The Plant Signaling Mechanisms Laboratory, Department of Plant Molecular Biology, University of Lausanne, 1015 Lausanne, Switzerland.
Department of Plant and Microbial Biology, University of Zurich, 8008 Zurich, Switzerland.
Proc Natl Acad Sci U S A. 2020 Mar 31;117(13):7494-7503. doi: 10.1073/pnas.2000100117. Epub 2020 Mar 12.
Plant reproduction relies on the highly regulated growth of the pollen tube for sperm delivery. This process is controlled by secreted RALF signaling peptides, which have previously been shown to be perceived by RLK1-like (RLK1Ls) membrane receptor-kinases/LORELEI-like GLYCOLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEINS (LLG) complexes, or by leucine-rich repeat (LRR) extensin proteins (LRXs). Here, we demonstrate that RALF peptides fold into bioactive, disulfide bond-stabilized proteins that bind the LRR domain of LRX proteins with low nanomolar affinity. Crystal structures of LRX2-RALF4 and LRX8-RALF4 complexes at 3.2- and 3.9-Å resolution, respectively, reveal a dimeric arrangement of LRX proteins, with each monomer binding one folded RALF peptide. Structure-based mutations targeting the LRX-RALF4 complex interface, or the RALF4 fold, reduce RALF4 binding to LRX8 in vitro and RALF4 function in growing pollen tubes. Mutants targeting the disulfide-bond stabilized LRX dimer interface fail to rescue infertility phenotypes. Quantitative biochemical assays reveal that RALF4 binds LLGs and LRX cell-wall modules with drastically different binding affinities, and with distinct and mutually exclusive binding modes. Our biochemical, structural, and genetic analyses reveal a complex signaling network by which RALF ligands instruct different signaling proteins using distinct targeting mechanisms.
植物繁殖依赖于花粉管的高度调控生长来输送精子。这个过程受到分泌的 RALF 信号肽的控制，先前的研究表明，RALF 信号肽被 RLK1 样（RLK1Ls）膜受体激酶/LORELEI 样糖基磷脂酰肌醇（GPI）-锚定蛋白（LLG）复合物，或富含亮氨酸的重复（LRR）伸展蛋白（LRXs）感知。在这里，我们证明 RALF 肽折叠成具有生物活性的、二硫键稳定的蛋白质，这些蛋白质以低纳摩尔亲和力结合 LRX 蛋白的 LRR 结构域。LRX2-RALF4 和 LRX8-RALF4 复合物的晶体结构分辨率分别为 3.2 和 3.9 Å，分别揭示了 LRX 蛋白的二聚体排列方式，每个单体结合一个折叠的 RALF 肽。针对 LRX-RALF4 复合物界面或 RALF4 折叠的基于结构的突变，减少了 RALF4 在体外与 LRX8 的结合以及 RALF4 在生长花粉管中的功能。针对二硫键稳定的 LRX 二聚体界面的突变体不能挽救不育表型。定量生化分析表明，RALF4 以截然不同的结合亲和力与 LLGs 和 LRX 细胞壁模块结合，并且具有不同的和相互排斥的结合模式。我们的生化、结构和遗传分析揭示了一个复杂的信号网络，其中 RALF 配体使用不同的靶向机制指示不同的信号蛋白。
