Thioredoxin VdTrx1, an unconventional secreted protein, is a virulence factor in Verticillium dahliae

Abstract

Understanding how plant pathogenic fungi adapt to their hosts is of critical importance to securing optimal crop productivity. In response to pathogenic attack, plants produce reactive oxygen species (ROS) as part of a multipronged defense response. Pathogens, in turn, have evolved ROS scavenging mechanisms to undermine host defense. Thioredoxins (Trx) are highly conserved oxidoreductase enzymes with a dithiol-disulfide active site, and function as antioxidants to protect cells against free radicals, such as ROS. However, the roles of thioredoxins in Verticillium dahliae, an important vascular pathogen, are not clear. Through proteomics analyses, we identified a putative thioredoxin (VdTrx1) lacking a signal peptide. VdTrx1 was present in the exoproteome of V. dahliae cultured in the presence of host tissues, a finding that suggested that it plays a role in host-pathogen interactions. We constructed a VdTrx1 deletion mutant ΔVdTrx1 that exhibited significantly higher sensitivity to ROS stress, H₂O₂, and tert-butyl hydroperoxide (t-BOOH). In vivo assays by live-cell imaging and in vitro assays by western blotting revealed that while VdTrx1 lacking the signal peptide can be localized within V. dahliae cells, VdTrx1 can also be secreted unconventionally depending on VdVps36, a member of the ESCRT-II protein complex. The ΔVdTrx1 strain was unable to scavenge host-generated extracellular ROS fully during host invasion. Deletion of VdTrx1 resulted in higher intracellular ROS levels of V. dahliae mycelium, displayed impaired conidial production, and showed significantly reduced virulence on Gossypium hirsutum, and model plants, Arabidopsis thaliana and Nicotiana benthamiana. Thus, we conclude that VdTrx1 acts as a virulence factor in V. dahliae.

Keywords: ROS scavenging; Verticillium dahliae; thioredoxin; unconventional secreted protein; virulence factor.

Figure 1

Gene cloning and bioinformatic analysis of VdTrx1 from Verticillium dahliae. (A) Structure of VdTrx1. Exons are black boxes and introns are white boxes. (B) Conserved domain of VdTrx1 predicted by CD-Search of NCBI. DB represent disulfide bond. (C) Alignment of conserved region of thioredoxins. Thioredoxin-specific redox-active sites are marked by asterisks. GenBank accession number of aligned sequences are V. dahliae (VEDA_00080); Verticillium alfalfae (XP_003005517.1); Fusarium oxysporum (EGU81922.1); Fusarium graminearum (ESU16261.1); Magnaporthe oryzae (EHA47211.1); Botrytis cinerea (EMR86794.1); Aspergillus fumigatus (XP_753517.1); Colletotrichum gloeosporioides (EQB49288.1); Candida albicans (XP_719372.1); Saccharomyces cerevisiae (YLR043C). (D) Signal peptide prediction of VdTrx1 using SignalP 5.0 program. OutCyte 1.0 (E) and SecretomeP 2.0 (F) were used to predict unconventional secretion characteristic of VdTrx1. UPS, unconventional protein secretions; Network 1, score of amino acid composition; Network 2, score of secondary structure prediction; Network 3, score of transmembrane helix prediction; SepP score, the average of three network scores.

Figure 2

VdTrx1 of V. dahliae mediates the response to oxidative stress. (A) Radial growth of VdTrx1 deletion strains, complemented strains (EC#1 and #2) and wild-type strain (Vd991) on CM (complete medium) supplemented with H₂O₂ at specified concentrations for 9 days. (B) Colony diameters of various V. dahliae strains on CM plates containing different concentration of H₂O₂ following 9 days incubation. Means and standard deviations of the mean from three biological replicates are shown. Asterisks (**) and (***) denote significant differences p < 0.01 and p < 0.001, respectively, according to Student’s t-test. (C) Reverse transcription-quantitative PCR (RT-qPCR) of VdTrx1 transcripts in V. dahliae hyphae treated with 1.0 mM H₂O₂ or incubated in CM only for 3 h. Total cDNA abundance in the samples was normalized to using VdEF-1α gene as a control. In RT-qPCR, expression of VdTrx1 in the strain without H₂O₂ treatment was set as 1. Bars indicate standard deviations from three biological replicates, *** denotes significant differences at p < 0.001 (Student’s t-test).

