The study employed the highly pathogenic Vd080 strain of V. dahliae (Zhang et al., 2015; Zhu et al., 2021). The fungus was cultured on potato dextrose agar medium and incubated in a dark environment at 25°C. To collect conidia, fungal plugs were grown in liquid Czapek-Dox broth and shaken at 200 rpm for 5 days at 25°C. The virulence assessment was conducted on cotton plants of the Jimian-11 variety, which is known to be susceptible. The cotton plants were cultivated in a greenhouse under controlled conditions with a 16 h light/8 h dark cycle at 25°C.

To generate the knockout mutant strain, upstream and downstream flanking sequences of the target gene were amplified from the genomic DNA of V. dahliae. An hygromycin resistance cassette (HPH) was amplified from the B303 vector and cloned into the B303 vector (Lv et al., 2023). The constructed vector was then transformed into Agrobacterium tumefaciens AGL1, and the knockout mutant was generated through Agrobacterium-mediated transformation (ATMT) (Wang et al., 2016a). For the construction of the complementation vector, a 1.5 kb upstream region of the genomic region of Vdsti1 was amplified and cloned into the pCAM-BIA1302 vector. The constructed vector was transformed into A. tumefaciens AGL1. The complementation strain was then generated through ATMT (Paz et al., 2011; Li et al., 2012; Zhou et al., 2013). NCBI’s Primer Blast tool was used for primer design.¹ DNAMAN was used to align multiple protein sequences. The primers used in this experiment are listed in Supplementary Table S1.

The spore suspension should be inoculated into Potato Dextrose Broth (PDB) culture medium and incubated at 25°C for 3 days. Follow the specific extraction method for ergosterol (Liu et al., 2013). The experiment should be conducted using a Waters 2,695 High Performance Liquid Chromatography system equipped with a Waters 2,996 UV detector. Ergosterol is separated on a Waters C18 column (5 μm, 4.6 mm × 250 mm) at 30°C, using 100% methanol (chromatographic grade) as the mobile phase. The detection wavelength is set at 282 nm (Dushkov et al., 2023; Fernandes et al., 2023; Manita et al., 2024). The standard for ergosterol is purchased from Shanghai YuanYue Biotechnology Co., Ltd. The experiment is repeated three times.

To determine temperature stress, activate all strains on PDA mediumat 16°C, 25°C, and 30°C for 14 days. Then, cultivate spores in a chaotrophic medium to prepare the spore suspension. Subject the spore suspensions of each strain to a heat shock at 42°C for 30 min. Observe the morphology of conidia under an electron scanning microscope. Afterward, subject the spore suspensions of each strain to a heat shock at 50°C for 30 min. Evenly spread the spore suspensions onto PDA plates and incubate them in a 25°C incubator. Calculate the spore viability after 3 days of incubation (Tang et al., 2017).

To determine 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 (Lv et al., 2022; Liu et al., 2023). The control group was grown on normal PDA medium.

For oxidative stress determination, 100 μL of spore suspension was spread onto PDA plates, and filter paper discs soaked with 5 μL of 15% hydrogen peroxide (H₂O₂) was placed in the center of each plate. Measure the diameter of the inhibition zones after 3 days of growth (Tang et al., 2017; Rehman et al., 2018; Tang et al., 2020). Ensure to repeat all experiments three times. The primers utilized in this experiment are available in Supplementary Table S2.

To study the production of microsclerotia, fresh spore suspensions were incubated flat on solid basal medium (BM) containing cellulose membranes for 15 days. Microsclerotia were then observed using a stereomicroscope (Xiong et al., 2014). The experiment was repeated three times.

For the investigation of the impact of VdSti1 knockout on the expression of genes related to other microsclerotia and melanin formation, the mycelium from different strains was collected. Total RNA was extracted from each mycelium using an RNA extraction kit (YPHBio, Tianjin, China). The extracted RNA was then used for cDNA synthesis using the HiScript II QRT SuperMix (+gDNAwiaper) kit (Vazyme). The obtained cDNA was subjected to qPCR analysis to determine the relative expression levels of genes VDH1, Vayg1, and VaflM (Klimes and Dobinson, 2006; Klimes et al., 2008; Fan et al., 2017; Li et al., 2020). The primers used in this experiment are listed in Supplementary Table S3.

