CRISPR/Cas9-mediated generation of fls2 mutant in Nicotiana benthamiana for investigating the flagellin recognition spectrum of diverse FLS2 receptors.
CRISPR/Cas9-mediated generation of fls2 mutant in Nicotiana benthamiana for investigating the flagellin recognition spectrum of diverse FLS2 receptors.
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CRISPR/Cas9介导在本塞姆氏烟草中产生fls2突变体，用于研究不同FLS2受体的鞭毛蛋白识别谱
DOI：
10.1111/pbi.13898
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发表时间：
2022-10
影响因子：
13.8
通讯作者：
Cheng, Qiang
中科院分区：
文献类型：
--
作者：
Wu, Ling;Xiao, Hongju;Zhao, Lijuan;Cheng, Qiang
关键词：
植物细胞表面模式识别受体（PRRs）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））来建立模式触发免疫（PTI），为抵御各种植物病原体提供第一道防线。拟南芥鞭毛蛋白传感2 （FLS2）在细菌鞭毛蛋白n端感知保守表位（flg22），是第一个被发现的PRR （Gomez-Gomez and 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可以识别来自大肠杆菌的flg15，但拟南芥FLS2不能（Robatzek et al., 2007）。农杆菌的Flg22避免了大多数植物的感知，而最近在野生葡萄中发现的FLS2可以感知这种顽固的鞭毛蛋白表位。FLS2的种间转移可以改变受体植物对鞭毛蛋白感知的特异性，增强其对瘤胃拟南杆菌的抗性（F€first et al., 2020）。同种异体四倍体烟草Nicotiana benthamiana基因组具有两个高度相似的FLS2基因，NbFLS2-1 （Niben101Scf03455g01008）和NbFLS22 （Niben101Scf01785g10011; Bombarely et al., 2012）。我们设计了三种单导rna （sgRNAs）来靶向NbFLS2-1和NbFLS2-2 （sgRNA1和sgRNA3）或NbFLS2-1 （sgRNA2）。将AtU6::sgRNAs序列与35S:: Cas9结合插入到pCambia1300载体中（附录S1）。对benthamiana进行了遗传转化。选择3个T1系，KO1和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)，因为它们不含cas9并且携带纯合的移框突变。虽然sgRNA2也靶向NbFLS2-2，并且sgRNA3与NbFLS2-1只有两个错配，但这些sgRNAs并没有分别导致KO1中NbFLS2-2和KO2中NbFLS2-1的突变。CRISPR/ Cas9基因编辑产生的移框突变导致相应NbFLS2s的n端（第102 - 254个氨基酸）的翻译终止，表明其功能完全丧失（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NbFLS2s的功能丧失，我们在野生型和KO系的叶片或幼苗上进行了三个典型的鞭毛蛋白响应实验。经过flg22（丁香假单胞菌）处理后，野生型和KO1产生活性氧（ROS）爆发（图1f），积累活化的MPK3/6（图1g），并表现出明显的生长抑制（图1h， i）。而ko1、KO2和KO2无明显反应。此外，用35S::gNbFLS2和35S::gNbFLS2:GFP （NbFLS2s基因组全长DNA序列gNbFLS2；绿色荧光蛋白编码序列GFP）瞬时表达发现，经过flg22处理后，35S::gNbFLS2-2和35S::gNbFLS2-2:GFP可以恢复ko1和ko2中产生ROS爆发的能力，而35S::gNbFLS2-1和35S::gNbFLS2-1:GFP不能（图1j）。此外，免疫印迹检测到NbFLS2-2-GFP的积累（~210 kDa），但未检测到NbFLS2-1GFP（图1k）。RT-PCR和qRT-PCR结果显示瞬时检测中有两个gNbFLS2s的表达（图S1a-c）。此外，NbFLS2-1编码序列的瞬时表达未观察到靶蛋白的积累（图1k）。因此，NbFLS2-1功能缺失可能与翻译水平调控有关。利用瞬时表达异种FLS2s的benthamiana叶片进行鞭毛蛋白诱导的ROS爆发试验是鉴定FLS2s功能的一种可靠且方便的实验方法，但功能性内源FLS2s的存在限制了该方法的应用，这些FLS2s可以识别一系列鞭毛蛋白表位和/或可能与下游元件相互作用。这里产生的NbFLS2双突变体可以帮助克服这一限制。我们克隆了多种植物FLS2同源物的基因组DNA序列，并构建了35S::gFLS2:GFP二元载体。它们在ko1和ko2中的瞬时表达显示29个gfp融合的FLS2s (GenBank登录号：成功积累了分子量约为200 ~ 210 kDa的ON556647-ON556668、MH079052、MH079054、MH079055、MH079056和MH079058（图11）。用3个鞭毛蛋白表位（flg22、flg15和flg22）刺激表达异种FLS2s的KO1&2叶片。4个FLS2同源物未能使ko1和ko2对flg22产生应答能力，其中莲叶、松叶和银杏的FLS2分别缺乏14 - 17、4 - 6、26和28个LRR基序，而桑树FLS2缺乏第15个LRR基序，胡杨FLS2缺乏第26个LRR基序，但仍能识别flg22（图1m）。此外，还有
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 derived from Escherichia coli, but Arabidopsis FLS2 cannot (Robatzek et al., 2007). Flg22 of Agrobacterium tumefaciens avoids perception by most plants, whereas FLS2 recently identified in wild grape can perceive this obstinate flagellin epitope. The interspecies transfer of FLS2 can alter the specificity of flagellin perception in the recipient plant and enhance its resistance to A. tumefaciens (F€ urst 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 NbFLS22 (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 wildtype and KO lines. After flg22 (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 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 NbFLS2-1GFP (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, flg15 and flg22) in ROS burst assays. Four FLS2 homologues failed to confer KO1&2 the ability to respond to flg22, 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 (Figure 1m). In addition, there was
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影响因子：
18
作者：
Fuerst, Ursula;Zeng, Yi;Felix, Georg
通讯作者：
Felix, Georg
影响因子：
3.5
作者：
Bombarely, Aureliano;Rosli, Hernan G.;Martin, Gregory B.
通讯作者：
Martin, Gregory B.
影响因子：
16
作者：
Gómez-Gómez, L;Boller, T
通讯作者：
Boller, T
影响因子：
5.1
作者：
Robatzek, Silke;Bittel, Pascal;Boller, Thomas
通讯作者：
Boller, Thomas