Structure-Function Analysis of Barley NLR Immune Receptor MLA10 Reveals Its Cell Compartment Specific Activity in Cell Death and Disease Resistance

Plant intracellular immune receptors comprise a large number of multi-domain proteins resembling animal NOD-like receptors (NLRs). Plant NLRs typically recognize isolate-specific pathogen-derived effectors, encoded by avirulence (AVR) genes, and trigger defense responses often associated with localized host cell death. The barley MLA gene is polymorphic in nature and encodes NLRs of the coiled-coil (CC)-NB-LRR type that each detects a cognate isolate-specific effector of the barley powdery mildew fungus. We report the systematic analyses of MLA10 activity in disease resistance and cell death signaling in barley and Nicotiana benthamiana. MLA10 CC domain-triggered cell death is regulated by highly conserved motifs in the CC and the NB-ARC domains and by the C-terminal LRR of the receptor. Enforced MLA10 subcellular localization, by tagging with a nuclear localization sequence (NLS) or a nuclear export sequence (NES), shows that MLA10 activity in cell death signaling is suppressed in the nucleus but enhanced in the cytoplasm. By contrast, nuclear localized MLA10 is sufficient to mediate disease resistance against powdery mildew fungus. MLA10 retention in the cytoplasm was achieved through attachment of a glucocorticoid receptor hormone-binding domain (GR), by which we reinforced the role of cytoplasmic MLA10 in cell death signaling. Together with our data showing an essential and sufficient nuclear MLA10 activity in disease resistance, this suggests a bifurcation of MLA10-triggered cell death and disease resistance signaling in a compartment-dependent manner.

植物胞内免疫受体是一类大量存在的多结构域蛋白，其结构与动物核苷酸结合寡聚化结构域样受体（NOD-like receptors, NLRs）相似。植物NLR通常识别由无毒（avirulence, AVR）基因编码的、分离株特异性的病原菌效应因子，并触发往往伴随宿主局部细胞死亡的防卫反应。大麦MLA基因天然具有多态性，其所编码的卷曲螺旋（coiled-coil, CC）-NB-LRR型NLR，各自可识别大麦白粉病菌的同源分离株特异性效应因子。本研究对MLA10在大麦与本氏烟草中的抗病性及细胞死亡信号通路中的活性进行了系统分析。MLA10的CC结构域触发的细胞死亡，受到CC结构域与NB-ARC结构域中的高度保守基序，以及该受体的C端富亮氨酸重复序列（LRR）的调控。通过融合核定位序列（nuclear localization sequence, NLS）或核输出序列（nuclear export sequence, NES）来强制改变MLA10的亚细胞定位，结果显示MLA10在细胞死亡信号通路中的活性在细胞核中被抑制，而在细胞质中被增强。与之相反，细胞核定位的MLA10足以介导对大麦白粉病菌的抗病性。通过融合糖皮质激素受体激素结合结构域（glucocorticoid receptor hormone-binding domain, GR）实现MLA10的细胞质滞留，进一步证实了细胞质MLA10在细胞死亡信号通路中的作用。结合我们关于细胞核定位的MLA10是介导抗病性所必需且足够的实验数据，上述结果表明MLA10触发的细胞死亡与抗病性信号通路以依赖于亚细胞区室的方式发生了分支。