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

Authors: TIAN, LI - Qufu Normal University; ZHUANG, JING - Qufu Normal University; LI, JUNJIAO - Chinese Academy Of Agricultural Sciences; ZHU, HE - Liaoning Academy Of Agricultural Sciences; Klosterman, Steven; DAI, XIAOFENG - Chinese Academy Of Agricultural Sciences; CHEN, JIEYIN - Chinese Academy Of Agricultural Sciences; SUBBARAO, KRISHNA - University Of California; ZHANG, DANDAN - Chinese Academy Of Agricultural Sciences

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2023
Publication Date: 3/31/2023
Citation: Tian, L., Zhuang, J., Li, J.J., Zhu, H., Klosterman, S.J., Dai, X.F., Chen, J.Y., Subbarao, K.V., Zhang, D.D. 2023. Thioredoxin VdTrx1, an unconventional secreted protein, is a virulence factor in Verticillium dahliae. Frontiers in Microbiology. 14. Article 1130468. https://doi.org/10.3389/fmicb.2023.1130468.
DOI: https://doi.org/10.3389/fmicb.2023.1130468

Interpretive Summary: The fungus Verticillium dahliae causes vascular wilt disease on hundreds of plant species. As part of a multifaceted defense response by the plant, reactive oxygen species (ROS), which cause cell damage, are produced after challenge with the fungus. In this study, we identified a V. dahliae gene encoding a protein named VdTrx1 that protects the fungus against antioxidants such as ROS. Deletion of VdTrx1 to study its function revealed the importance of VdTrx1 in scavenging ROS within the fungus and its important role in virulence. This study sheds light on the importance of another ROS scavenging protein in V. dahliae. Because of its importance in virulence, VdTrx1 and similar proteins may be targeted for development of novel disease control measures for Verticillium wilt.

Technical 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 'VdTrx1 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 'VdTrx1 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.