FLS2-BAK1 Extracellular Domain Interaction Sites Required for Defense Signaling Activation

Signaling initiation by receptor-like kinases (RLKs) at the plasma membrane of plant cells often requires regulatory leucine-rich repeat (LRR) RLK proteins such as SERK or BIR proteins. The present work examined how the microbe-associated molecular pattern (MAMP) receptor FLS2 builds signaling complexes with BAK1 (SERK3). We first, using in vivo methods that validate separate findings by others, demonstrated that flg22 (flagellin epitope) ligand-initiated FLS2-BAK1 extracellular domain interactions can proceed independent of intracellular domain interactions. We then explored a candidate SERK protein interaction site in the extracellular domains (ectodomains; ECDs) of the significantly different receptors FLS2, EFR (MAMP receptors), PEPR1 (damage-associated molecular pattern (DAMP) receptor), and BRI1 (hormone receptor). Repeat conservation mapping revealed a cluster of conserved solvent-exposed residues near the C-terminus of models of the folded LRR domains. However, site-directed mutagenesis of this conserved site in FLS2 did not impair FLS2-BAK1 ECD interactions, and mutations in the analogous site of EFR caused receptor maturation defects. Hence this conserved LRR C-terminal region apparently has functions other than mediating interactions with BAK1. In vivo tests of the subsequently published FLS2-flg22-BAK1 ECD co-crystal structure were then performed to functionally evaluate some of the unexpected configurations predicted by that crystal structure. In support of the crystal structure data, FLS2-BAK1 ECD interactions were no longer detected in in vivo co-immunoprecipitation experiments after site-directed mutagenesis of the FLS2 BAK1-interaction residues S554, Q530, Q627 or N674. In contrast, in vivo FLS2-mediated signaling persisted and was only minimally reduced, suggesting residual FLS2-BAK1 interaction and the limited sensitivity of co-immunoprecipitation data relative to in vivo assays for signaling outputs. However, Arabidopsis plants expressing FLS2 with the Q530A+Q627A double mutation were impaired both in detectable interaction with BAK1 and in FLS2-mediated responses, lending overall support to current models of FLS2 structure and function.

植物细胞质膜上的类受体激酶（receptor-like kinases, RLKs）启动信号转导，通常需要诸如SERK或BIR这类具有调节功能的富亮氨酸重复（leucine-rich repeat, LRR）RLK蛋白。本研究针对微生物相关分子模式（microbe-associated molecular pattern, MAMP）受体FLS2如何与BAK1（SERK3）构建信号复合物展开了探究。首先，我们采用能够验证其他学者独立研究结果的体内实验方法，证实了flg22（鞭毛蛋白表位）配体触发的FLS2-BAK1胞外结构域相互作用，可不依赖于胞内结构域相互作用而发生。随后，我们针对FLS2、EFR（均为MAMP受体）、PEPR1（损伤相关分子模式（damage-associated molecular pattern, DAMP）受体）以及BRI1（激素受体）这些结构差异显著的受体的胞外结构域（ectodomains, ECDs），探索了潜在的SERK蛋白相互作用位点。重复序列保守性分析显示，在折叠后的LRR结构域模型的C端附近，存在一簇保守的溶剂暴露残基。但后续实验发现，对FLS2中该保守位点进行定点诱变，并未削弱FLS2与BAK1的ECD相互作用；而在EFR的同源位点进行诱变，则会导致受体成熟缺陷。由此可见，该保守LRR C端区域的功能显然并非介导与BAK1的相互作用。紧接着，我们针对后续发表的FLS2-flg22-BAK1 ECD共晶结构开展体内实验，以功能评估该晶体结构预测的部分意外构象。为验证该晶体结构数据，我们对FLS2中与BAK1互作的残基S554、Q530、Q627或N674进行定点诱变后，在体内免疫共沉淀实验中便无法再检测到FLS2与BAK1的ECD相互作用。与之相对的是，体内FLS2介导的信号转导仍可正常进行，仅出现轻微减弱，这提示仍存在残留的FLS2-BAK1相互作用，且免疫共沉淀实验相较于信号输出的体内检测方法，灵敏度相对有限。不过，表达携带Q530A+Q627A双突变FLS2的拟南芥植株，不仅无法检测到与BAK1的相互作用，其FLS2介导的响应也受到了损害，整体上为当前FLS2的结构与功能模型提供了支持。