AUTHOR=Tian Li , Zhuang Jing , Li Jun-Jiao , Zhu He , Klosterman Steven J. , Dai Xiao-Feng , Chen Jie-Yin , Subbarao Krishna V. , Zhang Dan-Dan 

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

JOURNAL=Frontiers in Microbiology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2023.1130468

DOI=10.3389/fmicb.2023.1130468

ISSN=1664-302X

ABSTRACT=<p>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 <italic>Verticillium dahliae</italic>, 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 <italic>V. dahliae</italic> cultured in the presence of host tissues, a finding that suggested that it plays a role in host-pathogen interactions. We constructed a <italic>VdTrx1</italic> deletion mutant Δ<italic>VdTrx1</italic> that exhibited significantly higher sensitivity to ROS stress, H<sub>2</sub>O<sub>2</sub>, and <italic>tert</italic>-butyl hydroperoxide (<italic>t</italic>-BOOH). <italic>In vivo</italic> assays by live-cell imaging and <italic>in vitro</italic> assays by western blotting revealed that while VdTrx1 lacking the signal peptide can be localized within <italic>V. dahliae</italic> cells, VdTrx1 can also be secreted unconventionally depending on VdVps36, a member of the ESCRT-II protein complex. The Δ<italic>VdTrx1</italic> strain was unable to scavenge host-generated extracellular ROS fully during host invasion. Deletion of <italic>VdTrx1</italic> resulted in higher intracellular ROS levels of <italic>V. dahliae</italic> mycelium, displayed impaired conidial production, and showed significantly reduced virulence on <italic>Gossypium hirsutum</italic>, and model plants, <italic>Arabidopsis thaliana</italic> and <italic>Nicotiana benthamiana</italic>. Thus, we conclude that VdTrx1 acts as a virulence factor in <italic>V. dahliae</italic>.</p>