Plant cell surface pattern-recognition receptors (PRRs) mount pattern-triggered immunity (PTI) by recognizing the typical molecular structures of pathogens, termed pathogen-associated molecular patterns (PAMPs), providing the first line of defence against various phytopathogens. Flagellin-sensing 2 (FLS2) of Arabidopsis thaliana, which perceives conserved epitopes (flg22) in the N-terminus of bacterial flagellin, was the first PRR to be identified (Gomez-Gomez and Boller, 2000). FLS2 homologues exist in most higher plants, but they differ in their recognition specificity. For example, tomato FLS2 can recognize flg15^Eco derived from Escherichia coli, but Arabidopsis FLS2 cannot (Robatzek et al., 2007). Flg22^agro of Agrobacterium tumefaciens avoids perception by most plants, whereas FLS2^XL recently identified in wild grape can perceive this obstinate flagellin epitope. The interspecies transfer of FLS2^XL can alter the specificity of flagellin perception in the recipient plant and enhance its resistance to A. tumefaciens (Fürst et al., 2020).

The genome of allotetraploid tobacco Nicotiana benthamiana possesses two highly similar FLS2 genes (95.2% identity in coding sequences), NbFLS2-1 (Niben101Scf03455g01008), and NbFLS2-2 (Niben101Scf01785g10011; Bombarely et al., 2012). We designed three single-guide RNAs (sgRNAs) to target both NbFLS2-1 and NbFLS2-2 (sgRNA1 and sgRNA3) or NbFLS2-1 (sgRNA2). The sequences of AtU6::sgRNAs combined with 35S::Cas9 were inserted into the pCambia1300 vector (Appendix S1). Genetic transformations of N. benthamiana were performed. Three T1 lines, KO1&2 (transgenic sgRNA1 line, knockout of NbFLS2-1 and NbFLS2-2), KO1 (sgRNA2, knockout of NbFLS2-1), and KO2 (sgRNA3, knockout of NbFLS2-2) were chosen because they were Cas9-free and carried homozygous frame-shift mutations. Although sgRNA2 also targeted NbFLS2-2, and sgRNA3 had only two mismatches with NbFLS2-1, these sgRNAs did not result in mutations of NbFLS2-2 in KO1 and NbFLS2-1 in KO2, respectively. The frame-shift mutations generated by CRISPR/Cas9 gene-editing lead to translation termination at the N-termini (102nd–254th amino acids) of the corresponding NbFLS2s, suggesting their complete loss of function (Figure 1a–d). Furthermore, qRT-PCR results showed that the expression levels of mutated FLS2 genes were lower than that of wild type (Figure 1e).

To verify the NbFLS2s' loss of function, we performed three typical flagellin response experiments with leaf discs or seedlings of wild- type and KO lines. After flg22^Psy (Pseudomonas syringae) treatments, wild type and KO1 generated reactive oxygen species (ROS) bursts (Figure 1f), accumulated activated MPK3/6 (Figure 1g), and exhibited significant growth inhibition (Figure 1h, i). In contrast, there were no obvious responses by KO1&2 and KO2. In addition, transient expression with 35S::gNbFLS2 and 35S::gNbFLS2:GFP (gNbFLS2, the full-length genomic DNA sequences of NbFLS2s; GFP, coding sequence of green fluorescent protein) revealed that 35S::gNbFLS2-2 and 35S::gNbFLS2-2:GFP can recover the ability to generate ROS bursts in KO1&2 after flg22^Psy treatment, but 35S::gNbFLS2-1 and 35S::gNbFLS2-1:GFP cannot (Figure 1j). Moreover, immunoblotting detected the accumulation of NbFLS2-2-GFP (~210 kDa) but did not detect NbFLS2-1-GFP (Figure 1k). RT-PCR and qRT-PCR results demonstrated the expression of two gNbFLS2s in transient assay (Figure S1a–c). Furthermore, no accumulation of target protein was observed in transient expression of the coding sequence of NbFLS2-1 (Figure 1k). Therefore, the lack of function of NbFLS2-1 may be due to translational level regulation.

