The majority of cloned disease-resistance genes (R-genes) encode proteins with nucleotide-binding leucine-rich repeat domains (NLRs). R-genes tend to be physically clustered, and the structure of the cluster often facilitates expansion and sequence exchange amongst R-gene homologues (Luo et al., 2012). NLR proteins interact directly or indirectly with pathogens effectors, often triggering programmed cell death, also known as hypersensitive response (HR) at the infected sites (Dangl and Jones, 2001). HR may be triggered by pesticide molecules rather than pathogen effectors. For example, some tomato cultivars are sensitive to fenthion, developing toxic lesions after exposure to fenthion (Martin et al., 1994). Similarly, some lettuce germplasms are highly sensitive to triforine, an active ingredient in some commercial fungicides, with leaves showing wilting and necrosis 24 h after exposure to triforine (Figure 1a). Sensitivity to triforine in lettuce is controlled by a single locus (Tr) (Simko et al., 2011); however, the causal gene and its molecular mechanism remain unknown.

In this study, we used genome-wide SNPs to perform genome-wide association studies (GWAS) on sensitivity to triforine. The results showed a significant signal on chromosome 1 (Chr1) (Figure 1b). The candidate region spans 5087 kb and the prominent candidate genes include a TNL-encoding family. To confirm the GWAS results and to clone the Tr gene, we constructed an F₂ population by crossing a sensitive genotype (PI344074, Romaine) with an insensitive genotype (PI536839, Crisphead). Using Bulk Segregant Analysis + RNA-seq, the Tr gene was mapped to Chr1 (Figure 1c). Then, a total of 4639 individuals from an F₃ family were first screened using two far-end flanking markers and recombinants were further genotyped using markers in the candidate region. The Tr gene was ultimately fine mapped between markers AGH372 (F-primer:AACTTGACATTCTTCGGTG/R-primer:CTTCTGTTTAGTACAACATT) and AGH371 (F-primer:TTTAGATACCTATGACAACTT/R-primer:GTATATGTATCTATGTCTATGT), with an interval of approximately 140 kb (Figure 1d). This region of the reference genome (Lactuca sativa cv Salinas V8) has five genes, and all of them belong to a TIR type NLR-encoding family (TNL). Thus, we hypothesize that the Tr gene in sensitive parents was a homologue of this R-gene family.

To obtain the Tr gene, we used conserved primers to PCR amplify homologues of the R-gene family from the two parental genotypes, PCR products were cloned, and individual colonies were sequenced. Twenty-one and nine distinct Tr homologues were obtained from the sensitive and insensitive parents, respectively. Markers specific to each homologue were designed and used to screen the recombinants. The genetic analysis showed that only one (Tr-like109) homologue from the sensitive parent co-segregated with sensitivity to triforine. We also used the same pair of conserved primers to amplify homologues of the R-gene family from 29 sensitive accessions, PCR products were pooled and sequenced using Illumina Hiseq2500 platform. Similarly, PCR products from 46 insensitive individuals were also sequenced. Nine SNPs specific to the sensitive pool were found, which all present in the homologue Tr-like109. Therefore, the Tr-like109 gene is very likely the candidate for the Tr. Indeed, transformation of the Tr-like109 gene into the insensitive accession changed its reaction to triforine from insensitive to sensitive in the transformants (Figure 1e). On the other hand, three CRISPR knock-out lines of the Tr-like109 gene in the sensitive accession changed its sensitive reaction to insensitive, confirming that Tr-like109 is the Tr gene encoding sensitivity (susceptibility) to triforine (Figure 1f).

Tr transcripts were detected at multiple developmental stages in sensitive individuals. The expression of the Tr gene increased with leaf age in the first month after germination, then the increase slowed and the expression peaked in the second month, and maintained a high expression level for at least one more month (Figure 1h). We also analysed the expression of genes associated with disease resistance in parents, complementary and knock-out line after treatment with triforine. The Tr gene was rapidly up-regulated after treatment with triforine, and similar upregulation was also found for some genes associated with disease-resistance response (Figure 1i–j).

