The fungal-specific transcription factor Vdpf influences conidia production, melanized microsclerotia formation and pathogenicity in Verticillium dahliae

Abstract

Verticillium dahliae is a soil-borne, hemibiotrophic phytopathogenic fungus that causes wilting in crop plants. Here, we constructed a random insertional mutant library using Agrobacterium tumefaciens-mediated transformation to study the pathogenicity and regulatory mechanisms of V. dahliae. The fungal-specific transcription factor-encoding gene Vdpf was shown to be associated with vegetative growth and virulence, with the highest transcript expression occurring during conidia formation in the V991 strain. The deletion mutants (ΔVdpf) and insertion mutants (IMΔVdpf) produced fewer conidia than did the wild-type (WT) fungi, which contributed to the reduced virulence. Unlike the WT, the complemented strains and IMΔVdpf, ΔVdpf formed swollen, thick-walled and hyaline mycelium rather than melanized microsclerotia. The ΔVdpf mutants were melanin deficient, with undetectable expression of melanin biosynthesis-related genes (Brn1, Brn2 and Scd1). The melanin deficiency was related to cyclic adenosine monophosphate (cAMP) and the G-protein-coupled signalling pathways in this study. Similar to the WT and complemented strains, the ΔVdpf and IMΔVdpf mutants could also successfully penetrate into cotton and tobacco roots, but displayed reduced virulence because of lower biomass in the plant roots and significantly reduced expression of pathogenicity-related genes in V. dahliae. In conclusion, these results provide insights into the role of Vdpf in melanized microsclerotia formation, conidia production and pathogenicity.

Keywords: Verticillium dahliae; conidiation; fungal-specific transcription factor; melanized microsclerotia; pathogenicity.

Figure 1

Insertion site of T‐DNA in Vd.M.525 (IMΔVdpf). The T‐DNA cassette was inserted at 1095 bp of the Vdpf (VDAG_08521.1) open reading frame (ORF). The Zn(II)₂Cys₆ binuclear cluster domain covered the range from 279 to 395 bp, and the fungal‐specific transcription factor (TF) domain covered the range from 1415 to 1715 bp.

Figure 2

Verification of mutants. (A) The Vdpf (VDAG_08521.1) coding region was replaced with the HygB cassette. (B) Gel electrophoresis of Vdpf and HygB. HygB and the Zn(II)₂Cys₆ binuclear cluster domain region of Vdpf (VDAG_08521.1) were amplified from all of the mutants with the F‐hph/R‐hph and P7/P8 primers individually. (C) The fungal‐specific transcription factor (TF) domain region amplified with the P9/P10 primer. (D) The mutants were confirmed using nest primers. (E) Southern blotting was performed using the hph, ORF and D2 probes. Four biological replicates (n = 4) were used for this study. (F) Quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis of Vdpf (VDAG_08521.1) expression in the wild‐type (WT), ΔVdpf, IMΔVdpf and complemented strains. Three biological replicates (n = 3) were used for this study. The dots (•) in (B) indicate the lanes of mutants used in this study. M, 2000‐bp marker in (B) and (C); M, 15 000‐bp marker in (D); T1, ΔVdpf‐11; T2, ΔVdpf‐12; T3, ΔVdpf‐13; IM, insertion mutant; Com, complemented strain; E, EcΔVdpf.

Figure 3

Representative phenotypes of ΔVdpf deletion mutants and the IMΔVdpf insertion mutant. (A) Colony morphology of each strain. (B) Colony diameter comparisons between the mutants and wild‐type (WT) at 20 days post‐inoculation (dpi) on potato dextrose agar (PDA) medium. (C) Morphology of microsclerotia. MM, melanized microsclerotia; SM, specialized mycelium. Scale bars, 20 μm. (D) Conidia produced by each strain. (E, F) Extended hyphae and conidia germination rate/length of WT, ΔVdpf, IMΔVdpf and ΔVdpf:Vdpf‐phleo at 24 h after incubation in potato dextrose broth (PDB). Vdpf, VDAG_08521.1. A one‐way analysis of variance (ANOVA) and Student's t‐test were performed. Different letters indicate a significant difference (P < 0.05). The error bars represent the standard deviation of three replicates.

