Functional Domain Analysis of the Remorin Protein LjSYMREM1 in Lotus japonicus

In legumes rhizobial infection during root nodule symbiosis (RNS) is controlled by a conserved set of receptor proteins and downstream components. MtSYMREM1, a protein of the Remorin family in Medicago truncatula, was shown to interact with at least three receptor-like kinases (RLKs) that are essential for RNS. Remorins are comprised of a conserved C-terminal domain and a variable N-terminal region that defines the six different Remorin groups. While both N- and C-terminal regions of Remorins belonging to the same phylogenetic group are similar to each other throughout the plant kingdom, the N-terminal domains of legume-specific group 2 Remorins show exceptional high degrees of sequence divergence suggesting evolutionary specialization of this protein within this clade. We therefore identified and characterized the MtSYMREM1 ortholog from Lotus japonicus (LjSYMREM1), a model legume that forms determinate root nodules. Here, we resolved its spatio-temporal regulation and showed that over-expression of LjSYMREM1 increases nodulation on transgenic roots. Using a structure-function approach we show that protein interactions including Remorin oligomerization are mainly mediated and stabilized by the Remorin C-terminal region with its coiled-coil domain while the RLK kinase domains transiently interact in vivo and phosphorylate a residue in the N-terminal region of the LjSYMREM1 protein in vitro. These data provide novel insights into the mechanism of this putative molecular scaffold protein and underline its importance during rhizobial infection.在豆科植物中，根瘤共生（RNS）过程中的根瘤菌侵染受到一套保守的受体蛋白及下游组分的调控。蒺藜苜蓿（*Medicago truncatula*）中瑞莫林（Remorin）蛋白家族成员MtSYMREM1，已被证实可与至少3种对根瘤共生至关重要的类受体激酶（RLKs）发生相互作用。瑞莫林蛋白由保守的C端结构域与可变的N端区域构成，这一特征也是划分6个不同瑞莫林蛋白类群的依据。尽管在整个植物界中，隶属于同一系统发育类群的瑞莫林蛋白的N端与C端区域均具有较高相似性，但豆科特异性2类瑞莫林蛋白的N端结构域却呈现出极高的序列分化程度，暗示该蛋白在该演化分支中发生了适应性特化。因此，本研究从定定型根瘤形成的模式豆科植物百脉根（*Lotus japonicus*）中鉴定并表征了MtSYMREM1的直向同源基因LjSYMREM1。本研究明确了LjSYMREM1的时空表达调控模式，并证实LjSYMREM1过表达可提升转基因发根的结瘤数量。通过结构-功能研究方法，本研究证实包括瑞莫林蛋白寡聚化在内的蛋白质相互作用，主要由带有卷曲螺旋结构域的瑞莫林C端区域介导并维持稳定；而类受体激酶的激酶结构域可在体内发生瞬时相互作用，并在体外磷酸化LjSYMREM1蛋白N端区域的特定氨基酸残基。本研究数据为这一潜在分子支架蛋白的作用机制提供了新的见解，并凸显了其在根瘤菌侵染过程中的重要性。