In this experiment, the sensitivity of Verticillium dahliae to the amine-type drug Tridemorph and the non-amine type drugs Triadimefon and Tebuconazole was evaluated. The EC50, which represents the half-effective concentration of each drug against V. dahliae, was determined following a previously established method (Gu et al., 2016). Different strains of V. dahliae were inoculated onto PDA medium supplemented with the respective drugs. After 14 days of incubation, the growth diameter of colonies was measured to assess the inhibitory effect of each drug on fungal growth. Each test was repeated at least three times.

To prepare spore suspensions of various strains, adjust the concentration to 1 × 10⁷ CFU/mL. Immerse water-cultured cotton seedlings (4 weeks old) in the spore suspension for 2 min. Then, transfer the seedlings back into the nutrient soil and place them in a cotton greenhouse for planting. After 21 days of inoculation, record the disease index based on the previous method and take photographs to document the disease occurrence. The symptoms of the cotyledons and true leaves are graded on a scale of 0 to 4 (Gong et al., 2017; Zhang et al., 2023). Using a surgical knife, make a longitudinal cut at the same location on the stems and roots of cotton plants harvested after 21 days of inoculation. Observe the extent of browning on the cotton stems under a stereomicroscope (Leica, M165 FC, Germany).

The stems were harvested 21 days after inoculation and ground to powder. Total DNA was extracted from the cotton plants using the Plant Genome Extraction Kit (Vazyme). The biomass of V. dahliae in the stalks was determined using the 2^−ΔΔCT method. The target gene for the assay was Vdβt, and GhUBQ7 served as the internal reference gene. The primers used in this experiment can be found in Supplementary Table S4.

A 1 μL conidial suspension (1 × 107 CFU/mL) of both the wild type and VdSti1 knockout mutant strains were inoculated in 200 mL of PDB and incubated on a 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 instruction. qRT-PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme).s. The relative expression of each gene was determined using the 2^−∆∆Ct method, as previously described. All experiments were repeated three times. The primers used in RT-qPCR are listed in Supplementary Table S5.

PGBKT7-VdSti1 was used as bait to screen interacting proteins from the yeast AD-cDNA library of V. dahliae (provided by our laboratory). The screened proteins were sequenced using NCBI ² and then cloned into pGADT7 for yeast heterozygosity verification with pGBKT7-VdSti1. Transfer the recombinant plasmids of the two interacting proteins into yeast receptive cells. Coat them in two deficient culture media (SD Leu Trp) and dilute the single colony with sterile water after growth. Then, transfer them to cells containing X-α-gal dye four deficient media (SD His Leu Trp Ade) and observe their growth after 1 week.

Agrobacterium tumefaciens containing plasmids VdSti1-nLUC and VdExt2-cLUC were mixed and uniformly injected in a 1:1 ratio into 4 week-old tobacco leaves. The leaves were then cultivated in a dark environment for 1 day, followed by transfer to normal cultivation conditions for one day. The fluorescence signal of the tobacco leaves was detected using a weak light cooled charge coupled device Nightshade LB985 (BERTHOLDTECHNOLOGIES, Germany). The primers used in PCR are listed in Supplementary Table S6.

A homolog of Sti1/Hop, referred to as Sti1, was discovered in the genome of V. dahliae strain VdS.17. ³ The VdSti1 gene (VDAG_06242) spans 1755 bp and codes for a 584 amino acid protein. A comparison of VdSti1’s amino acid sequences with other Sti1/Hop homologs using DNAMAN software indicated a high level of conservation of the Sti1/Hop protein from yeast to V. dahliae (Supplementary Figure S1). To investigate the specific function of the Hsp90 co-chaperone VdSti1, this study utilized homologous recombination techniques to create knockout and complementation mutants (Figure 1A). Two knockout mutants, ΔVdSti1-1 and ΔVdSti1-2, were produced, alongside two complementation mutants, C-ΔVdSti1-1 and C-ΔVdSti1-2. PCR validation confirmed the successful generation of these mutants (Figures 1B–D).

In order to investigate the role of Hsp90 co-chaperones in the stress response induced by amines and azoles, the half effective concentration (EC50) of each drug for inhibiting the growth of V. dahliae. Results showed EC50 concentrations of 0.1 mg/L for tebuconazole, 20 mg/L for triadimefon, and 2 mg/L for tridemorph, compared to the control group (Supplementary Figure S2). Notably, the growth inhibition of the VdSti1 knockout mutant was more significant than that of the wild type and complementary mutant when exposed to these drugs. Tebuconazole exhibited the strongest inhibitory effect on the knockout mutant among the tested drugs. These results strongly indicate that the Hsp90 co-chaperone VdSti1 plays a vital role in the stress response of V. dahliae induced by azoles (Figures 2A–D). The heightened sensitivity of the VdSti1 knockout mutant to these drugs underscores the importance of VdSti1 in modulating the stress response of V. dahliae.

