LAUSR

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