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Structure-function analysis of barley NLR immune receptor MLA10 reveals its cell compartment specific activity in cell death and disease resistance.

Bai S, Liu J, Chang C, Zhang L, Maekawa T, Wang Q, Xiao W, Liu Y, Chai J, Takken FL, Schulze-Lefert P, Shen QH - PLoS Pathog. (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

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.

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Tightly regulated MLA10 cell death inducing activity.(A) Analysis of cell death triggering activity of MLA10 variants harboring the K207R P-loop mutation. The indicated N-terminal MLA10 fragments containing the K207R mutation, or FL variants harboring K207R alone or K207R combined with an MHD mutation (H501, D502V), were expressed in N. benthamiana leaves by Agro-infiltration. Cell death triggered by each fusion was assessed by trypan blue staining at 42 hpi (upper panel). Protein expression levels of indicated MLA10 mutant fragments are shown (lower panel). Proteins were extracted at 40 hpi and detection was done by immunoblotting with anti-HA antibody. (B) Analysis of cell death activity of MLA10 variants harboring mutations in the EDVID motif. The EDVID motif (EDVVD in MLA10) was mutated to AAVVD, and the indicated MLA10 fragments or FL variants harboring indicated mutation(s) were expressed in N. benthamiana leaves by Agro-infiltration. Cell death triggered by each variant was assessed by trypan blue staining at 42 hpi (upper panel). Protein expression levels of indicated MLA10 mutant fragments are shown (lower panel). Proteins were extracted at 40 hpi and detection was done by immunoblotting with anti-HA antibody. (C) Analysis of cell death activity of F99E containing MLA10 fragments or FL variants. Indicated MLA10 fragments or FL variants harboring indicated mutation(s) were expressed in N. benthamiana leaves by Agro-infiltration. Cell death triggered by each protein was scored by trypan blue staining at 42 hpi (upper panel); Protein were extracts from N. benthamiana leaves and subjected to immunoblotting with anti-HA antibody (lower panel).
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ppat-1002752-g004: Tightly regulated MLA10 cell death inducing activity.(A) Analysis of cell death triggering activity of MLA10 variants harboring the K207R P-loop mutation. The indicated N-terminal MLA10 fragments containing the K207R mutation, or FL variants harboring K207R alone or K207R combined with an MHD mutation (H501, D502V), were expressed in N. benthamiana leaves by Agro-infiltration. Cell death triggered by each fusion was assessed by trypan blue staining at 42 hpi (upper panel). Protein expression levels of indicated MLA10 mutant fragments are shown (lower panel). Proteins were extracted at 40 hpi and detection was done by immunoblotting with anti-HA antibody. (B) Analysis of cell death activity of MLA10 variants harboring mutations in the EDVID motif. The EDVID motif (EDVVD in MLA10) was mutated to AAVVD, and the indicated MLA10 fragments or FL variants harboring indicated mutation(s) were expressed in N. benthamiana leaves by Agro-infiltration. Cell death triggered by each variant was assessed by trypan blue staining at 42 hpi (upper panel). Protein expression levels of indicated MLA10 mutant fragments are shown (lower panel). Proteins were extracted at 40 hpi and detection was done by immunoblotting with anti-HA antibody. (C) Analysis of cell death activity of F99E containing MLA10 fragments or FL variants. Indicated MLA10 fragments or FL variants harboring indicated mutation(s) were expressed in N. benthamiana leaves by Agro-infiltration. Cell death triggered by each protein was scored by trypan blue staining at 42 hpi (upper panel); Protein were extracts from N. benthamiana leaves and subjected to immunoblotting with anti-HA antibody (lower panel).

Mentions: To further examine the regulation of the MLA10-mediated cell death activity by the P-loop motif, we introduced the K207R P-loop mutation into the MLA10 N-terminal fragments (CC-NB, CC-NB-ARC), and also combined this mutation with two autoactive MHD mutations (H501A or D502V) in MLA10 FL. The resulting K207R containing variants were then assessed for their cell death-inducing activity in N. benthamiana (Figure 4A). Unexpectedly, both CC-NB(K207R) and CC-NB-ARC(K207R) triggered cell death, and similar to the CC-NB and CC-NB-ARC fragments described above, the activity of the latter is weaker (Figure 4A, and 1B). However, just as the FL(K207R), the two FL variants, FL(K207R/H501A) and FL(K207R/D502V), were no longer able to induce cell death (Figure 4A). This indicates that the loss-of-function P-loop motif mutation in the receptor counteracts the autoactivating effect of the MHD motif mutations.