To verify that the sensitive response to triforine followed the same pathway as the HR in disease-resistance, we knocked out the EDS1 (LG1_140621) gene in the sensitive accession, which is required in the resistance pathway for TNL proteins. The homozygous eds1 mutants were insensitive to triforine (Figure 1g). We, therefore, conclude that the Tr gene triggers the HR response through the disease-resistance pathway. Next, we investigated whether the accumulation of ROS was associated with sensitivity to triforine. DAB, NBT as well as H₂O₂ content showed that the accumulation of ROS in the leaves originating from triforine-sensitive individuals were higher than leaves originating from triforine-insensitive individuals after triforine treatment (Figure 1k–l). The enzymatic activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were considerably higher in the sensitive genotype than in the insensitive genotypes (Figure 1m). Sensitivity to triforine was alleviated if the sensitive plants were pretreated with the ROS-inhibitor diphenyleneiodonium chloride (Figure 1k).

We used primers specific to the Tr gene to amplify PCR products from 817 lettuce accessions (203 cultivated lettuce (Lactuca sativa) and 614 wild lettuce (Lactuca serriola)). As expected, PCR products were obtained from all sensitive accessions, including 26 cultivated and six wild accessions. PCR products were also obtained from two insensitive accessions. The Tr sequences from all sensitive lettuce cultivars and two sensitive L. serriola accessions (CGN17427, CGN21383) were identical (Figure 1n). Therefore, the Tr gene likely originated from L. serriola and underwent at least five sequence exchanges (SE) during domestication or introgression (Figure 1o).

In this study, the Tr gene was identified through GWAS and map-based cloning. The candidate region contains a large R-gene family, which made the identification of the candidate gene challenging. We exhaustively sequenced the R-gene family in the two parents, and a large segregating population was used to narrow down the candidate gene, which facilitated the following process of gene verification. The Tr gene, encoding extreme sensitivity to triforine, has potential applications in plant biotechnology. For example, the Tr gene, if included in a transformation vector, can be used for larger-scale selection for marker-free individuals. It can be also used as a selection marker in mutagenesis to study the HR signalling transduction pathway.

大多数克隆的抗病基因（R基因）编码具有核苷酸结合富亮氨酸重复结构域（NLR）的蛋白质。R-基因倾向于在物理上成簇，簇的结构通常有利于R-基因同源物之间的扩展和序列交换（Luo et al ., 2012）。NLR 蛋白直接或间接与病原体效应器相互作用，通常在感染部位引发程序性细胞死亡，也称为过敏反应 (HR) (Dangl and Jones, 2001）。HR 可能由农药分子而非病原体效应物触发。例如，一些番茄品种对倍硫磷敏感，在接触倍硫磷后会出现毒性损伤（Martin等人，1994 年）。同样，一些莴苣种质对曲福林高度敏感，曲福林是一些商业杀菌剂中的一种活性成分，叶片在接触曲福林后 24 小时出现萎蔫和坏死（图 1a）。生菜中对三福林的敏感性由一个基因座 ( Tr ) 控制 (Simko et al ., 2011 )；然而，致病基因及其分子机制仍然未知。

在这项研究中，我们使用全基因组 SNP 对三福林敏感性进行全基因组关联研究 (GWAS)。结果显示1号染色体（Chr1）上有显着信号（图1b）。候选区域跨越 5087 kb，突出的候选基因包括一个 TNL 编码家族。为了确认GWAS结果并克隆Tr基因，我们通过将敏感基因型（PI344074，Romaine）与不敏感基因型（PI536839，Crisphead）杂交来构建F _2群体。使用 Bulk Segregant Analysis + RNA-seq，将Tr基因定位到 Chr1（图 1c）。然后，共有 4639 个人来自 F ₃首先使用两个远端侧翼标记筛选家族，并使用候选区域中的标记进一步对重组体进行基因分型。Tr基因最终在标记 AGH372（F-引物：AACTTGACATTCTTCGGTG/R-引物：CTTCTGTTTAGTACAACATT）和 AGH371（F-引物：TTTAGATACTATGACAACTT/R-引物：GTATATGTATCTATGTCTATGT）之间精细定位，间隔约为 140 kb（图 1d） . 参考基因组（ Lactuca sativa cv Salinas V8）的这个区域有五个基因，它们都属于TIR型NLR编码家族（TNL）。因此，我们假设敏感父母中的Tr基因是这个R基因家族的同源物。

