VdPT1 Encoding a Neutral Trehalase of Verticillium dahliae Is Required for Growth and Virulence of the Pathogen

Abstract

Verticillum dahliae is a soil-borne phytopathogenic fungus causing destructive Verticillium wilt disease. We previously found a trehalase-encoding gene (VdPT1) in V. dahliae being significantly up-regulated after sensing root exudates from a susceptible cotton variety. In this study, we characterized the function of VdPT1 in the growth and virulence of V. dahliae using its deletion-mutant strains. The VdPT1 deletion mutants (ΔVdPT1) displayed slow colony expansion and mycelial growth, reduced conidial production and germination rate, and decreased mycelial penetration ability and virulence on cotton, but exhibited enhanced stress resistance, suggesting that VdPT1 is involved in the growth, pathogenesis, and stress resistance of V. dahliae. Host-induced silencing of VdPT1 in cotton reduced fungal biomass and enhanced cotton resistance against V. dahliae. Comparative transcriptome analysis between wild-type and mutant identified 1480 up-regulated and 1650 down-regulated genes in the ΔVdPT1 strain. Several down-regulated genes encode plant cell wall-degrading enzymes required for full virulence of V. dahliae to cotton, and down-regulated genes related to carbon metabolism, DNA replication, and amino acid biosynthesis seemed to be responsible for the decreased growth of the ΔVdPT1 strain. In contrast, up-regulation of several genes related to glycerophospholipid metabolism in the ΔVdPT1 strain enhanced the stress resistance of the mutated strain.

Keywords: RNA-seq; RNA-sequencing; Verticillium dahliae; cotton; trehalase.

Figure 1

Functional structural domains of VdPT1, phylogenetic tree, and multiple sequence alignment of trehalases. (A) The trehalase domains in VdPT1 predicted based on Pfam. (B) The phylogenetic tree of trehalases from nine microbes constructed by MEGA 7.0 through the Neighbor-joining method (NJ) and the bootstrap test repeated 1000 times. The trehalases were grouped into three clades, which were distinguished by different colors. The nine species are as follows: V. dahliae—Verticillium dahliae; T. volcanicus—Thermoplasma volcanicus (bacteria); A. niger—Aspergillus niger; N. crassa—Neurospora crassa; S. cerevisiae—Saccharomyces cerevisiae; S. pastorianus—Saccharomyces pastorianus; T. harzianum—Trichoderma harzianum; F. oxysporum—Fusarium oxysporum; F. graminearum—Fusarium graminearum. (C) Alignment of four selected trehalases of the GH37 subfamily, including VdPT1 (VDAG_03038). The conserved trehalase labeling domain 1 (PGGRFXEXYXWDXY) and domain 2 (QWDXPX[G/A]W[P/A/S]P) are marked by green and magenta bars, respectively.

Figure 2

The expression levels of VdPT1, trehalase activity, and trehalose content in different V. dahliae strains. (A) The expression levels of VdPT1 in different strains as determined by qRT-PCR. (B) Trehalase activity in different strains. (C) Trehalose content in different strains. Data were statistically analyzed using IBM SPSS Statistics 26.0. Significant differences between different treatments were analyzed using Duncan’s multiple-range tests (different letters above the error bars indicate statistically different at p < 0.01) for one-way ANOVA.

Figure 3

Colony morphology and growth rates of different V. dahliae strains. All strains were incubated on PDA, CM, or BMM medium. (A) Colony morphology of Vd991, deletion mutants (ΔVdPT1-1, ΔVdPT1-2), and a complementary strain (ΔVdPT1-C) after 21 days of incubation on media. (B) Colony growth rates of different strains on PDA, CM, or BMM medium. Values were means ± SD from three replicates. The data were statistically analyzed using IBM SPSS statistics 26.0. Significant differences between different treatments were analyzed using Duncan’s multiple-range tests. Letters above the error bars indicate statistically significant differences at p < 0.05 from one-way ANOVA tests.

Figure 4

Detection of mycelial growth and penetration of different V. dahliae strains. (A) Mycelial growth of different strains grown on PDA medium after 12, 24 and 48 h of inoculation. (B) Cellophane penetration assay. The top panel shows different strains grown on PDA medium covered with cellophane at 7 days post inoculation (7 dpi), and the bottom panel shows the growth of different strains at 7 days after removal of cellophane (14 dpi).

Figure 5

Comparison of monoconidial germination, conidial production, and germination rate of different V. dahliae strains. (A) Micrographs of monoconidial growth of different strains on PDA medium. (B) Conidial germination rates of different strains on PDA medium after 12 h of incubation. (C) Conidial production of different strains in CM liquid medium after 24, 48, and 72 h of incubation. The results were based on at least three independent experiments. Data were statistically analyzed using IBM SPSS statistics 26.0 and significance analysis was performed using Duncan’s multiple range tests. Different letters above the error bars indicate statistically significant differences at p < 0.01 from one-way ANOVA tests.