Figure 4

Reduced pathogenicity in ΔVdpf. (A) Necrosis and defoliation in intact cotton plants (Gossypium hirsutum L.) at 20 days post‐inoculation with 4 × 10⁷ conidia of WT, ΔVdpf mutants, IMΔVdpf mutants and complemented strains. (B) Virulence measurements on tobacco plants (Nicotiana benthamiana). (C) Infection of WT, ΔVdpf mutants, IMΔVdpf mutants, complemented strains and CK in root cortical cells and the vascular bundle. (D) Detection of fungal biomass in infected plant roots. WT, wild‐type Verticillium dahlia; ΔVdpf, deletion mutant; IMΔVdpf, insertion mutant; Com, ΔVdpf:Vdpf‐phleo mutant; CK, plant roots inoculated with sterile water. Scale bars, 50 μm. Vdpf, VDAG_08521.1.

Figure 5

Expression of vegetative growth‐ and pathogenicity‐related genes. The relative transcription abundance of each gene was determined in comparison with the 18srRNA gene transcripts in the same tissue. The y axes illustrate the normalized relative quantity of the transcripts compared with the housekeeping gene 18srRNA. Three biological replicates (n = 3) were used for this study. The error bars indicate the standard deviation. Statistical analysis was performed using t‐test. *P < 0.05. **P < 0.01. WT, wild‐type; KO, knockout mutants; IM, insertion mutants; Com, complemented strains. Vdpf, VDAG_08521.1.

Figure 6

Normalized expression of Vdpf at different stages and in the electrophoretic mobility shift assay (EMSA) of Vdpf. (A) The relative transcript amount at each stage was determined in comparison with the 18srRNA transcripts in the same tissue. The y axes illustrate the relative quantity of the transcripts compared with the housekeeping gene: 18srRNA. Three biological replicates (n = 3) were used for this study. The error bars indicate the standard deviation. The statistical analysis was performed using the t‐test. 0 h, conidia; 6–12 h, formation of germ tube; 12–24 h, conidia germination; 24–48 h, hyphal growth; 48–120 h, conidium formation. (B) The Zn(II)₂Cys₆ domain expressed in Pichia pastoris (Gs115). The protein expressed in Pichia pastoris was transformed with purified pPIC9k and served as the control. Vdpf (EGY18187.1) was purified using His‐tag. (C) Vdpf binds to its own promoter. A 1000‐bp promoter fragment of Vdpf was used for binding assays with 0, 2, 4 or 6 μL of purified Vdpf. The concentration of purified Vdpf was 513.636 μg/mL. The pure promoter was used as a probe control. The red arrow points to the shift bands. Three biological replicates (n = 3) were used for this study.

Figure 7

Real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) and electrophoretic mobility shift assay (EMSA) of fungal DHN‐melanin biosynthesis‐related genes. (A) RT‐PCR analysed the expression of Pks1, Brn1, Brn2 and Scd1 at 0, 18, 48 and 120 h. WT, wild‐type; KO, knockout mutants; IM, insertion mutants; Com, complemented strains. (B) EMSA for Vdpf (EGY18187.1) and the promoters of melanin synthesis‐related genes. The promoters of Brn1, Brn2, Pks1 and Scd1 were used for the binding assays with 0, 3, 4 or 5 μL of purified Vdpf. The concentration of purified Vdpf was 513.636 μg/mL. The red arrows point to the shift bands. Four biological replicates (n = 4) were used for this study.

Figure 8

Electrophoretic mobility shift assay (EMSA) and the predicted model for the process of melanin biosynthesis. (A) EMSA for Vdpf (EGY18187.1) and the promoters of Ac, Pka, Mkk1, Mkh1, Gp1, Gp2 and Gp3. Ac represents the adenylate cyclase‐encoding gene, and Pka represents the cyclic adenosine monophosphate (cAMP)‐dependent protein kinase A (PKA)‐encoding gene; these genes are involved in the cAMP signalling pathway. Mkk1 and Mkh1 represent the mitogen‐activated protein (MAP) kinase kinase MKK1/SSP32 gene and the MAP kinase kinase kinase mkh1 gene, respectively, which play important roles in the MAP kinase signalling pathway. Gp1, Gp2 and Gp3 are genes that encode the G‐protein‐coupled receptor; they participate in the G‐protein‐mediated signalling pathway. The binding assays used 0, 3, 4 or 5 μL of purified Vdpf (EGY18187.1). The red arrow points to the shift bands. (B) Real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) analysis of the signalling genes. The expression level was measured by Quantity One software. (C) Deducible signalling pathways in melanin synthesis. Chk1 and Mps1 are two MAP kinase genes involved in the MAP kinase signalling pathway. They have been demonstrated to be involved in melanin synthesis through the transcription factor Cmr1/Amr1. The purple arrows represent known results, and the black arrows represent results in this research study. The broken lines represent unconfirmed results. Three biological replicates (n = 3) were used for this study.