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.

The Coiled-Coil and Nucleotide Binding Domains of BROWN PLANTHOPPER RESISTANCE14 Function in Signaling and Resistance against Planthopper in Rice

“…However, the CC domain of BPH14 conferred P-loop-independent BPH resistance, implying that it might mimic a postactivation state of BPH14 such that the P-loop functionality is no longer required for resistance induction. We speculate that the C-terminal LRR domain of BPH14 plays a negative regulatory role in BPH14-mediated BPH resistance, as has been reported for the LRR domains of some NB-LRR disease resistance proteins (Bai et al, 2012;Takken and Goverse, 2012;Slootweg et al, 2013). The isolated CC and NB domains do not appear to be limited by interaction with the LRR domain or nucleotide hydrolysis, which may also play a negative role in signaling activation.…”

The CC domain structure from the wheat stem rust resistance protein Sr33 challenges paradigms for dimerization in plant NLR proteins

“…These truncations were transiently expressed in Nicotiana benthamiana under the control of the 35S promoter and fused to a C-terminal HA tag. Fragments truncated at, or beyond, the equivalent of MLA10 residue 142 induced cell death similar to the autoactive 1-160 fragments (16,25), whereas shorter fragments were inactive (Fig. 5A).…”

“…Both CC and TIR domains are implicated in downstream signaling, and have been shown to be necessary and sufficient for cell death responses in a number of systems (6,(13)(14)(15)(16)(17)(18).…”

“…Self-association in the absence of effector proteins has been observed in CC-NLR proteins from Arabidopsis RPS5 (24), barley (MLA10) (14), maize (Rp1-D) (18), and wheat (Sr33 and Sr50) (25). However, MLA10, Sr33, and Sr50 also display autoactivity when overexpressed (16,25), suggesting autoassociation may mimic the activated state.…”

The Chloroplastic Protein THF1 Interacts with the Coiled-Coil Domain of the Disease Resistance Protein N′ and Regulates Light-Dependent Cell Death

“…The HR involves plant-specific characteristics, including vacuolization and chloroplast disruption (Coll et al, 2011). Several reports suggest that activation of different effector-triggered immunity features are mediated by NB-LRR proteins depending on their cytoplasmic or nuclear localization, and that the activation of the HR requires NB-LRR activity in the cytoplasm Tameling et al, 2010;Heidrich et al, 2011;Bai et al, 2012). Development of the HR is also dependent on reactive oxygen species (ROS) produced by plasma membrane NADPH oxidases, as well as within organelles such as chloroplasts, mitochondria, and peroxisomes (Coll et al, 2011).…”