Figure 6

Comparison of colony morphology and growth rates of different V. dahliae strains under various stress conditions. (A) Colony morphology of different strains under various stress conditions. (B) Colony growth rates of different strains under various stress conditions. HT and LT indicate high and low temperature, respectively. Data were statistically analyzed using IBM SPSS Statistics 26.0. Significant differences between different treatments were analyzed using Duncan’s multiple range tests (different letters above the error bars indicate statistically significant differences at p < 0.01) from one-way ANOVA tests.

Figure 7

Comparison of pathogenicity of different V. dahliae strains. (A) Disease symptoms of cotton plants infected with different strains at 18 dpi and 30 dpi. Vascular browning observation was performed at 14 dpi. (B) Disease index of cotton plants infected with different strains at 18 dpi and 30 dpi. (C) qRT-PCR assay of fungal biomass in cotton plants treated with different strains at 21 dpi. Data were statistically analyzed using IBM SPSS statistics 26.0. Significant differences between different treatments were analyzed using Duncan’s multiple-range tests. Different letters above the error bars indicate statistically significant differences at p < 0.01 from one-way ANOVA tests.

Figure 8

Functional assessment of VdPT1 in the pathogenicity of V. dahliae by TRV-based HIGS. (A) Fungal infection symptoms of cotton seedlings treated with pTRV2-00 or pTRV2-VdPT1 at 14 and 21 dpi. (B) Comparison of vascular browning in the stem segments of HIGS cotton seedlings at 14 dpi. (C) Disease index of HIGS cotton seedlings at 14 and 21 dpi. (D) qRT-PCR assay of VdPT1 expression in root, stem and leaf of HIGS cotton seedlings at 21 dpi. Cotton tubulin gene was used as the internal reference gene. (E) qRT-PCR assay of fungal biomass in HIGS cotton seedlings at 21 dpi. The data were statistically analyzed used IBM SPSS statistics 26.0. Statistical significance was determined using Student’s t-test. ** and * above the error line indicate a significant difference between HIGS cotton seedlings and pTRV2-00 control at p < 0.01 and p < 0.05, respectively.

Figure 9

Enrichment analysis of the genes differentially expressed between wild-type (Vd991) and ΔVdPT1 strains of V. dahliae. (A) GO enrichment analysis of up-regulated DEGs in the ΔVdPT1-1 strain. (B) GO enrichment analysis of down-regulated DEGs in the ΔVdPT1-1 strain. (C) KEGG pathway analysis of up-regulated DEGs in the ΔVdPT1-1 strain. (D) KEGG pathway analysis of down-regulated DEGs in the ΔVdPT1-1 strain.

Figure 10

Heatmap showing the expression levels of DEGs related to hydrolase activity and the hydrolysis of O-glycosyl compounds.

Figure 11

Heatmap showing the expression levels of DEGs related to carbon metabolism and related pathways.(A) Heatmap of DEGs related to glycolysis, the pentose phosphate pathway, and the glyoxalate cycle. (B) A diagram showing carbon metabolism-related pathways. The inset shows the glucose content in different V. dahliae strains. Data were statistically analyzed using IBM SPSS Statistics 26.0. Significant differences between different treatments were analyzed using Duncan’s multiple range tests. Different letters above the error bars indicate statistically significant differences at p < 0.01 from one-way ANOVA tests.

Figure 12

Heatmap and network diagram of DEGs related to glycerophospholipid metabolism and steroid biosynthesis. (A) Heatmap of DEGs related to glycerophospholipid metabolism. (B) Heatmap of DEGs related to steroid biosynthesis. (C) A diagram showing the network of DEGs related to glycerophospholipid metabolism. (D) A diagram showing the network of DEGs related to steroid biosynthesis.

Figure 13

Comparison of glycerophospholipid content and cell membrane integrity between wild-type (Vd991) and deletion mutant ΔVdPT1-1. (A) The content of glycerophospholipids in wild-type and the ΔVdPT1-1 mutant. (B) Membrane integrity of wild-type and the ΔVdPT1-1 mutant at 0 M NaCl and 0.7 M NaCl examined by flow cytometry analysis. The boxed area is the percentage of PI-stained cells or dead cells caused by impaired cell membranes. (C) Quantification of membrane integrity in the wild-type and the ΔVdPT1-1 mutant at 0 M NaCl and 0.7 M NaCl. All data are the mean values of three independent experiments. Data were statistically analyzed using IBM SPSS statistics 26.0. Statistical significance was determined using Student’s t-test. Asterisks (*/**) above the error bars indicated significant difference at p < 0.05 or p < 0.01 between wild-type (Vd991) and deletion mutant (ΔVdPT1-1). Significant differences in different treatments were analyzed using Duncan’s multiple-range tests. Different letters above the error bars indicate statistically significant differences at p < 0.01 from one-way ANOVA tests.

Figure 14

A working model for the role of VdPT1 in the growth and development, pathogenicity, and stress resistance of V. dahliae.