Verticillium dahliae secreted protein Vd424Y is required for full virulence, targets the nucleus of plant cells, and induces cell death

Abstract

Fungal pathogens secrete effector proteins that regulate host immunity and can suppress basal defence mechanisms against colonization in plants. Verticillium dahliae is a widespread and destructive soilborne fungus that can cause vascular wilt disease and reduces plant yields. However, little is currently known about how the effectors secreted by V. dahliae function. In this study, we analysed and identified 34 candidate effectors in the V. dahliae secretome and found that Vd424Y, a glycoside hydrolase family 11 protein, was highly upregulated during the early stages of V. dahliae infection in cotton plants. This protein was located in the nucleus and its deletion compromised the virulence of the fungus. The transient expression of Vd424Y in Nicotiana benthamiana induced BAK1- and SOBIR1-dependent cell death and activated both salicylic acid and jasmonic acid signalling. This enhanced its resistance to the oomycetes Phytophthora capsici in a way that depended on its nuclear localization signal and signal peptides. Our results demonstrate that Vd424Y is an important effector protein targeting the host nucleus to regulate and activate effector-triggered immunity in plants.

Keywords: Verticillium dahliae; cell death; effector; nuclear localization signal; virulence.

FIGURE 1

Vd424Y is an elicitor of cell death. (a) Vd424Y induces cell death in Nicotiana benthamiana. Four‐week‐old plants were used to express PVX‐GFP‐HA, PVX‐Vd424Y‐HA, and PVX‐BAX. Photographs were taken 7 days after Agrobacterium infiltration. (b) Quantification analysis of cell death via electrolyte leakage measurement. The data shown represent the mean ± SE estimated from three biological replicates. Significant p values (p < .05) for a Student's t test are represented by *. (c) Western blotting (WB) analysis of protein levels in N. benthamiana transiently expressing green fluorescent protein (GFP) control (left) and Vd424Y fused with HA tag (right). Proteins were stained with Coomassie brilliant blue R‐250 (CBB) to confirm equal loading

FIGURE 2

Signal peptide and nuclear localization signal are required for Vd424Y‐induced cell death. (a) Schematic illustration of the Vd424Y deletion mutants: Vd424Y, the full‐length sequence of the candidate effector; Vd424N, effector variant lacking the signal peptide (SP) sequence; Vd424Y‐nls, effector variant where the predicted nuclear localization signal (NLS) was mutated to alanine residues; Vd424Y‐nls‐NES, the variant Vd424Y‐nls was fused with a NES at C‐terminus; Vd424Y‐NES, effector variant where the NES at C‐terminus was tagged to the wildtype Vd424Y allele; Vd424N‐SPNbPR1, effector variant with a fused SP of Nicotiana benthamiana pathogenesis‐related protein (NbPR1). (b) N. benthamiana leaves were infiltrated with Agrobacterium tumefaciens carrying Vd424Y, Vd424N, Vd424Y‐nls, Vd424Y‐nls‐NES, Vd424Y‐NES, Vd424N‐SP_NbPR1 , positive control BAX, and control green fluorescent protein (GFP). Representative photographs of N. benthamiana leaves were taken after 7 days. (c) Subcellular localizations of Vd424Y‐YFP, Vd424Y‐nls‐YFP, Vd424Y‐nls‐NES‐YFP, and Vd424Y‐NES‐YFP in N. benthamiana on A. tumefaciens‐mediated transient expression. Fluorescence was detected by confocal microscopy 48 hr postinfiltration. Bars, 40 µm

FIGURE 3

Vd424Y induces plant defence responses in Nicotiana benthamiana. Relative expression of hypersensitive‐response‐specific and defence‐related marker genes in N. benthamiana infiltrated with Agrobacterium tumefaciens carrying Vd424Y, Vd424N, and Vd424Y‐nls. At 3 days postinfiltration (dpi), total RNA was extracted and transcript levels were detected by quantitative reverse transcription PCR. NbActin was used as the internal reference gene. The data shown represents the mean across three independent experiments. Bars indicate ± SE. Significance levels p < 0.05 and p < 0.01 are represented by * and **, respectively

FIGURE 4

Vd424Y promotes transcription of PAMP‐PTI marker genes in Nicotiana benthamiana. Relative transcript levels of NbCYP71D20, NbPti5, NbWRKY7, and NbWRKY8 were analysed in N. benthamiana infiltrated with Agrobacterium tumefaciens carrying Vd424Y, Vd424N, and Vd424Y‐nls. At 3 days postinfiltration, total RNA was extracted and transcript levels were detected by quantitative reverse transcription PCR. NbActin was used as the internal reference gene. The data shown represent the mean across three independent experiments. Significance levels p < 0.05 and p < 0.01 are represented by * and **, respectively

FIGURE 5

NbBAK1 and NbSOBIR1 are required for Vd424Y‐induced cell death. (a) Virus‐induced gene silnecing (VIGS) technology was used to silence NbBAK1 and NbSOBIR1 by inoculation with TRV constructs (pTRV2:GFP, pTRV2:NbBAK1, and NbSOBIR1) in Nicotiana benthamiana plants. Three weeks after inoculation, GFP, BAX, Vd424Y, and Vd424Y mutations were transiently expressed in NbBAK1‐ and NbSOBIR1‐silenced N. benthamiana plant leaves. Photographs were taken 7 days after agroinfiltration. The experiment was carried out three times with five plants for each TRV construct. (b) The expression levels of NbBAK1 and NbSOBIR1 after VIGS treatment as evaluated by quantitative reverse transcription PCR. NbActin was used as the internal reference gene. Mean and SE were calculated from three independent experiments. Bars indicate ± SE. Significance level p < 0.01 is represented by *. (c) Western blot (WB) analysis of green fluorescent protein (GFP), Vd424Y, and Vd424Y mutations protein fused with HA tags after transient expression in N. benthamiana leaves. Proteins were stained with Coomassie brilliant blue (CBB) to confirm equal loading

FIGURE 6

Expression of Vd424Y in Nicotiana benthamiana enhanced resistance to an oomycete pathogen. Leaf regions transiently expressing Vd424Y, Vd424N, and Vd424Y‐nls were inoculated with zoospores of Phytophthora capsici strain 35. (a) Resulting lesions visualized using trypan blue staining. (b) Quantitative PCR analysis of relative Phytophthora biomass following P. capsici infection. Infected leaves (n = 10) were collected 48 hr after infection, after which DNA was isolated and qPCR analysis was performed. (c) Size of the lesions caused by P. capsici infection on plant leaves expressing Vd424Y and Vd424Y mutations

FIGURE 7

Vd424Y contributes to the virulence of Verticillium dahliae. (a) Images of Vd991 (wild type), ΔVd424Y mutant, and ΔVd424Y/VD424Y‐complementation transformants cultured on potato dextrose agar plates at 25 °C for 7 days in the dark. (b) Colony diameter of Vd991, ΔVd424Y mutant, and ΔVd424Y/VD424Y transformants. The data shows the mean across three independent replicates. (c) Images of disease symptoms on cotton 26 days postinoculation (dpi). The cotton cultivar Jimian 11 was inoculated with Vd991, ΔVd424Y mutant, and ΔVd424Y/VD424Y transformants. Images are representative of three independent experiments. (d) Disease index of the cotton plants at 26 dpi. The disease index (DI) was calculated as the following formula: DI = [(Ʃdisease grades × number of infected plants)/(total checked plants × 4)] × 100%. Seedlings were classified into five grades (0, 1, 2, 3, and 4). The data represent the mean across three independent experiments