Transcription Factor VdCf2 Regulates Growth, Pathogenicity, and the Expression of a Putative Secondary Metabolism Gene Cluster in Verticillium dahliae

Abstract

Transcription factors (TFs) bind to the promoters of target genes to regulate gene expression in response to different stimuli. The functions and regulatory mechanisms of transcription factors (TFs) in Verticillium dahliae are, however, still largely unclear. This study showed that a C2H2-type zinc finger TF, VdCf2 (V. dahliae chorion transcription factor 2), plays key roles in V. dahliae growth, melanin production, and virulence. Transcriptome sequencing analysis showed that VdCf2 was involved in the regulation of expression of genes encoding secreted proteins, pathogen-host interaction (PHI) homologs, TFs, and G protein-coupled receptors (GPCRs). Furthermore, VdCf2 positively regulated the expression of VdPevD1 (VDAG_02735), a previously reported virulence factor. VdCf2 thus regulates the expression of several pathogenicity-related genes that also contribute to virulence in V. dahliae. VdCf2 also inhibited the transcription of the Vd276-280 gene cluster and interacted with two members encoding proteins (VDAG_07276 and VDAG_07278) in the gene cluster. IMPORTANCE Verticillium dahliae is an important soilborne phytopathogen which can ruinously attack numerous host plants and cause significant economic losses. Transcription factors (TFs) were reported to be involved in various biological processes, such as hyphal growth and virulence of pathogenic fungi. However, the functions and regulatory mechanisms of TFs in V. dahliae remain largely unclear. In this study, we identified a new transcription factor, VdCf2 (V. dahliae chorion transcription factor 2), based on previous transcriptome data, which participates in growth, melanin production, and virulence of V. dahliae. We provide evidence that VdCf2 regulates the expression of the pathogenicity-related gene VdPevD1 (VDAG_02735) and Vd276-280 gene cluster. VdCf2 also interacts with VDAG_07276 and VDAG_07278 in this gene cluster based on a yeast two-hybrid and bimolecular fluorescence complementation assay. These results revealed the regulatory mechanisms of a pivotal pathogenicity-related transcription factor, VdCf2 in V. dahliae.

Keywords: VdCf2; Verticillium dahliae; gene cluster; pathogenicity; transcription factor; transcriptome.

FIG 1

Sequence analysis and subcellular localization of VdCf2. (A) Dendrogram of VdCf2 and its homologues from Verticillium longisporum, Verticillium nonalfalfae, Verticillium alfalfae, Colletotrichum siamense, Plectosphaerella cucumerina, M. oryzae, Fusarium oxysporum, Valsa mali, Puccinia striiformis, Ustilago maydis, N. crassa, Beauveria bassiana, Metarhizium anisopliae, and Saccharomyces cerevisiae. The phylogenetic tree was constructed using MEGA 7.0 software based on the neighbor-joining method. The number of bootstrap replications was set as 1,000. (B) Schematic representation of the VdCf2 structure containing conserved C2H2 zinc finger domain and coiled-coil region according to the prediction from SMART as well as the nuclear localization signal predicted by cNLS Mapper. Multiple sequence alignment of the C2H2-type zinc finger domain of VdCf2 orthologs from V. dahliae, Colletotrichum siamense, F. oxysporum, and N. crassa was completed using MEGA 7.0 software and displayed using the multiple sequence comparison display online website. (C) The subcellular localization was performed in V. dahliae (first line, bars = 20 μm) and tobacco (N. benthamiana) cells (last two lines, bars = 40 μm). pBin-GFP was used as the control. (D) Western blot analysis of GFP and VdCf2-GFP fusion proteins in tobacco (N. benthamiana) cells.

