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.\nLineage: The data deposited here are the raw long-read RNA sequencing results of MCU-5 (resistant to V. dahliae) and Siokra 1-4 (susceptible to V. dahliae). The tissues used were root and leaf of MCU-5, and 1-dpi root of Vd-infected MCU-5 and Siokra 1-4 plants. The RNA used in the experiment was isolated using the Maxwell® RSC Plant RNA Kit (Promega, Madison, USA) by following the manufacture’s instruction. Sequencing was done by Australian Genome Research Facility (AGRF, Victoria, Australia) using the Sequel Sequencing Kit 3.0 on the PacBio Sequel instrument, with a 20-hour movie time and 4-hour pre-extension time.

黄萎病（Verticillium wilt）是由大丽轮枝菌（Verticillium dahliae）引发的广谱真菌病害，对全球棉花生产造成了严峻挑战。核苷酸结合域与亮氨酸重复序列（Nucleotide-binding domain and Leucine-rich Repeat，简称NLR）基因在植物的效应子触发免疫（effector-trigged immunity，简称ETI）中发挥关键调控作用，但目前棉花中功能得到解析的NLR基因寥寥无几。本研究通过多种方法全基因组鉴定陆地棉（Gossypium hirsutum）中的NLR基因，系统性开展以下研究：与陆地棉的祖先物种——草棉（G. arboreum）和雷蒙德氏棉（G. raimondii）的NLR基因进行比较分析以解析其演化历程；利用第三代长读长测序技术获取全长NLR转录本异构体；分析NLR基因的组织特异性、品种特异性以及大丽轮枝菌诱导的可变剪接事件；同时检测不同抗病/感病棉花品种中NLR基因响应大丽轮枝菌侵染的表达模式。
本研究分别在草棉、雷蒙德氏棉和陆地棉中鉴定到302、289和511个NLR基因；陆地棉经异源四倍体化事件后，其At和Dt两个亚基因组中均出现了明显的NLR基因得失动态。尽管约四分之一的NLR基因为经典类型，但近半数NLR基因仅包含NBS和LRR结构域。可变剪接现象在NLR基因中广泛存在，大量全长NLR转录本和可变剪接事件呈现组织或品种特异性，且可被大丽轮枝菌侵染诱导。
相较于其他基因，响应大丽轮枝菌侵染时差异表达的NLR基因数量更多，表明NLR基因在棉花抗大丽轮枝菌侵染过程中具有重要意义。超过半数的部分同源NLR基因对偏好Dt亚基因组的同源拷贝表达，且该偏好性似乎不受大丽轮枝菌侵染的影响。共有95个NLR基因定位于与大丽轮枝菌抗性相关的数量性状基因座（quantitative trait locus，简称QTL）区域，其中21个基因在抗病与感病品种间存在显著表达差异。通过沉默Ghir_A04G002130和Ghir_D01G011600后棉花植株的病害症状加重，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响应大丽轮枝菌侵染的防御反应中NLR基因的全貌提供了宝贵的理论依据。
数据集说明：本数据集所存储的原始数据为抗大丽轮枝菌品种MCU-5和感病品种Siokra 1-4的长读长RNA测序结果。测序所用组织包括MCU-5的根和叶，以及经大丽轮枝菌侵染1天（1-dpi）的MCU-5和Siokra 1-4的根。实验所用RNA采用Maxwell® RSC Plant RNA Kit（Promega公司，美国麦迪逊），严格按照制造商说明书进行分离纯化。测序工作由澳大利亚基因组研究设施（Australian Genome Research Facility, AGRF，澳大利亚维多利亚州）完成，使用PacBio Sequel测序平台及Sequel Sequencing Kit 3.0，测序时采用20小时的测序时长与4小时的预延伸时长。