Verticillium dahliae is a soil-borne hemi-biotrophic fungal pathogen that inflicts major losses in agricultural production worldwide. In this study, reference genomes are determined for more aggressive and defoliating isolate V991 and non-defoliating isolate 1cd3-2. Transcriptome analysis combined with comparative genomics showed that genes potentially associated with pathogen virulence are induced at the late stage of infection (Stage II), accompanied by the burst of reactive oxygen species (ROS), and more genes involved in defense response in cotton. A V991-specific virulence gene SP3 was identified and highly expressed during the Stage II. Knock-out of SP3 attenuates pathogenicity, with less ROS produced in cotton seedlings. To further investigate this, polyethyleneimine coated MXene quantum dots (PEI-MQDs), which possess the ability to remove ROS, are employed. Cotton seedlings maintain ROS homeostasis with enhanced peroxidase, catalase, and glutathione peroxidase activities after exogenous treatment with PEI-MQDs and show significantly improved tolerance to V.dahliae.