FIG 2

VdCf2 plays key roles in V. dahliae growth and melanin production. (A) The identification electrophoretogram of the ΔVdCf2 and ΔVdCf2/VdCf2 strains. M, 2,000 bp marker; 1, ΔVdCf2 strain; 2, ΔVdCf2/VdCf2 strain; 3, wild-type strain; 4, negative control (sterile water). (B) Confirmation of VdCf2 deletion by Southern blot analysis. Total genomic DNA of the wild-type and ΔVdCf2 strains was digested with BamHI and subjected to Southern blot analysis. The probe in the VdCf2 gene region amplified with primer pair NF/NR was used to confirm the presence or absence of the VdCf2 gene in the wild-type and ΔVdCf2 strains. The probe in the hygromycin B resistance gene region amplified by primer pair HYG-F/HYG-R was used to verify the copy number of the hygromycin B resistance gene in the wild-type and ΔVdCf2 strains. (C) Colony morphology of the XJ592, ΔVdCf2, and ΔVdCf2/VdCf2 strains on PDA, MM, and WA plates. Pictures were taken from the upper side of 14-day-old cultures. (D) Colony diameter of the XJ592, ΔVdCf2, and ΔVdCf2/VdCf2 strains on three media. Error bars are standard errors calculated from three replicates, and different letters represent statistically significant differences (P = 0.05). The SNK test was executed among each strain in each medium. (E) VdCf2 was not required for osmotic stress response. The inhibition rate of colony growth was quantified. Strains XJ592 and ΔVdCf2 were cultured on CM plates containing osmotic stress reagents (1.2 M sorbitol, 0.8 M KCl, or 0.4 M NaCl) for 14 days. Error bars indicate standard errors calculated from three replicates, and different letters represent the statistical significance (P = 0.05). Student’s t test was executed between strains in each osmotic stress reagent. (F) VdCf2 participated in oxidative stress response. The inhibition zones of the XJ592 and ΔVdCf2 strains were quantified. Error bars indicate standard errors calculated from three replicates (Student’s t test, P = 0.05). (G) VdCf2 curtails melanin production. The conidial suspensions of strains XJ592, ΔVdCf2, and ΔVdCf2/VdCf2 were cultured on PDA and MM plates for 3 days and BMM plates for 4 days. The mycelia on the BMM plates were observed under a light microscope (bars = 20 μm).

FIG 3

VdCf2 was required for full virulence on cotton. (A) VdCf2 was not involved in cellophane membrane penetration of V. dahliae. Strains XJ592, ΔVdCf2, and ΔVdCf2/VdCf2 were cultured on top of cellophane membrane overlaid onto MM plates for 3, 4, or 5 days. The cellophane membranes were removed, and all strains were cultured for additional 3 days. (B) Verticillium wilt symptoms on susceptible cotton plants (JM11) inoculated with the wild-type, ΔVdCf2, and ΔVdCf2/VdCf2 strains at 35 days postinoculation. (C) The vascular discoloration in cotton plants inoculated with the wild-type, ΔVdCf2, and ΔVdCf2/VdCf2 strains. (D) Disease index of cotton plants inoculated with the wild-type, ΔVdCf2, and ΔVdCf2/VdCf2 strains. Error bars represent standard errors calculated from three replicates, and asterisks represent the statistical significance (SNK test, P = 0.05).

FIG 4

KEGG and GO enrichment analysis of differentially expressed genes in the transcriptome analysis. (A) Sample preparation of RNA-seq and the number of differentially expressed genes between the wild-type strain and ΔVdCf2 strain. Three fungus blocks (6-mm diameter) were inoculated into the Czapek-Dox medium, and then the mycelia were collected at 8 days postinoculation. WT represents the XJ592 strain and Mut represents the ΔVdCf2 strain. The gene expression was analyzed in the mycelia of the wild-type XJ592 strain compared to that of strain ΔVdCf2. Differentially expressed genes were defined by a log₂ fold change of ≥1.0 and P value of <0.05. Three repeats were performed in this transcriptome sequencing assay. (B) KEGG pathway enrichment analysis of upregulated differentially expressed genes in the transcriptome data. The percentage of genes represents the proportion of upregulated differentially expressed genes in this pathway. (C) GO enrichment analysis of all differentially expressed genes in RNA-seq analysis in the mycelia of strain XJ592 compared to that of strain ΔVdCf2.

