Cu/Zn superoxide dismutase (VdSOD1) mediates reactive oxygen species detoxification and modulates virulence in Verticillium dahliae

Abstract

The accumulation of reactive oxygen species (ROS) is a widespread defence mechanism in higher plants against pathogen attack and sometimes is the cause of cell death that facilitates attack by necrotrophic pathogens. Plant pathogens use superoxide dismutase (SOD) to scavenge ROS derived from their own metabolism or generated from host defence. The significance and roles of SODs in the vascular plant pathogen Verticillium dahliae are unclear. Our previous study showed a significant upregulation of Cu/Zn-SOD1 (VdSOD1) in cotton tissues following V. dahliae infection, suggesting that it may play a role in pathogen virulence. Here, we constructed VdSOD1 deletion mutants (ΔSOD1) and investigated its function in scavenging ROS and promoting pathogen virulence. ΔSOD1 had normal growth and conidiation but exhibited significantly higher sensitivity to the intracellular ROS generator menadione. Despite lacking a signal peptide, assays in vitro by western blot and in vivo by confocal microscopy revealed that secretion of VdSOD1 is dependent on the Golgi reassembly stacking protein (VdGRASP). Both menadione-treated ΔSOD1 and cotton roots infected with ΔSOD1 accumulated more $O_2^{-}$ and less H₂ O₂ than with the wildtype strain. The absence of a functioning VdSOD1 significantly reduced symptom severity and pathogen colonization in both cotton and Nicotiana benthamiana. VdSOD1 is nonessential for growth or viability of V. dahliae, but is involved in the detoxification of both intracellular ROS and host-generated extracellular ROS, and contributes significantly to virulence in V. dahliae.

Keywords: Verticillium dahliae; ROS detoxification; reactive oxygen species; superoxide dismutase; unconventional secretion; virulence.

FIGURE 1

Bioinformatic analysis of VdSOD1. (a) Structure of VdSOD1. Exons are black boxes and introns are white boxes. (b) Alignment of VdSOD1 and homologs from other fungi. Highly conserved residues are marked by black boxes. The Cu/Zn‐SOD motifs are underlined, Cu²⁺ and Zn²⁺ binding sites are marked by asterisks and hashtags, respectively. The unconventional secretion motif is double‐underlined. GenBank accession number of aligned sequences are Verticillium dahliae (VEDA_03436), Verticillium alfalfae (XP_003007974.1), Fusarium oxysporum (EGU80493.1), Fusarium verticillioides (EWG38836.1), Fusarium graminearum (CEF77131.1), Magnaporthe oryzae (XP_003721165.1), Botrytis cinerea (XP_001560530.1), Aspergillus fumigatus (Q9Y8D9.3), Candida albicans (EEQ45246.1), and Saccharomyces cerevisiae (NP_012638.1)

FIGURE 2

Growth rate, carbon source utilization, and conidia production by VdSOD1 deletion strains. (a) Radial growth of VdSOD1 deletion strains, complemented strains (EC), and wildtype strain Vd991 on potato dextrose agar (PDA) and Czapek medium supplemented with carbon sources sucrose, starch, pectin, or cellulose, respectively, after incubation at 25 °C for 12 days. A 2 μl conidial suspension (5 × 10⁶ conidia/ml) was placed in the centre of the plates as inoculum. (b) Colony diameters on different growth media after 12 days’ incubation. Means and standard deviations from three biological replicates are shown. Double asterisks indicate significant differences at p < 0.01. (c) Conidia production after 7 days’ incubation on PDA. Conidia counts were from 5‐mm‐diameter PDA plugs from the colony edge. Error bars represent the standard deviations of the mean from three independent experiments

FIGURE 3

Impact of intracellular superoxide generator menadione on growth rate and superoxide dismutase (SOD) activity. (a) The wildtype strain Vd991, VdSOD1 deletion strains, and the complemented (EC) strains were cultured on potato dextrose agar (PDA) supplemented with menadione at specified concentrations for 9 days. (b–d) Colony diameters on PDA plates containing different concentration of menadione following 5 days (b), 7 days (c), and 9 days (d) of incubation. Means and standard deviations of the mean from three biological replicates are shown. Asterisks (**) denote significant differences (p < 0.01) according to Student's t test. (e) The total SOD activity of mycelial protein extract of the wildtype strain, ΔSOD1 strains, and complemented strains were detected using the nitroblue tetrazolium (NBT) reduction method. All strains were stressed with 10 μM menadione for 24 hr. Means and error bars (represent standard deviation) are from three independent experiments. Asterisks (**) mean significant differences (p < 0.01) according to Student's t test