Structure-function analysis of barley NLR immune receptor MLA10 reveals its cell compartment specific activity in cell death and disease resistance.

Bai S, Liu J, Chang C, Zhang L, Maekawa T, Wang Q, Xiao W, Liu Y, Chai J, Takken FL, Schulze-Lefert P, Shen QH - PLoS Pathog. (2012)

Tightly regulated MLA10 cell death inducing activity.(A) Analysis of cell death triggering activity of MLA10 variants harboring the K207R P-loop mutation. The indicated N-terminal MLA10 fragments containing the K207R mutation, or FL variants harboring K207R alone or K207R combined with an MHD mutation (H501, D502V), were expressed in N. benthamiana leaves by Agro-infiltration. Cell death triggered by each fusion was assessed by trypan blue staining at 42 hpi (upper panel). Protein expression levels of indicated MLA10 mutant fragments are shown (lower panel). Proteins were extracted at 40 hpi and detection was done by immunoblotting with anti-HA antibody. (B) Analysis of cell death activity of MLA10 variants harboring mutations in the EDVID motif. The EDVID motif (EDVVD in MLA10) was mutated to AAVVD, and the indicated MLA10 fragments or FL variants harboring indicated mutation(s) were expressed in N. benthamiana leaves by Agro-infiltration. Cell death triggered by each variant was assessed by trypan blue staining at 42 hpi (upper panel). Protein expression levels of indicated MLA10 mutant fragments are shown (lower panel). Proteins were extracted at 40 hpi and detection was done by immunoblotting with anti-HA antibody. (C) Analysis of cell death activity of F99E containing MLA10 fragments or FL variants. Indicated MLA10 fragments or FL variants harboring indicated mutation(s) were expressed in N. benthamiana leaves by Agro-infiltration. Cell death triggered by each protein was scored by trypan blue staining at 42 hpi (upper panel); Protein were extracts from N. benthamiana leaves and subjected to immunoblotting with anti-HA antibody (lower panel).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369952&req=5

ppat-1002752-g004: Tightly regulated MLA10 cell death inducing activity.(A) Analysis of cell death triggering activity of MLA10 variants harboring the K207R P-loop mutation. The indicated N-terminal MLA10 fragments containing the K207R mutation, or FL variants harboring K207R alone or K207R combined with an MHD mutation (H501, D502V), were expressed in N. benthamiana leaves by Agro-infiltration. Cell death triggered by each fusion was assessed by trypan blue staining at 42 hpi (upper panel). Protein expression levels of indicated MLA10 mutant fragments are shown (lower panel). Proteins were extracted at 40 hpi and detection was done by immunoblotting with anti-HA antibody. (B) Analysis of cell death activity of MLA10 variants harboring mutations in the EDVID motif. The EDVID motif (EDVVD in MLA10) was mutated to AAVVD, and the indicated MLA10 fragments or FL variants harboring indicated mutation(s) were expressed in N. benthamiana leaves by Agro-infiltration. Cell death triggered by each variant was assessed by trypan blue staining at 42 hpi (upper panel). Protein expression levels of indicated MLA10 mutant fragments are shown (lower panel). Proteins were extracted at 40 hpi and detection was done by immunoblotting with anti-HA antibody. (C) Analysis of cell death activity of F99E containing MLA10 fragments or FL variants. Indicated MLA10 fragments or FL variants harboring indicated mutation(s) were expressed in N. benthamiana leaves by Agro-infiltration. Cell death triggered by each protein was scored by trypan blue staining at 42 hpi (upper panel); Protein were extracts from N. benthamiana leaves and subjected to immunoblotting with anti-HA antibody (lower panel).
Mentions: To further examine the regulation of the MLA10-mediated cell death activity by the P-loop motif, we introduced the K207R P-loop mutation into the MLA10 N-terminal fragments (CC-NB, CC-NB-ARC), and also combined this mutation with two autoactive MHD mutations (H501A or D502V) in MLA10 FL. The resulting K207R containing variants were then assessed for their cell death-inducing activity in N. benthamiana (Figure 4A). Unexpectedly, both CC-NB(K207R) and CC-NB-ARC(K207R) triggered cell death, and similar to the CC-NB and CC-NB-ARC fragments described above, the activity of the latter is weaker (Figure 4A, and 1B). However, just as the FL(K207R), the two FL variants, FL(K207R/H501A) and FL(K207R/D502V), were no longer able to induce cell death (Figure 4A). This indicates that the loss-of-function P-loop motif mutation in the receptor counteracts the autoactivating effect of the MHD motif mutations.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

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.

Show MeSH