Deciphering the transcriptional landscape of NLR genes responding to Verticillium dahliae infection in cotton (Gossypium hirsutm L.)

Nucleotide-binding domain and Leucine-rich Repeat (NLR) proteins play crucial roles in activating the innate immune system by recognizing pathogen effectors. Although several cotton NLRs have been implicated in resistance to Verticillium dahliae (Vd), a devastating fungal pathogen threatening global cotton production, the comprehensive landscape of NLRs response to Vd-infection, particularly across resistant and susceptible cotton cultivars, remaining largely unexplored. Here, we identified 511 NLR s in upland cotton ( Gossypium hirsutum L.) that were supported by at least two prediction algorithms. All 511 NLRs were transcriptionally active based on RNA sequencing (RNA-seq) data generated from roots of MCU5 (Vd-resistant) and Siokra1–4 (Vd-susceptible). Among them, 207 (40.5 %) were further supported by full-length transcripts obtained from the two cultivars via third-generation long-read RNA-seq. While many full-length NLRs were shared between MCU5 and Siokra1–4, a substantial number were cultivar-specific, including unique alternative splicing events, implying cultivar-specific transcriptional regulation and post-transcriptional modification of NLRs in response to Vd-infection. Analysis of homoeologous NLR pairs revealed that A t homoeologs were under relaxing purifying selection, whereas their corresponding D t homoeologs were more conserved and showed higher expression, possibly balancing the need for rapid immune response with evolutionary adaptability. Notably, NLRs might contribute more to basal resistance in MCU5 than in Siokra1–4. Functional validation demonstrated that silencing two specific NLRs significantly increased disease severity, underscoring their positive role in Vd-resistance. Collectively, this work provides a comprehensive overview of the cotton NLR repertoire, highlights cultivar-specific immune response, and offers a valuable resource for further functional and breeding studies of NLRs aiming at enhancing Vd-resistance in cotton.