Figure 3

VdTrx1 can be secreted by V. dahliae to extracellular spaces. (A) Validation of the non-secretory function of the 25 aa N-terminal peptide and full-length sequence of VdTrx1 by a yeast signal trap assay. The region encoding the 25 aa N-terminal peptide and the full-length sequence of VdTrx1 were fused in-frame to the invertase sequence in the pSUC2 vector and transformed into yeast strain YTK12. The untransformed YTK12 strain and empty pSUC2 vector transformed into YTK12 were used as negative controls, while the pSUC2-Avr1b^SP vector (integrating known functional signal peptide Avr1b^SP into pSUC2) transformed YTK12 was used as a positive control. Only yeast strains that can secrete invertase converted 2,3,5- triphenyl tetrazolium chloride (TTC) to red triphenylformazan. (B) Western blotting assay demonstrates VdTrx1 secretion into culture filtrates. VdTrx1-HA was expressed in V. dahliae wild-type strain Vd991 to produce strain WT::VdTrx1-HA. Proteins extracted from mycelia (M) and culture supernatants (S) of strain WT::VdTrx1-HA were analyzed using western blotting with anti-HA or anti-β-actin antibodies, as indicated. (C) Live-cell imaging by confocal microscopy of VdTrx1. V. dahliae strains WT::VdTrx1-GFP, WT::GFP and WT::VdEG1-GFP were used to infect onion epidermal cells (the latter two strains were used as controls), respectively. Images were taken at 5 dpi using confocal laser scanning microscopy to perform a subcellular localization assay. Bar, 50 μm.

Figure 4

Secretion mechanism of VdTrx1 from V. dahliae. (A) Proteins extracted from mycelia (M) and culture supernatants (S) of strain WT::VdTrx1-HA, ΔVdGRASP::VdTrx1-HA, ΔVdATG1::VdTrx1-HA and ΔVdVps36::VdTrx1-HA were analyzed using western blotting with anti-HA or anti-β-actin antibodies, as indicated. (B) The V. dahliae strains WT::VdTrx1-GFP, ΔVdGRASP::VdTrx1-HA, ΔVdATG1::VdTrx1-HA and ΔVdVps36::VdTrx1-HA were used to inoculate onion epidermal cells, respectively, at room temperature for 5 days followed by confocal microscopy imaging. Bar, 50 μm.

Figure 5

The role of VdTrx1 from V. dahliae in the degradation of ROS. (A) Hyphae from wild-type strain Vd991, VdTrx1 deletion strains, and the complemented strains were grown in liquid complete medium for 4 days. The fluorescent dye 2′,7′–dichlorofluorescin diacetate (DCFH-DA) was used to visualize the production of H₂O₂. Bar, 10 μm. (B) Accumulation of H₂O₂ in infected cotton roots. Detection of H₂O₂ accumulation in cotton roots inoculated with sterile water, the wild-type strain Vd991, ΔVdTrx1 and complemented strains at 2 days and 5 days post-inoculation, respectively, by 3,3′- diaminobenzidine staining.

Figure 6

VdTrx1 is essential for sulfite assimilation and conidiation in V. dahliae. (A) Radial growth of VdTrx1 deletion strains, complemented strains (EC#1 and #2) and wild-type strain (Vd991) on different media after incubation at 25°C for 12 days. A 2 μl conidial suspension (5 × 10⁶ conidia/ml) was placed in the center of the plates as inoculum. (B) Colony diameter of the different strains on different media after 12 days of incubation. Means and standard deviations from three biological replicates are shown. Double asterisks indicate significant differences at p < 0.01. (C) Growth of V. dahliae strains on plates with different source of sulfate. The various V. dahliae strains were inoculated on MM plates, or MM plates supplemented with 2 mM sulfate (${\mathrm{SO}}_3^{2-}$) and 1.4 mM cysteine (distribution of strains on the plate are indicated in figure). The strains were cultured at 25°C for 4 days. (D) Quantification of conidial production was based on a 5-mm-diameter PDA agar plug from the edge of 9-days-old fungal culture colonies in 1 ml water. The error bars represent the standard deviations of the mean (n = 3). Student’s t-test was employed to determine treatment differences and double asterisks indicate significant differences at p < 0.01.

Figure 7

VdTrx1 is required for the full virulence of V. dahliae. (A) The disease symptoms of cotton seedlings inoculated with sterile water (CK) or the VdTrx1 deletion strains, complemented strains (EC#1 and #2) and wild-type strain (Vd991) at 21 days post-inoculation (Top). The discoloration of the inoculation shoot longitudinal sections is shown at the bottom. Phenotypes of N. benthamiana (C) and A. thaliana (E) plants inoculated with indicated strains. The fungal biomasses of each fungal strain in cotton (B), N. benthamiana (D) and A. thaliana (F) plants were determined by qPCR. Error bars represent standard deviations (n = 3). Asterisks *** indicate significant differences at (p < 0.001) based on the Student’s t-test.

Figure 8

Proposed model of VdTrx1 function during V. dahliae-host interactions.