Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN, 55455, USA.
Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, 55455, USA.
Sci Rep. 2022 Jan 11;12(1):501. doi: 10.1038/s41598-021-04259-8.
Plant immune receptors are often difficult to express heterologously, hindering study of direct interactions between these receptors and their targets with traditional biochemical approaches. The cell-free method ribosome display (RD) enables expression of such recalcitrant proteins by keeping each nascent polypeptide chain tethered to its ribosome, which can enhance protein folding by virtue of its size and solubility. Moreover, in contrast to an in planta readout of receptor activity such as a hypersensitive response that conflates binding and signaling, RD enables direct probing of the interaction between plant immune receptors and their targets. Here, we demonstrate the utility of this approach using tomato recognition of Trichoderma viride ethylene-inducing xylanase (EIX) as a case study. Leveraging the modular nature of the tomato LeEIX2 and LeEIX1 leucine-rich repeat (LRR) receptors, we applied an entropy-informed algorithm to maximize the information content in our receptor segmentation RD experiments to identify segments implicated in EIX binding. Unexpectedly, two distinct EIX-binding hotspots were discovered on LeEIX2 and both hotspots are shared with decoy LeEIX1, suggesting that their contrasting receptor functions are not due to differential modes of ligand binding. Given that most plant immune receptors are thought to engage targets via their LRR sequences, this approach should be of broad utility in rapidly identifying their binding hotspots.
植物免疫受体通常难以异源表达，这阻碍了传统生化方法研究这些受体与其靶标的直接相互作用。无细胞方法核糖体展示（RD）通过将每个新生多肽链与其核糖体连接在一起，从而保持其连接状态，使核糖体能够通过其大小和溶解度增强蛋白质折叠，从而使这些顽固蛋白的表达成为可能。此外，与植物中受体活性的读出（例如将结合和信号传导混为一谈的过敏反应）相比，RD 能够直接探测植物免疫受体与其靶标的相互作用。在这里，我们通过使用番茄对木霉绿色木霉乙烯诱导木聚糖酶（EIX）的识别作为案例研究来证明该方法的实用性。利用番茄 LeEIX2 和 LeEIX1 富含亮氨酸重复（LRR）受体的模块化性质，我们应用了一种熵信息算法，以使我们的受体分段 RD 实验中的信息含量最大化，以鉴定与 EIX 结合相关的片段。出乎意料的是，在 LeEIX2 上发现了两个不同的 EIX 结合热点，并且这两个热点都与诱饵 LeEIX1 共享，这表明它们相反的受体功能不是由于配体结合的不同模式所致。鉴于大多数植物免疫受体被认为通过其 LRR 序列与靶标结合，因此该方法应该在快速识别其结合热点方面具有广泛的用途。