High-performance liquid chromatography (HPLC) analysis of ergosterol extracted from various hyphae strains, including wild-type, ΔVdSti1-1, ΔVdSti1-2, C-ΔVdSti1-1, and C-ΔVdSti1-2, showed a distinct ergosterol-specific absorption peak with a retention time of 13.7 min across all strains. Notably, the ergosterol content in the ΔVdSti1-1 and ΔVdSti1-2 mutants was notably lower than in the wild type and complementation mutants. Intriguingly, the introduction of 5 mg/L of geldanamycin, an Hsp90 inhibitor, to the wild-type strain led to reduced ergosterol production compared to the control. Despite this decrease, the wild type still exhibited higher ergosterol levels than the knockout mutants (Figure 3). These results indicate a potential crucial role for VdSti1 in the ergosterol synthesis pathway, potentially interacting with Hsp90. The decrease in ergosterol production in the knockout mutants and with geldanamycin treatment in the wild type further supports the involvement of VdSti1 and Hsp90 in the regulation of this essential fungal lipid.

Previous studies have shown that gene expression of the Hsp90 co-chaperone Sti1/Hop is induced under heat stress conditions in yeast and soybean. Furthermore, research on Hsp90 in Paracocidioides brasiliensis has revealed its role in inhibiting reactive oxygen species (ROS) production and promoting antioxidant defenses during heat-induced stress (Matos et al., 2013). To investigate the involvement of VdSti1 in stress response, the growth rate of the VdSti1 knockout mutant was evaluated at different temperatures. The results indicated that at 25°C, there were no significant differences in growth rate and conidial morphology between the VdSti1 knockout mutant strain and the wild-type or complementation mutant strains (Figures 4A–C). However, at both low temperatures (16°C) and high temperatures (30°C), the growth rate of the VdSti1 knockout mutant strain was notably lower compared to the wild type and complementation mutants (Figures 4A,B). Moreover, when exposed to high-temperature stress (42°C), the VdSti1 knockout mutant displayed conidial crumpling (Figure 4C). Additionally, the knockout mutant showed compromised conidial germination at 50°C following high-temperature treatment (Supplementary Figure S3).

To evaluate cell wall and cell membrane integrity, VdSti1 knockout mutant, wild type, and complementation mutant strains were grown on plates containing CR and SDS. Results showed that the VdSti1 knockout mutant exhibited slower growth compared to the wild type under CR and SDS stress. However, the complementation mutant displayed similar sensitivity to CR and SDS as the wild type strain (Figures 4D–F). Furthermore, to assess the response of the VdSti1 knockout mutant to osmotic stress, all strains were cultured on plates supplemented with KCl, NaCl, and Sorbitol. The growth rate of the VdSti1 knockout mutant under osmotic stress resembled that of the wild type and complementation mutants (Supplementary Figure S4). These results suggest that the deletion of VdSti1 in V. dahliae leads to temperature sensitivity and impairment in fungal cell wall and cell membrane integrity.

To evaluate the sensitivity of the ΔVdSti1 mutant to H2O2, an H2O2 sensitivity assay was performed. The results demonstrated that the ΔVdSti1 mutant displayed significantly higher sensitivity to H2O2 compared to the wild-type and complementation mutant strains. The zone of inhibition caused by H2O2 was notably larger for the ΔVdSti1 mutant (Figures 5A,B). Furthermore, the expression levels of genes related to reactive oxygen species (ROS) indicators, including VdSOD1, VdCAT1, and VdGSS, were assessed using real-time fluorescence quantitative PCR (Figure 5C). The analysis revealed a down-regulation in the expression of these genes in the ΔVdSti1 mutant in comparison to the wild type and complementation mutants. These results strongly indicate that VdSti1 is involved in V. dahliae’s response to H2O2-induced oxidative stress. The heightened sensitivity to H2O2 and the reduced expression of ROS indicator genes in the ΔVdSti1 mutant highlight the importance of VdSti1 in regulating oxidative stress responses in this fungus.

