Data from: TNL genes in peach: insights into the post-LRR domain

Background: Plants develop sustainable defence responses to pathogen attacks through resistance (R) genes contributing to effector-triggered immunity (ETI). TIR-NB-LRR genes (TNL genes) constitute a major family of ETI R genes in dicots. The putative functions or roles of the TIR, NB and LRR domains of the proteins they encode (TNLs) are well documented, but TNLs also have a poorly characterised C-terminal region, the function of which is unknown in most cases. We characterised this prevalent stress-response protein family in a perennial plant, using the genome of peach (Prunus persica), the model Prunus species. The first TNL gene from this genus to be cloned, the Ma gene, confers complete-spectrum resistance to root-knot nematodes (RKNs) and encodes a protein with a huge C-terminal region with five duplicated post-LRR (PL) domains. This gene was the cornerstone of this study. Results: We investigated the role of this C-terminal region, by first describing the frequency, distribution and structural characteristics of i) TNL genes and ii) their PL domains in the peach genome, using the v1.0 Sanger sequence together with the v2.0 sequence, which has better genome annotation due to the incorporation of transcriptomic data. We detected 195 predicted TNL genes from the eight peach chromosomes: 85 % of these genes mapped to chromosomes 1, 2, 7 and 8. We reconstructed the putative structure of the predicted exons of all the TNL genes identified, and it was possible to retrieve the PL domains among two thirds of the TNL genes. We used our predicted TNL gene sequences to develop an annotation file for use with the Gbrowse tool in the v2.0 genome. The use of these annotation data made it possible to detect transcribed PL sequences in two Prunus species. We then used consensus sequences defined on the basis of 124 PL domains to design specific motifs, and we found that the use of these motifs significantly increased the numbers of PL domains and correlative TNL genes detected in diverse dicot genomes. Based on PL signatures, we showed that TNL genes with multiple PL domains were rare in peach and the other plants screened. The five-PL domain pattern is probably unique to Ma and its orthologues within Prunus and closely related genera from the Rosaceae and was probably inherited from the common ancestor of these plants in the subfamily Spiraeoideae. Conclusions: The first physical TNL gene map for Prunus species can be used for the further investigation of R genes in this genus. The PL signature motifs are a complementary tool for the detection of TNL R genes in dicots. The low degree of similarity between PL domains and the neighbouring LRR exons and the specificity of PL signature motifs suggest that PL and LRR domains have different origins, with PL domains being specific to TNL genes, and possibly essential to the functioning of these genes in some cases. Investigations of the role of the oversized Ma PL region, in ligand binding or intramolecular interactions for example, may help to enrich our understanding of NB-LRR-mediated plant immunity to RKNs.

背景：植物通过参与效应子触发的免疫（effector-triggered immunity, ETI）的抗病（R）基因，构建针对病原菌侵染的可持续防御响应。TIR-NB-LRR基因（TNL genes）是双子叶植物中一类主要的ETI相关R基因家族。这类蛋白（TNLs）的TIR、NB和LRR结构域的推定功能已有充分研究，但TNL还存在一个研究不足的C端区域，多数情况下其功能仍未明确。本研究以蔷薇科李属模式物种桃（Prunus persica）的基因组为研究对象，解析了这一广泛存在的胁迫响应蛋白家族。该属首个被克隆的TNL基因——Ma基因，可赋予植物对根结线虫（root-knot nematodes, RKNs）的广谱抗性，其编码的蛋白带有一个巨大的C端区域，包含5个重复的LRR后（post-LRR, PL）结构域。该基因是本研究的核心基础。
结果：本研究首先借助整合了转录组数据、基因组注释更完善的桃基因组v2.0版本，同时结合v1.0 Sanger测序序列，解析了桃基因组中TNL基因及其PL结构域的分布频率、位置特征与结构特点，以此探究该C端区域的功能。本研究从桃的8条染色体中共鉴定得到195个预测的TNL基因，其中85%的基因定位在1、2、7、8号染色体上。我们对所有鉴定到的TNL基因的预测外显子结构进行了推定重构，其中三分之二的TNL基因可被检测到PL结构域。我们利用预测得到的TNL基因序列，为桃基因组v2.0版本开发了适配Gbrowse工具的注释文件。借助该注释数据，我们在2个李属物种中检测到了转录的PL序列。随后，我们基于124个PL结构域的保守序列设计了特异性基序，实验发现，使用该基序可显著提升在多种双子叶植物基因组中检测到的PL结构域及关联TNL基因的数量。基于PL结构域的特征序列，我们发现带有多个PL结构域的TNL基因在桃及其他筛选的植物中极为罕见。含有5个PL结构域的模式仅存在于Ma基因及其在李属、蔷薇科近缘属中的同源基因中，该特征大概率由这些植物在绣线菊亚科的共同祖先处遗传而来。
结论：本研究构建的首个李属植物TNL基因物理图谱，可用于该属抗病基因的后续研究。PL结构域特征基序可作为双子叶植物TNL型抗病基因检测的辅助工具。PL结构域与其相邻的LRR外显子之间的相似性极低，且PL特征基序具有特异性，这表明PL与LRR结构域起源不同，PL结构域为TNL基因所特有，且在部分场景中可能对TNL基因的功能至关重要。例如针对Ma基因超大PL区域的功能（如配体结合或分子内相互作用）开展研究，将有助于加深我们对NB-LRR介导的植物抗根结线虫免疫机制的理解。