FIG 5

VdCf2 regulated the expression of potential pathogenicity-related genes. (A) Number of SP_Cs, SP_NCs, PHIs, TFs, and GPCRs in differentially expressed genes. SP_Cs, classical secretory proteins; SP_NCs, nonclassical secretory proteins; PHIs, pathogen-host interaction-related proteins; TFs, transcription factors; GPCRs, G protein-coupled receptors. (B) The expression level of VDAG_02735 and VDAG_06155 was determined by an RT-qPCR experiment. Conidial suspensions (10⁷ conidia/mL) of the wild-type and ΔVdCf2 strains were cultured on the Czapek-Dox medium for 5 days before being treated with cotton root extracts for 2 days. The β-tubulin gene (VDAG_10074) was used as an internal reference. The expression level of each gene in the wild-type strain in the control was standardized as 1. Error bars represent standard errors calculated from three replicates, and different letters indicate the statistical significance of Tukey’s test at a P value of 0.05. (C) Schematic diagrams of VdCf2-F1 and VdCf2-F2 used in the EMSA. (D) VdCf2, VdCf2-F1, and VdCf2-F2 proteins bound to the promoter of the VDAG_02735 gene according to the EMSA.

FIG 6

VdCf2 regulated the expression of several PHI genes. Sample preparation and data analysis are consistent with Fig. 5B. Error bars represent standard errors calculated from three replicates, and different letters indicate the statistical significance by Tukey’s test at a P value of 0.05.

FIG 7

VdCf2 regulated the expression of other TFs. (A) The value of the log₂ fold change of genes encoding TFs regulated by VdCf2 according to transcriptome data. (B) The relative expression level of VDAG_00592 and VDAG_05292 genes in the wild-type and ΔVdCf2 strains. Conidial suspensions (10⁷ conidia/mL) were cultured in the Czapek-Dox medium for 5 days before being treated with cotton root extracts for 2 days. The β-tubulin gene (VDAG_10074) was used as the internal reference. The expression level of each gene in the wild-type strain in the control was standardized as 1. Error bars represent standard errors calculated from three replicates, and different letters indicate the statistical significance of Tukey’s test at a P value of 0.05.

FIG 8

VdCf2 regulated the expression of a putative secondary metabolism gene cluster. (A) Visualization of RNA-seq coverage of this putative secondary metabolism gene cluster in the wild-type and ΔVdCf2 strains. The blue lines indicate read coverage. (B) Gene expression level of the Vd276-280 gene cluster. The conidial suspensions (10⁷ conidia/mL) were cultured in the Czapek-Dox medium for 5 days before being treated with cotton root extracts for 2 days. The β-tubulin gene (VDAG_10074) was used as the internal reference. The expression level of each gene in the wild-type strain in the control was standardized as 1. Error bars represent standard errors calculated from three replicates, and different letters indicate the statistical significance of Tukey’s test at a P value of 0.05.

FIG 9

VdCf2 interacted with VDAG_07276 and VDAG_07278. (A) VdCf2 interacted with VDAG_07276 and VDAG_07278 in a yeast two-hybrid system. For analysis of interaction, the full-length cDNA of VdCf2 was inserted into the pGBKT7 vector to generate the bait construct, and the full-length CDS of VDAG_07276 and VDAG_07278 was inserted into the pGADT7 vector to generate the prey construct. The Y2HGold yeast cells containing the bait construct and pGADT7 vector were used to detect self-activation. The Y2HGold yeast cells containing the bait and prey constructs were used to detect interaction. The Y2HGold yeast cells with 10-fold serial dilutions (10⁰, 10⁻¹, and 10⁻²) were simultaneously cultured on SD plates –LW (lacking Leu and Trp), –LWH (lacking Leu, Trp, and His), and –LWH (lacking Leu, Trp, and His) supplemented with 40 mg/L X-α-Gal. The Y2HGold yeast cells containing the pGBKT7-53 and pGADT7-T vectors were regarded as the positive control, and the Y2HGold yeast cells containing the pGBKT7-Lam and pGADT7-T vectors were regarded as the negative control. (B) VdCf2 interacted with VDAG_07276 and VDAG_07278 as determined by a bimolecular fluorescence complementation assay. The epidermal cells of N. benthamiana leaves coinfiltrated with nYFP and cYFP-07276, nYFP and cYFP-07278, and nYFP-VdCf2 and cYFP were negative controls. Scale bars = 20 μm.