Verticillium wilt (VW), a widespread disease caused by the fungal pathogen Verticillium dahliae, poses a significant challenge to global cotton production. Nucleotide-binding domain and Leucine-rich Repeat (NLR) genes play crucial roles in effector-trigged immunity (ETI) in plants. However, very few NLRs have been functionally characterised in cotton. In this study, we comprehensively investigate NLRs in Gossypium hirsutum by genome-wide identification of NLRs using multiple methodologies, exploring their evolution through comparative analysis with the NLRs identified in the ancestor species (G. arboreum and G. raimondii) of G. hirsutum, generating full-length NLR transcript isoforms using the third-generation long-read sequencing technology, analysing tissue- and cultivar-specific as well as V. dahliae induced alternative splicing events, and profiling the expression patterns of NLRs in response to V. dahliae infection in resistant or susceptible cotton cultivars. A total of 302, 289, and 511 NLRs were identified in G. arboreum, G. raimondii, and G. hirsutum, respectively, with evident loss and gain of NLRs in both At and Dt subgenomes of G. hirsutum after the tetraploidisation event. Despite about a quarter of the NLRs are canonical ones, about half of the NLRs contain only the NBS and LRR domains. Alternative splicing (AS) was frequently observed in NLRs. Many full-length NLR transcripts and AS events were found to be tissue- or cultivar-specific and induced by V. dahliae infection. Upon V. dahliae infection, more NLRs than other genes were differentially expressed, indicating the importance of NLRs in response to V. dahliae infection. More than half homoeologous NLR pairs showed a preferential expression of the Dt subgenome homoeolog, which seemed to be not impacted by V. dahliae infection. A total of 95 NLRs are located at the QTLs associated with V. dahliae resistance, 21 of them showed significant differences between resistant and susceptible cultivars. The function of NLRs in cotton resistance to V. dahliae was further demonstrated by enhanced disease symptoms observed in cotton plants with silenced Ghir_A04G002130 or Ghir_D01G011600. These findings provide invaluable insights into the landscape of cotton NLRs in defence responses against V. dahliae infection.
