Secreted Protein VdCUE Modulates Virulence of Verticillium dahliae Without Interfering with BAX-Induced Cell Death

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Verticillium wilt, caused by Verticillium dahliae, severely threatens various crops and trees worldwide. This study aimed to characterize the function of a CUE (coupling of ubiquitin conjugation to endoplasmic reticulum (ER) degradation)-domain-containing protein, VdCUE, in V. dahliae, which exhibits sequence divergence between the defoliating strain XJ592 and the non-defoliating strain XJ511. We generated ∆VdCUE-knockout mutants and evaluated their phenotypes in growth and virulence. Functional analyses included verifying the signal peptide activity of VdCUE, testing its ability to induce cell death or inhibit BAX-induced cell death in Nicotiana benthamiana leaves, and identifying host targets via yeast two-hybrid screening. The ∆VdCUE mutants showed reduced formation of melanized microsclerotia but no other obvious growth defects. Cotton plants infected with the ∆VdCUE mutants exhibited a significantly lower disease index and defoliation rate. VdCUE was confirmed to be secreted via a functional signal peptide, but it neither triggered cell death nor inhibited BAX-induced cell death. Three putative host targets were identified and supported by AI-based three-dimensional structural modeling, including tRNA-specific 2-thiouridylase, peptidyl-prolyl cis-trans isomerase, and 40S ribosomal protein, which may mediate VdCUE-dependent virulence regulation. These findings reveal VdCUE as a key virulence factor in V. dahliae, contributing to our understanding of its pathogenic mechanism.