为了获得Tr基因，我们使用保守引物对来自两个亲本基因型的R基因家族的同源物进行 PCR 扩增，克隆 PCR 产物，并对单个菌落进行测序。分别从敏感和不敏感的父母获得了21 个和 9 个不同的Tr同源物。对每个同源物特异的标记被设计并用于筛选重组体。遗传分析表明，来自敏感亲本的只有一个（Tr-like109）同源物对曲福林敏感共分离。我们还使用同一对保守引物来扩增R的同源物- 来自 29 个敏感种质的基因家族，使用 Illumina Hiseq2500 平台汇集和测序 PCR 产物。同样，还对来自 46 个不敏感个体的 PCR 产物进行了测序。发现了九个特定于敏感池的 SNP，它们都存在于同源物Tr-like109中。因此，Tr-like109基因很可能是Tr的候选者。实际上，将Tr-like109基因转化为不敏感种质将其对三福林的反应从转化体中的不敏感变为敏感（图 1e）。另一方面，敏感种质中Tr-like109基因的三个CRISPR敲除系将其敏感反应变为不敏感，证实了Tr-like109是编码对三福林敏感性（易感性）的Tr基因（图 1f）。

在敏感个体的多个发育阶段检测到Tr转录物。Tr基因的表达在萌发后的第一个月随着叶龄的增加而增加，然后在第二个月增加放缓并达到峰值，并在至少一个月内保持高表达水平（图1h）。我们还分析了用曲福林治疗后父母、互补系和敲除系中与抗病性相关的基因的表达。Tr基因在用 triforine 治疗后迅速上调，并且对于一些与抗病反应相关的基因也发现了类似的上调（图 1i-j）。

为了验证对曲福林的敏感反应在抗病性中遵循与 HR 相同的途径，我们敲除敏感种质中的EDS1 ( LG1_140621 ) 基因，这是 TNL 蛋白抗性途径所必需的。纯合eds1突变体对三福林不敏感（图1g）。因此，我们得出结论，Tr基因通过抗病途径触发 HR 反应。接下来，我们研究了 ROS 的积累是否与对曲福林的敏感性有关。DAB、NBT 以及 H ₂ O ₂含量表明，在triforine处理后，来自triforine敏感个体的叶子中ROS的积累高于来自triforine不敏感个体的叶子（图1k-l）。超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）的酶活性在敏感基因型中显着高于在不敏感基因型中（图1m）。如果敏感植物用 ROS 抑制剂二苯碘鎓氯化物预处理，则对三福林的敏感性得到缓解（图 1k）。

我们使用特异于Tr基因的引物从 817 个莴苣种质（203 个栽培莴苣 ( Lactuca sativa ) 和 614 个野生莴苣 ( Lactuca serriola )）中扩增 PCR 产物。正如预期的那样，PCR 产物来自所有敏感种质，包括 26 个栽培种质和 6 个野生种质。PCR产物也从两个不敏感的种质中获得。来自所有敏感莴苣品种和两个敏感L. serriola种质（CGN17427，CGN21383）的Tr序列是相同的（图 1n）。因此，Tr基因可能起源于L. serriola并且在驯化或渗入期间经历了至少五次序列交换（SE）（图1o）。

在本研究中，通过 GWAS 和基于图谱的克隆鉴定了Tr基因。候选区域包含一个大的R基因家族，这使得候选基因的鉴定具有挑战性。我们对两个亲本中的R基因家族进行了详尽的测序，并使用了一个大的分离群体来缩小候选基因的范围，这有利于后续的基因验证过程。编码对三福林极度敏感的Tr基因在植物生物技术中具有潜在应用。例如，Tr基因，如果包含在转化载体中，可用于大规模选择无标记个体。它也可以用作诱变中的选择标记，以研究 HR 信号转导途径。