Deciphering the transcriptional landscape of NLR genes responding to Verticillium dahliae infection in cotton

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.

黄萎病（Verticillium wilt, VW）是由大丽轮枝菌（Verticillium dahliae）引发的全球性真菌病害，对全球棉花生产造成了严重威胁。核苷酸结合域与富亮氨酸重复（Nucleotide-binding domain and Leucine-rich Repeat, NLR）基因在植物效应子触发免疫（effector-trigged immunity, ETI）过程中发挥核心调控作用，但目前棉花中已完成功能表征的NLR基因数量极少。本研究针对陆地棉（Gossypium hirsutum）中的NLR基因开展系统性解析：通过多种方法完成全基因组NLR基因鉴定；与陆地棉的祖先物种亚洲棉（G. arboreum）和雷蒙德氏棉（G. raimondii）的已鉴定NLR基因进行比较分析，以揭示其演化规律；利用第三代长读长测序技术获取全长NLR转录本亚型；分析组织特异性、品种特异性以及大丽轮枝菌诱导的可变剪接（alternative splicing, AS）事件；同时解析不同抗病与感病棉花品种中，NLR基因在大丽轮枝菌侵染后的表达模式。本研究共在亚洲棉、雷蒙德氏棉和陆地棉中分别鉴定出302、289和511个NLR基因；陆地棉在异源四倍体化事件后，其At与Dt亚基因组中均出现了显著的NLR基因得失动态。尽管约四分之一的NLR基因为典型结构型NLR，仍有近半数NLR基因仅包含NBS与LRR结构域。可变剪接在NLR基因中频繁发生，大量全长NLR转录本及可变剪接事件呈现组织或品种特异性，并可被大丽轮枝菌侵染诱导。相较于其他功能基因，大丽轮枝菌侵染后差异表达的NLR基因数量显著更多，印证了NLR基因在棉花响应大丽轮枝菌侵染过程中的关键作用。超过半数的部分同源NLR基因对偏好表达Dt亚基因组的同源拷贝，且该表达偏好性似乎不受大丽轮枝菌侵染的影响。共有95个NLR基因位于与大丽轮枝菌抗性相关的数量性状位点（quantitative trait locus, QTL）区域内，其中21个在抗病与感病品种间存在显著表达差异。通过沉默Ghir_A04G002130与Ghir_D01G011600基因后，棉花植株的病害症状显著加重，进一步验证了NLR基因在棉花抗大丽轮枝菌侵染中的功能。本研究结果为解析棉花NLR基因介导的抗大丽轮枝菌防御反应提供了极具价值的理论参考。