**Background:** Reactive oxygen species (ROS) are a key component of plant defense against pathogens. To successfully colonize hosts, pathogens must neutralize host-derived ROS. Thioredoxins are small, ubiquitous redox-active proteins that play critical roles in ROS detoxification. In Verticillium dahliae, a destructive soilborne phytopathogen causing wilt disease in many crops, the mechanisms by which it copes with oxidative stress, particularly the removal of excess H2O2, are not fully understood. Previous exoproteome analysis identified a predicted thioredoxin, VdTrx1, lacking a signal peptide, suggesting it may be an unconventionally secreted protein involved in host-pathogen interactions.
**Methods:** The VdTrx1 gene (VEDA_00080) was characterized bioinformatically using SignalP 5.0, SecretomeP 2.0, and OutCyte 1.0. Gene deletion mutants (ΔVdTrx1) were generated in the Vd991 background via homologous recombination, and complemented strains (EC-1/2) were created by reintroducing VdTrx1 with its native promoter. Secretion was assessed using a yeast signal sequence trap assay, western blotting of culture supernatants (with β-actin as a lysis control), and confocal microscopy of VdTrx1-GFP in onion epidermal cells. The secretion mechanism was investigated by expressing VdTrx1-HA in ΔVdGRASP, ΔVdATG1, and ΔVdVps36 deletion backgrounds. Oxidative stress tolerance was tested on CM plates with H2O2 (1.0, 1.5, 2.0 mM) and t-BOOH (0.2, 0.3, 0.4 mM). Intracellular ROS were visualized with DCFH-DA staining, and host ROS accumulation in cotton roots was detected by DAB staining at 2 and 5 days post-inoculation (dpi). Sulfite assimilation was tested on MM medium with or without Na2SO3 and cysteine. Pathogenicity was assessed on cotton, N. benthamiana, and A. thaliana, with disease symptoms recorded at 21 dpi and fungal biomass quantified by qPCR targeting VdEF-1α normalized to host reference genes.
**Key Results:** VdTrx1 encodes a 118-amino-acid protein (12.89 kDa, pI 4.77) with a conserved thioredoxin domain (pfam00085) and the redox-active site WCGPC. SignalP analysis confirmed the absence of a signal peptide (probability 0.9987), but OutCyte (UPS score 0.598) and SecretomeP (SecP score 0.599) predicted unconventional secretion. The N-terminal 25 amino acids did not function as a signal peptide in the yeast trap assay. Western blotting detected VdTrx1-HA in both mycelial extracts and culture supernatants, while β-actin was only in mycelia, confirming secretion without cell lysis. Confocal microscopy showed VdTrx1-GFP fluorescence in hyphae and around onion epidermal cell plasma membranes, similar to the known secreted effector VdEG1-GFP. Deletion of VdVps36 (ESCRT-II component) abolished VdTrx1 secretion, while deletion of VdGRASP or VdATG1 did not. ΔVdTrx1 strains showed significantly increased sensitivity to H2O2 (colony diameter reduced by 10% at 1.5 mM and 26% at 2.0 mM; growth halted at 2.5 mM) and t-BOOH. VdTrx1 expression was upregulated 5-fold after 3 h of 1 mM H2O2 treatment. ΔVdTrx1 mycelia showed stronger DCFH-DA fluorescence, indicating higher intracellular ROS. DAB staining of cotton roots at 5 dpi showed increased H2O2 accumulation in ΔVdTrx1-inoculated roots compared to wild-type. ΔVdTrx1 strains produced 81–85% fewer conidia than wild-type and showed a 20% reduction in radial growth on MM medium (containing only SO42−), which was rescued by supplementation with Na2SO3 or cysteine. No defects in cell-wall or hyperosmotic stress responses were observed. Pathogenicity assays revealed significantly reduced disease symptoms and fungal biomass in ΔVdTrx1-inoculated plants: fungal biomass was reduced by 61% in cotton, and similar reductions were observed in N. benthamiana and A. thaliana. VdTrx1 expression was upregulated in planta, peaking at 4 dpi.
**Clinical Implications:** This study identifies VdTrx1 as a novel unconventionally secreted virulence factor in V. dahliae that operates via a VdVps36-dependent, GRASP-independent secretion pathway—distinct from the CUPS-mediated pathway described in yeast. VdTrx1 plays a dual role: intracellularly in sulfite assimilation and endogenous ROS scavenging, and extracellularly in detoxifying host-derived H2O2 in the apoplast. The requirement of VdTrx1 for full virulence across multiple hosts (cotton, Arabidopsis, N. benthamiana) suggests it is a core pathogenicity factor. Targeting VdTrx1 or its secretion pathway could offer new strategies for controlling Verticillium wilt, a disease for which few effective management options exist. The findings also underscore that searches for pathogenicity factors in fungal secretomes should not be restricted to proteins bearing canonical signal peptides.