Table3_A large-scale screening identifies receptor-like kinases with common features in kinase domains that are potentially related to disease resistance in planta.xlsx

IntroductionThe plant genome encodes a plethora of proteins with structural similarity to animal receptor protein kinases, collectively known as receptor-like protein kinases (RLKs), which predominantly localize to the plasma membrane where they activate their kinase domains to convey extracellular signals to the interior of the cell, playing crucial roles in various signaling pathways. Despite the large number of members within the RLK family, to date, only a few have been identified as pattern-recognition receptors (PRRs), leaving many potential RLKs that could play roles in plant immunity undiscovered.
MethodsIn this study, a recombinant strategy was initially employed to screen the kinase domains of 133 RLKs in the Arabidopsis genome to determine their involvement in the pathogen-triggered immunity (PTI) pathway. Subsequently, 6 potential immune-related recombinant RLKs (rRLKs) were selected for the creation of transgenic materials and underwent functional characterization analysis. Finally, a sequence analysis was conducted on the kinase domains of these 133 RLKs as well as the known immune RLK receptor kinase domains from other species.
ResultsIt was found that 24 rRLKs activated the PTI response in Arabidopsis fls2 mutant protoplasts following flg22 treatment. Consistently, when 6 of these rRLKs were individually expressed in fls2 background, they exhibited diverse PTI signal transduction capabilities via different pathways while all retained membrane localization. Intriguingly, sequence analysis revealed multiple conserved amino acid sites within kinase domains of these experimentally identified immune-related RLKs in Arabidopsis. Importantly, these patterns are also preserved in RLKs involved in PTI in other species.
DiscussionThis study, on one hand, identifies common features that theoretically can enhance our understanding of immune-related RLKs and facilitate the discovery of novel immune-related RLKs in the future. On the other hand, it provides experimental evidence for the use of recombinant technique to develop diverse rRLKs for molecular breeding, thereby conferring high resistance to plants without compromising their normal growth and development.

引言
植物基因组编码大量结构与动物受体蛋白激酶相似的蛋白质，这类蛋白统称为类受体蛋白激酶（receptor-like protein kinases，RLKs）。这类蛋白主要定位于质膜，通过激活自身激酶结构域将胞外信号传递至细胞内部，在多种信号通路中发挥关键作用。尽管RLK家族成员数量庞大，但截至目前仅少数被鉴定为模式识别受体（pattern-recognition receptors，PRRs），仍有大量可能参与植物免疫的RLK尚未被发现。
方法
本研究首先采用重组策略，对拟南芥基因组中133个RLK的激酶结构域进行筛选，以确定其是否参与病原体触发的免疫（pathogen-triggered immunity，PTI）通路。随后，筛选得到6个潜在免疫相关的重组类受体蛋白激酶（recombinant RLKs，rRLKs），用于构建转基因材料并开展功能表征分析。最后，对这133个RLK的激酶结构域以及其他物种中已报道的免疫相关RLK激酶结构域进行了序列分析。
结果
研究发现，在flg22处理后，有24个rRLKs可在拟南芥fls2突变体原生质体中激活PTI应答。与之一致的是，将其中6个rRLKs分别在fls2遗传背景中表达后，它们可通过不同通路介导多样化的PTI信号转导功能，且所有蛋白均保留质膜定位特性。有趣的是，序列分析显示，拟南芥中经实验鉴定的免疫相关RLK的激酶结构域内存在多个保守氨基酸位点。重要的是，这些保守模式在其他物种中参与PTI的RLK中同样保守存在。
讨论
本研究一方面明确了免疫相关RLK的共有特征，理论上可加深我们对这类蛋白的认知，并助力未来新型免疫相关RLK的挖掘。另一方面，本研究为利用重组技术开发多样化rRLKs用于分子育种提供了实验依据，可在不影响植物正常生长发育的前提下赋予其高抗性。