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Despite the established significance of WRKY proteins and phenylpropanoid metabolism in plant immunity, how WRKY proteins modulate aspects of the phenylpropanoid pathway remains undetermined. To understand better the role of WRKY proteins in plant defence, we identified a cotton (Gossypium hirsutum) protein, GhWRKY41, that is, universally and rapidly induced in three disease-resistant cotton cultivars following inoculation with the plant pathogenic fungus, Verticillium dahliae. We show that overexpression of GhWRKY41 in transgenic cotton and Arabidopsis enhances resistance to V. dahliae, while knock-down increases cotton more susceptibility to the fungus. GhWRKY41 physically interacts with itself and directly activates its own transcription. A genome-wide chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), in combination with RNA sequencing (RNA-seq) analyses, revealed that 43.1% of GhWRKY41-binding genes were up-regulated in cotton upon inoculation with V. dahliae, including several phenylpropanoid metabolism master switches, receptor kinases, and disease resistance-related proteins. We also show that GhWRKY41 homodimer directly activates the expression of GhC4H and Gh4CL, thereby modulating the accumulation of lignin and flavonoids. This finding expands our understanding of WRKY-WRKY protein interactions and provides important insights into the regulation of the phenylpropanoid pathway in plant immune responses by a WRKY protein.

Verticillium dahliae is a soil-borne hemi-biotrophic fungal pathogen and inflicts major losses in agricultural production worldwide. Although the pathogenicity of the fungus and plant immunity have been extensively studied, how cotton leaf necrosis is induced by defoliating strains of V. dahliae is poorly understood. In this study, reference-grade genomes were determined for two representative V. dahliae isolates: V991, which is defoliating and aggressive, and 1cd3-2, which is non-defoliating and has weak pathogenicity. Transcriptome analysis showed that cotton resistance to V. dahliae mainly depends on cell wall structure or physical barrier generation in the early stage of infection (3-9 days post inoculation, dpi). However, a large number of pathogenic factors could be detected from 12 dpi and accumulated quickly in cotton stems, accompanied by the burst of reactive oxygen species (ROS), leaf necrosis and defoliation. A V991 specific virulence gene SP3 was identified through comparative genome analysis and found to be highly expressed after colonization. Knock-out of SP3 clearly attenuated pathogenicity, with less ROS produced. These results indicate that disease symptoms in cotton may be due to abnormal immune activation and excessive ROS induced by the pathogen. To further investigate this, Polyethyleneimine coated MXene quantum dots (PEI-MQDs), a type of nano material that possesses the ability to remove ROS in vitro, were used. Cotton seedlings maintain ROS homeostasis with enhanced peroxidase (POD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities after exogenous treatment with PEI-MQDs and show significant tolerance to V. dahliae isolate V991. Our results suggest that excessive ROS and constitutive immune activation in cotton induced by the defoliating strain of V. dahliae is responsible for disease symptoms such as leaf necrosis, and PEI-MQDs application improve cotton tolerance to V. dahliae by maintaining ROS homeostasis.