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

Abstract

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.

Author Summary

Plants utilize a multilayered immune system to protect themselves against pathogens. One layer of innate immunity is controlled by intracellular immune receptors called disease resistance (R) proteins. Plant R proteins are powerful molecules capable of triggering host cell suicide thereby restricting pathogen growth. Therefore, it is crucial for plants to control R protein activity in signaling cell death to avoid harmful autoimmune responses. The Barley MLA locus encodes a number of immune receptors that each recognizes a specific powdery mildew fungal strain. Upon pathogen recognition MLAs trigger host defenses concomitant with a rapid cell death response. We here show that MLA10 cell death-inducing activity is tightly regulated by conserved motifs located in two of its domains and by specific cellular chaperone components. Furthermore, we show distinct functions for the nuclear and cytoplasmic MLA10 pools in disease resistance and cell death signaling and provide evidence for a model uncoupling MLA10 cell death signaling from its disease resistance activity. Our results suggest that plant immune receptors integrate signals from multiple sub-cellular compartments to coordinate effective immune responses against pathogen attack.