Flagellin-induced ROS burst assays using N. benthamiana leaves that transiently express heterologous FLS2s represent a robust and convenient experimental method for identifying the function of FLS2s, but the presence of functional endogenous FLS2s, which can recognize a range of flagellin epitopes and/or may interact with downstream elements, limits the method's application. The NbFLS2 double-mutant generated here can help overcome this limitation. We cloned the genomic DNA sequences of FLS2 homologues from multiple plants and generated binary vectors with the 35S::gFLS2:GFP construct. Their transient expression in KO1&2 revealed that 29 GFP-fused FLS2s (GenBank accession No. ON556647–ON556668, MH079052, MH079054, MH079055, MH079056 and MH079058) with molecular weights of approximately 200 to 210 kDa were successfully accumulated (Figure 1l). The leaf discs of KO1&2 expressing heterologous FLS2s were challenged with three flagellin epitopes (flg22^Psy, flg15^Eco, and flg22^Agro) in ROS burst assays. Four FLS2 homologues failed to confer KO1&2 the ability to respond to flg22^Psy, among which FLS2 from Nelumbo nucifera, Kalanchoe laxiflora and Ginkgo biloba lacked the 14–17th, 4–6th, and 26 & 28th LRR motifs, respectively, whereas Morus alba FLS2, lacking the 15th LRR motif and Populus euphratica FLS2, lacking the 26th LRR motif, still recognized flg22^Psy (Figure 1m). In addition, there was a difference in flg15^Eco recognition among poplar FLS2s, i.e., FLS2 from P. trichocarpa and P. euphratica recognized flg15^Eco, but FLS2s from five other poplar species did not (Figure 1n). Furthermore, FLS2 from Quercus variabilis and Trachelospermum jasminoides are highly sensitive to flg22^Agro (Figure 1o).

Here, we used CRISPR/Cas9 technology to knock out two FLS2 genes in N. benthamiana both separately and together, and we found that only NbFLS2-2 contributed to the recognition of flg22^Psy. In addition, we combined transient expression and ROS burst assays to rapidly validate the FLS2 flagellin epitope recognition spectrum from 29 plant species in an N. benthamiana FLS2 double-mutant. This convenient approach, combined with a large number of FLS2 homologues currently revealed by plant genome sequencing, will facilitate screening of the FLS2s that can trigger broad-spectrum resistance or resistance targeting specific pathogens, and investigating co-evolutionary dynamics of plant FLS2 and bacterial flagellin in a given environment.

植物细胞表面模式识别受体（PRR）通过识别病原体的典型分子结构（称为病原体相关分子模式（PAMP））来启动模式触发免疫（PTI），提供针对各种植物病原体的第一道防线。拟南芥的鞭毛蛋白感应 2 (FLS2) 感知细菌鞭毛蛋白 N 末端的保守表位 (flg22)，是第一个被鉴定的 PRR（Gomez-Gomez 和 Boller， 2000 ）。 FLS2同源物存在于大多数高等植物中，但它们的识别特异性不同。例如，番茄FLS2可以识别源自大肠杆菌的flg15 ^Eco ，但拟南芥FLS2不能(Robatzek等， 2007 )。根癌农杆菌的 Flg22 ^agro避免被大多数植物感知，而最近在野生葡萄中发现的 FLS2 ^XL可以感知这种顽固的鞭毛蛋白表位。 FLS2 ^XL的种间转移可以改变受体植物中鞭毛蛋白感知的特异性，并增强其对根癌农杆菌的抗性（Fürst等人， 2020 ）。

异源四倍体烟草本塞姆氏烟草的基因组拥有两个高度相似的FLS2基因（编码序列同一性95.2%）， NbFLS2-1 （ Niben101Scf03455g01008 ）和NbFLS2-2 （ Niben101Scf01785g10011 ；Bombarely等， 2012 ）。我们设计了三种单向导RNA（sgRNA）来靶向NbFLS2-1和NbFLS2-2 （sgRNA1和sgRNA3）或NbFLS2-1 （sgRNA2）。将AtU6::sgRNA与35S::Cas9组合的序列插入 pCambia1300 载体（附录 S1）。对本塞姆氏烟草进行了遗传转化。选择三个 T1 系：KO1&2（转基因 sgRNA1 系，敲除NbFLS2-1和NbFLS2-2 ）、KO1（sgRNA2，敲除NbFLS2-1 ）和 KO2（sgRNA3，敲除NbFLS2-2 ），因为它们是Cas9 -游离并携带纯合移码突变。尽管sgRNA2也靶向NbFLS2-2 ，并且sgRNA3与NbFLS2-1仅存在两个错配，但这些sgRNA并未分别导致KO1中的NbFLS2-2和KO2中的NbFLS2-1的突变。 CRISPR/Cas9基因编辑产生的移码突变导致相应NbFLS2的N末端（第102-254个氨基酸）翻译终止，表明它们完全丧失功能（图1a-d）。此外，qRT-PCR结果显示突变FLS2基因的表达水平低于野生型（图1e）。