The ΔVdSti1 mutant exhibited elevated melanin production under both low and high temperature conditions. To explore the connection between VdSti1, melanin, and microsclerotia formation in V. dahliae, we assessed melanin and microsclerotia production after 15 days of incubation on solid basal medium (BM) with cellulose membranes. Results showed that the ΔVdSti1 mutant significantly increased melanin and micronuclei production compared to the wild type and complementation mutant strains (Figure 6A). Additionally, gene expression analysis of VaflM (VDAG_00183), Vayg1 (VDAG_04954), and VDH1 (VDAG_02273) associated with melanin and microsclerotia formation revealed significantly higher levels in the ΔVdSti1 mutant than in the wild type and complementation mutants (Figure 6B). These results suggest that VdSti1, as an Hsp90 co-chaperone, negatively regulates microsclerotia formation. The increased melanin production and upregulation of genes linked to melanin and microsclerotia formation in the ΔVdSti1 mutant imply the involvement of VdSti1 in modulating these processes in V. dahliae.

To further investigate the impact of the Hsp90 co-chaperone VdSti1 on the pathogenicity of V. dahliae, inoculation experiments were conducted using conidial suspensions of the wild type, ΔVdSti1 mutant, and C-ΔVdSti1 mutant strains, with pure water as a control. After a 21 days incubation period, clear symptoms of leaf yellow wilt and necrosis were observed in the wild-type and C-ΔVdSti1 mutant strains (Figure 7A). In contrast, the ΔVdSti1-1 and ΔVdSti1-2 mutant strains exhibited significantly reduced yellow wilt symptoms, leading to a substantial decrease in the disease index by approximately 0.35 and 0.38 times, respectively (Figure 7B). Examination of cotton stalks revealed less browning in those inoculated with the VdSti1 knockout mutants compared to the wild-type strain (Figure 7A). Furthermore, qRT-PCR analysis showed no significant difference in root fungal biomass between the C-ΔVdSti1-1 and C-ΔVdSti1-2 strains and the wild-type strain. However, both the ΔVdSti1-1 and ΔVdSti1-2 strains displayed a notable decrease in fungal biomass by approximately 0.32 and 0.40 times, respectively (Figure 7C). These results suggest that VdSti1 plays a critical role in positively regulating the pathogenicity of V. dahliae.

To gain a better understanding of the potential function of VdSti1 in V. dahliae strains, we analysed differentially expressed genes (DEGs) in ΔVdSti1 strains using RNA sequencing (RNA-seq). The RNA-seq results identified 2,627 DEGs in the ΔVdSti1 strain, including 1792 up-regulated and 835 down-regulated genes (Supplementary Figure S5). This result clarifies the gene expression changes resulting from the knockdown of VdSti1 in V. dahliae. To enhance comprehension of the down-regulated genes’ function, this study conducted Go term and KEGG enrichment analyses. The figure displays the top 15 significantly enriched Go term pathways, including integral component of membrane, intrinsic component of membrane, membrane, transmembrane transporter activity, transporter activity, oxidoreductase activity, carbohydrate metabolic process, and extracellular region pathways (Figure 8A). The pathways consist of differentially expressed genes (DEGs), with 186, 186, 194, 42, 42, 82, 30, and 8 genes, respectively. The figure shows the top 15 significantly enriched KEGG pathways, such as ABC transporters, MAPK signaling pathway-yeast, Glutathione metabolism, and Homologous recombination. It contains DEGs of 7, 6, 6, and 3, respectively (Figure 8B). We randomly selected ten down-regulated candidate genes in the enrichment pathway and analysed the expression levels of the wild-type and ΔVdSti1 strains. The ΔVdSti1 strain caused down-regulation of all 10 candidate genes in comparison to the wild-type and C-ΔVdSti1 strains (Figure 8C). The RT-qPCR results showed a correlation between the expression levels of the 10 genes and the RNA-Seq results (Figure 8D). These findings suggest that the knockout of VdSti1 in V. dahliae resulted in changes in the expression levels of other genes involved in different pathways with different biological functions.

To gain further insight into the role of VdSti1 in the pathogen, we cloned the CDS sequence of VdSti1 into the pGBKT7 vector as a bait. From a cDNA library of V. dahliae, which was subjected to cotton root baiting, we identified five proteins (VDAG_01750, VDAG_01827, VDAG_03209, VDAG_04454, and VDAG_10388) that may interact with VdSti1 (Supplementary Figure S6). Through yeast two-hybrid experiments, we confirmed that the exostosin-2 protein (VDAG_10388, named VdExt2) interacts with VdSti1 (Figure 8E). Furthermore, we confirmed the interaction between VdSti1 and VdExt2 by conducting LCI assays in N. benthamiana (Figure 8F).