The role of VdSti1 in Verticillium dahliae: insights into pathogenicity and stress responses

Abstract

Sti1/Hop, a stress-induced co-chaperone protein, serves as a crucial link between Hsp70 and Hsp90 during cellular stress responses. Despite its importance in stress defense mechanisms, the biological role of Sti1 in Verticillium dahliae, a destructive fungal pathogen, remains largely unexplored. This study focused on identifying and characterizing Sti1 homologues in V. dahliae by comparing them to those found in Saccharomyces cerevisiae. The results indicated that the VdSti1-deficient mutant displayed increased sensitivity to drugs targeting the ergosterol synthesis pathway, leading to a notable inhibition of ergosterol biosynthesis. Moreover, the mutant exhibited reduced production of microsclerotia and melanin, accompanied by decreased expression of microsclerotia and melanin-related genes VDH1, Vayg1, and VaflM. Additionally, the mutant's conidia showed more severe damage under heat shock conditions and displayed growth defects under various stressors such as temperature, SDS, and CR stress, as well as increased sensitivity to H2O2, while osmotic stress did not impact its growth. Importantly, the VdSti1-deficient mutant demonstrated significantly diminished pathogenicity compared to the wild-type strain. This study sheds light on the functional conservation and divergence of Sti1 homologues in fungal biology and underscores the critical role of VdSti1 in microsclerotia development, stress response, and pathogenicity of V. dahliae.

Keywords: Sti1/Hop; Verticillium dahliae; microsclerotia; pathogenicity; stress response.

Figure 1

Acquisition of knock-out and complementation mutants. (A) Gene knockout mechanism of V. dahliae VdSti1. (B–D) Knockout and complementation strains were determined by PCR. (M1:VdSti1, M2:HPH, M3:UP1000 + VdSti1, M4:DOWN1000 + VdSti1, M5:UP1000 + DOWN1000 + VdSti1, M6:VdSti1, M7:UP1500 + VdSti1).

Figure 2

VdSti1 deletion mutants exhibit distinct responses to different fungicides. (A) All strains were cultured on PDA plates supplemented with 0.1 mg/L tebuconazole, 20 mg/L triadimefon and 2 mg/L tridemorph at 25°C for 14 days. Scale = 1 cm. (B–D) Colony diameter of all strains. Values represent means ± standard deviation of three replicates. The asterisks represent statistical differences performed by a t-test (*p < 0.05, **p < 0.01, and ***p < 0.001) in comparison with the wild type strains.

Figure 3

Decrease of ergosterol biosynthesis in the VdSti1 knockout mutant. The ergosterol content in wild type, ΔVdSti1 and C-ΔVdSti1 strains were determined by high-performance liquid chromatography (HPLC). Commercial standard of ergosterol was used as the control. The ergosterol content of the wild type, the wild type (5 mg/L GA+), ΔVdSti1 and C-ΔVdSti1 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 < 0.05, **p < 0.01, and ***p < 0.001).

Figure 4

VdSti1 responds to temperature stress, cell wall stress, and cell membrane stress in V. dahliae. (A) Colony morphology of all strains cultured on PDA plates at 16°C, 25°C, 30°C in dark for 14 days. Scale = 1 cm. (B) Colony diameter of all strains. Values represent means ± standard deviation of three replicates. The asterisks represent statistical differences performed by a t-test (ns, p < 0.01, *p < 0.05, **p < 0.01, ***p < 0.001) in comparison with the wild type strains. (C) Morphology of conidia of all strains under scanning electron microscope. Scale = 4 μm. (D) All strains were cultured on PDA plates supplemented 0.02 %CR and 0.002% SDS at 25°C for 14 days. Scale = 1 cm. (E,F) Colony diameter of all strains. Values represent means ± standard deviation of three replicates. The asterisks represent statistical differences performed by a t-test (ns, p < 0.01, *p < 0.05, **p < 0.01, and ***p < 0.001) in comparison with the wild type strains.

Figure 5

VdSti1 contributes to the oxidative stress response. (A) The ΔVdSti1 mutants were compared with the wild type and the C-ΔVdSti1 strain. Equal conidial suspension (1.5 mL, 1*10¹⁰ spores/mL) of each strain was added on PDA plates. Sterile filter paper disks with 5 mm diameters were placed in the center of the plates, and 10 μL of 15% H2O2 were added to each paper disk, respectively. The plates were incubated at 25°C for 4 days. Scale = 1 cm. (B) Zones of growth inhibition in (A) were quantified. Error bars represent standard deviation. The asterisks represent statistical differences performed by a t-test (ns, p < 0.01,*p < 0.05, **p < 0.01, ***p < 0.001) in comparison with the wild type strains. (C) Downregulation of genes related to peroxidase in the ΔVdSti1 mutant. Relative expression levels of three genes VdSOD1 (VDAG_08724), VdCAT1 (VDAG_03661) and VdGSS1 (VDAG_06340), which encode peroxidases, were determined by qRT-PCR using the RNA from mycelium treated with 1 mM H2O2 for 30 min. Error bars represent standard deviation.

Figure 6

VdSti1 promoted the production of microsclerotia. (A) Growth of microsclerotia of wild type, ΔVdSti1 and C-ΔVdSti1 strains cultured on BM solid medium containing nitrocellulose membrane for 15 days. (B) Analysis using quantitative RT-qPCR to detect the expression of melanin-related genes VaflM, Vayg1, VDH1. 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 (ns, p < 0.01,*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 7

VdSti1 positively regulated the pathogenicity of V. dahliae in cotton. (A) Disease symptoms of cotton after the wild type, ΔVdSti1 and C-ΔVdSti1 strains infection. Vascular discoloration of the cotton stem tissue. Photographs were taken 21 days after fungal inoculation. (B) Disease index of cotton plants at 21 days after the wild type, ΔVdSti1 and C-ΔVdVdSti1 strains infection. (C) Fungal biomass in stems of cotton after the wild type, ΔVdSti1 and C-ΔVdSti1 strains infection at 21 days. Vdβt was used as the detection gene, and GhUBQ7 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 (ns, p < 0.01,*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 8

VdSti1 participates in multiple related pathways. (A) Gene ontology enrichment of DEGs from ΔVdSti1 versus WT. Gene ratio is the number of DEGs divided by the total number of genes associated with a specific pathway. (B) KEGG enrichment of down-regulated DEGs from ΔVdSti1 versus WT. (C) Heatmap of partially downregulated genes in knockout mutants. (D) Analysis using quantitative RT-qPCR to detect the expression of related genes. Values represent means ± standard deviation of three replicates. (E) Yeast two-hybrid assays of the interactions of VdSti1 with VdExt2. (F) The interaction of VdSti1 with VdExt2 was detected by LCI assay.