为了验证NbFLS2 的功能丧失，我们用野生型和 KO 品系的叶盘或幼苗进行了三个典型的鞭毛蛋白反应实验。经过 flg22 ^Psy （丁香假单胞菌）处理后，野生型和 KO1 产生活性氧 (ROS) 爆发（图 1f），积累激活的 MPK3/6（图 1g），并表现出显着的生长抑制（图 1h，i）。相比之下，KO1&2和KO2没有明显反应。此外，用35S::gNbFLS2和35S::gNbFLS2:GFP （ gNbFLS2 ， NbFLS2s的全长基因组 DNA 序列； GFP ，绿色荧光蛋白的编码序列）进行瞬时表达表明35S::gNbFLS2-2和35S： :gNbFLS2-2:GFP在 flg22 ^Psy处理后可以恢复在 KO1&2 中产生 ROS 爆发的能力，但35S::gNbFLS2-1和35S::gNbFLS2-1:GFP不能（图 1j）。此外，免疫印迹检测到 NbFLS2-2-GFP (~210 kDa) 的积累，但未检测到 NbFLS2-1-GFP（图 1k）。 RT-PCR 和 qRT-PCR 结果证明了瞬时测定中两个gNbFLS2的表达（图 S1a-c）。此外，在NbFLS2-1编码序列的瞬时表达中没有观察到靶蛋白的积累（图 1k）。因此， NbFLS2-1功能的缺失可能是由于翻译水平调节所致。

使用瞬时表达异源FLS2 的本塞姆氏烟草叶子进行的鞭毛蛋白诱导的 ROS 爆发测定代表了一种用于鉴定 FLS2 功能的稳健且方便的实验方法，但是功能性内源 FLS2 的存在（可以识别一系列鞭毛蛋白表位和/或可能）与下游元素交互，限制了该方法的应用。这里生成的NbFLS2双突变体可以帮助克服这一限制。我们克隆了来自多种植物的FLS2同源物的基因组 DNA 序列，并使用35S::gFLS2:GFP构建体生成了二元载体。它们在 KO1&2 中的瞬时表达表明，成功积累了 29 个分子量约为 200 至 210 kDa 的 GFP 融合 FLS2（GenBank 登录号 ON556647-ON556668、MH079052、MH079054、MH079055、MH079056 和 MH079058）（图 1l）。在 ROS 爆发测定中，用三个鞭毛蛋白表位（flg22 ^Psy 、 flg15 ^Eco和 flg22 ^Agro ）攻击表达异源 FLS2 的 KO1 和 2 的叶盘。四种FLS2同源物未能赋予KO1&2响应flg22 ^Psy的能力，其中来自荷花、长寿花和银杏的FLS2分别缺乏第14-17、4-6、26和28个LRR基序，而桑树FLS2 ，缺少第15个LRR基序和胡杨FLS2，缺少第26个LRR基序，仍然识别flg22 ^Psy （图1m）。此外，杨树FLS2（即毛果杨和杨树的FLS2）对flg15 ^Eco的识别存在差异。 胡杨树识别 flg15 ^Eco ，但其他五种杨树的 FLS2 不识别（图 1n）。此外，来自栓皮栎和络石的FLS2 对 flg22 ^Agro高度敏感（图 1o）。

在这里，我们利用CRISPR/Cas9技术分别敲除本塞姆氏烟草中的两个FLS2基因，发现只有NbFLS2-2有助于识别flg22 ^Psy 。此外，我们结合瞬时表达和 ROS 爆发测定来快速验证本塞姆氏烟草 FLS2双突变体中 29 种植物的 FLS2 鞭毛蛋白表位识别谱。这种便捷的方法，与目前通过植物基因组测序揭示的大量FLS2同源物相结合，将有助于筛选能够引发广谱抗性或针对特定病原体的抗性的FLS2，并研究植物FLS2和细菌鞭毛蛋白的共同进化动力学在给定的环境中。