FIGURE 4

VdSOD1 can be secreted to extracellular spaces and the putative mechanism of secretion. (a) In vitro assay to demonstrate VdSOD1 secretion into culture filtrates is dependent on VdGRASP. Proteins extracted from WT::OE^SOD1 and ΔGRASP::OE^SOD1 tissue and culture filtrate were analysed using western blotting with anti‐hemagglutinin (HA) or anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) antibodies. Total proteins of fungal tissues and culture supernatants on the membrane were determined by abundance of VdGAPDH. (b) In vivo assay to show VdSOD1 can be secreted extracellularly relying on VdGRASP. Verticillium dahliae strains Vd991::VdSOD1‐GFP, ΔGRASP::VdSOD1‐GFP, Vd8::VCR1‐GFP, and Vd991::GFP were used to infect onion epidermal cells (the latter two strains were used as controls), respectively. Images were taken at 72 hr postinoculation (hpi) using confocal laser scanning microscopy to perform a subcellular localization assay. Bar, 50 μm. (c) Validation of the nonsecretion function of the 26 amino acid N‐terminal peptide of VdSOD1 by a yeast signal trap assay. Fusion of the 26 amino acid N‐terminal peptide of VdSOD1 (VdSOD1^N26) in‐frame with signal‐peptide‐lacking yeast invertase could not enable YTK12::pSUC2‐VdSOD1^N26 to grow on YPRAA medium. YTK12::pSUC2‐VdSOD1^N26 cannot change TTC to red formazan in the formazan assay. The known functional signal peptide Avr1b was used as a positive control

FIGURE 5

Accumulation of superoxide under menadione treatment and in cotton roots inoculated with Vd991 and ΔSOD1. (a) Hyphae from wildtype strain Vd991, VdSOD1 deletion strain, and the complemented (EC) strain were grown in liquid complete medium for 4 days and treated with 5 μM menadione for 24 hr. Strains not treated with menadione were included as untreated control. H₂DCFDA was used as a fluorescent dye to visualize the production of H₂O₂. (b) Cotton roots inoculated with the wildtype strain, ΔSOD1, and complemented strains at 1 and 5 days postinoculation. The production of ${\mathrm{O}}_2^{‐}$ and H₂O₂ was visualized by p‐nitroblue tetrazolium (NBT) and 3,3′‐diaminobenzidine (DAB) staining, respectively

FIGURE 6

VdSOD1 is required for full virulence of Verticillium dahliae. (a) Quantitative reverse transcription PCR (RT‐qPCR) was used to detect the transcript levels of genes involved in the hyphopodium differentiation in the tested strains. The wildtype, mutants, and complemented strains were grown on minimal medium plates covered with the cellophane membrane (mem) for 2 days for hyphopodium induction. Error bars represent standard deviations (n = 3). Asterisks (*) indicate significant differences (p < 0.05) according to Student's t test. (b) Phenotypes of cotton seedlings inoculated with the wildtype strain, VdSOD1 gene deletion strains, and complemented (EC) strains (top). The discolouration in the longitudinal sections of the inoculated shoots is shown at the bottom. Plants treated with sterile water (Mock) were used as controls. Two‐week‐old seedlings of susceptible cotton (Gossypium hirsutum 'Junmian No. 1') were inoculated with indicated strains. Verticillium wilt symptoms were photographed 3 weeks after inoculation. (c) Phenotypes of Nicotiana benthamiana plants inoculated with the wildtype strain, the gene deletion mutants of VdSOD1, and corresponding complementation strains. Four‐week‐old seedlings of N. benthamiana plants were inoculated with the indicated strains. Verticillium wilt symptoms were photographed 3 weeks after inoculation