:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-10-07T01:26:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-10-07T01:22:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-10-05T06:52:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Current Genetics","date":"2022-10-04T12:35:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-genetics-and-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cuge","sideBox":"Learn more about [Current Genetics](https://www.springer.com/journal/294)","snPcode":"294","submissionUrl":"https://submission.nature.com/new-submission/294/3","title":"Discover Genetics and Evolution","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c3cb823d-7e0e-4e50-892e-95467dbda13d","owner":[],"postedDate":"October 10th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-11-01T12:59:18+00:00","versionOfRecord":[],"versionCreatedAt":"2022-10-10 17:42:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2131818","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2131818","identity":"rs-2131818","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}