Interaction Analysis of the Pattern Recognition Receptor FLS2-BAK1 in Pepper and Nicotiana benthmiana

doi:10.16420/j.issn.0513-353x.2024-0461

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (10): 2613-2624.doi: 10.16420/j.issn.0513-353x.2024-0461

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Interaction Analysis of the Pattern Recognition Receptor FLS2-BAK1 in Pepper and Nicotiana benthmiana

JIN Jinghao, SU Chao, CHEN Yi, SUN Shichao, CHEN Xinxin, CHEN Xiaoren^*()

College of Plant Protection，Yangzhou University，Yangzhou，Jiangsu 225000，China

Received:2025-05-04 Revised:2025-06-12 Online:2025-10-25 Published:2025-10-28
Contact: CHEN Xiaoren

Abstract

Abstract:

Through interacting with co-receptor kinase，plant receptor kinases play an important role in the activating of immune response. The interaction mechanism of pattern recognition receptor module FLS2-BAK1 in Arabidopsis has been revealed. While the interaction relationship between the function domain of FLS2 and BAK1 in pepper and tobacco is still unclear. In the present study，CaFLS2-CaBAK1 and NbFLS2-NbBAK1 were cloned from Capsicum annuum and Nicotiana benthamiana respectively. Then structure character analysis，subcellular location and interaction relationship of them were analyzed. Research results showed that CaFLS2，CaBAK1，NbFLS2s and NbBAK1 contain the character domain of LRR-type receptor kinase and NbFLS2 may has different type coding sequence. All of the receptor kinases are localized on the cell membrane. The BiFC experiment results indicated that there is an interaction relationship between the extracellular domain，intracellular domain of CaFLS2 and CaBAK1，and also NbFLS2s and NbBAK1. What’s more，the interaction between the full-length protein of NbFLS2s and NbBAK1 was detected with BiFC assay，and their co-expression could induce ROS production in N. benthamiana. These results not only increase the knowledge of FLS2-BAK1 module in pepper and N. benthamiana，but also provide a basis for revealing the immunity signal pathway medicated by receptor kinase in Solanaceae crops.

Key words: pepper, receptor kinase, interaction analysis, function domain, bimolecular fluorescence complementation

JIN Jinghao, SU Chao, CHEN Yi, SUN Shichao, CHEN Xinxin, CHEN Xiaoren. Interaction Analysis of the Pattern Recognition Receptor FLS2-BAK1 in Pepper and Nicotiana benthmiana[J]. Acta Horticulturae Sinica, 2025, 52(10): 2613-2624.

Figures/Tables 7

Table 1 Primers used for gene cloning and vector construction in this study

引物名称Prime name	引物序列（5′-3′）Primer sequence
NbFLS2-F	GGGGTACCATGTCACAGACAGTTTTATATGCA
NbFLS2-ECD-R	TACGATTCATCTGCAGCTCGAGTTAGTCGACGGTTTTCTTGGAAAACCCGTGAGT
NbFLS2-R	TACGATTCATCTGCAGCTCGAGTTAGTCGACAATTGCATCTTTTACCAAACAAGC
NbFLS2-ID-F	CGCCATATGGGTACCATGGAGGATATGATTCCAAAGTAT
NbBAK1-F	GGGGTACCATGGATCAATGGCTATTGGGGATC
NbBAK1-ECD-R	TACGATTCATCTGCAGCTCGAGTTAGTCGACTCCTCCAGCAATCGCCCCGGT
NbBAK1-R	TACGATTCATCTGCAGCTCGAGTTAGTCGACTCTTGGCCCTGATAACTCATC
NbBAK1-ID-F	GGCCATGGAGGCCAGTGAATTCGGTACCATGCACTTCTTTGATGTACCTGCT
CaFLS2-F	CGGGGTACCATGATGTCAAACACAGTTGCA
CaFLS2-ECD-R	ACGCGTCGACAGTTTTCTTGGAAAAACCATGAGA
CaFLS2-ID-F	CGGGGTACCATGACAGAGGATACAAATCCAAAT
CaFLS2-R	ACGCGTCGACATTGAGATTTGATGATACTCC
CaBAK1-F	CGGGGTACCATGATGGATCAATGGGTGTTG
CaBAK1-ECD-R	ACGCGTCGACACCACCAGCAATCGCCCCGGT
CaBAK1-ID-F	CGGGGTACCATGCCAGAAGACCACTTCTTTGAT
CaBAK1-R	ACGCGTCGACTCTTGGCCCTGATAACTCATC

Fig. 1 Sequence structure characteristics analysis of FLS2s and BAK1 in pepper and tobacco A：Sequence structure analysis of NbFLS2s and CaFLS2；B：Sequence structure analysis of NbBAK1 and CaBAK1. Key residues in the interaction between AtFLS2 and AtBAK1 are indicated by black triangle

Fig. 2 Subcellular location of NbFLS2-1，NbFLS2-2 and NbBAK1 in tobacco A：NbFLS2-1，NbFLS2-2 and NbBAK1 were localized in the cell membrane，AtPIP2A-RFP was used as cell membrane marker；B：Protein detection of NbFLS2-1，NbFLS2-2 and NbBAK1

Fig. 3 Subcellular location of CaFLS2 and CaBAK1 in tobacco A：CaFLS2 and CaBAK1 were localized in the cell membrane；B：Protein detection of CaFLS2 and CaBAK1

Fig. 4 Interaction analysis between CaFLS2 and CaBAK1 with BiFC assay A：Structure diagram of LRR-RLK；B：Observation of yellow fluorescent protein in tobacco leaves；C：Detection of target protein in tobacco leaves. nYFP fusion protein and cYFP fusion protein were indicated with red and blue arrow，respectively. The same below

Fig. 5 Interaction analysis of NbFLS2s and NbBAK1 with BiFC assay A：Observation of yellow fluorescent protein in tobacco leaves；B：Detection of target protein in tobacco leaves

Fig. 6 The co-expression of NbFLS2 and NbBAK1 could induce ROS production in tobacco leaves

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Interaction Analysis of the Pattern Recognition Receptor FLS2-BAK1 in Pepper and Nicotiana benthmiana

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