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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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MLA10 CC-NB or full-length trigger cell death signaling in the cytoplasm.(A) Analysis of cell death inducing activity of MLA10 CC-NB fusion proteins. MLA10 CC-NB fusion proteins, CC-NB-YFP, CC-NB-YFP-NES and CC-NB-YFP-NLS, were expressed in N. benthamiana leaves by Agro-infiltration; confocal images were taken at ∼22 hpi (upper panel) and cell-death triggered by each fusion protein was scored by trypan blue staining at ∼48 hpi (lower panel). Scale bar is 50 µm. (B) Analysis of cell death phenotype upon expression of MLA10 CC-NB-YFP-GR or co-expression of MLA10 CC-NB-YFP-NLS and CC-NB-YFP-GR fusions before and after Dex treatment. The indicated fusion(s) were expressed in N. benthamiana leaves by Agro-infiltration. Buffer with/or without Dex was sprayed onto N. benthamiana leafs 20 hpi before the confocal images were taken (upper panel). The cell-death phenotype of each fusion protein was scored by trypan blue staining at ∼48 hrs post treatment with/or without Dex (lower panel). GR: Steroid binding domain of the mammalian glucocorticoid receptor. Scale bar is 50 µm. (C) Analysis of cell death activity of GR fusions of MLA10 WT and autoactive variants. Indicated MLA10-GR fusion, and the C-terminal YFP-GR fusions of MLA10 WT and two autoactive mutant variants were expressed in N. benthamiana leaves by Agro-infiltration. The cell-death phenotype was revealed by trypan blue staining at ∼24 hrs post infiltration without Dex induction.
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ppat-1002752-g006: MLA10 CC-NB or full-length trigger cell death signaling in the cytoplasm.(A) Analysis of cell death inducing activity of MLA10 CC-NB fusion proteins. MLA10 CC-NB fusion proteins, CC-NB-YFP, CC-NB-YFP-NES and CC-NB-YFP-NLS, were expressed in N. benthamiana leaves by Agro-infiltration; confocal images were taken at ∼22 hpi (upper panel) and cell-death triggered by each fusion protein was scored by trypan blue staining at ∼48 hpi (lower panel). Scale bar is 50 µm. (B) Analysis of cell death phenotype upon expression of MLA10 CC-NB-YFP-GR or co-expression of MLA10 CC-NB-YFP-NLS and CC-NB-YFP-GR fusions before and after Dex treatment. The indicated fusion(s) were expressed in N. benthamiana leaves by Agro-infiltration. Buffer with/or without Dex was sprayed onto N. benthamiana leafs 20 hpi before the confocal images were taken (upper panel). The cell-death phenotype of each fusion protein was scored by trypan blue staining at ∼48 hrs post treatment with/or without Dex (lower panel). GR: Steroid binding domain of the mammalian glucocorticoid receptor. Scale bar is 50 µm. (C) Analysis of cell death activity of GR fusions of MLA10 WT and autoactive variants. Indicated MLA10-GR fusion, and the C-terminal YFP-GR fusions of MLA10 WT and two autoactive mutant variants were expressed in N. benthamiana leaves by Agro-infiltration. The cell-death phenotype was revealed by trypan blue staining at ∼24 hrs post infiltration without Dex induction.

Mentions: To further study the subcellular localization of MLA10 in N. benthamiana, we constructed MLA10 CC-NB-YFP and MLA10-YFP fusions with a C-terminal YFP sequence. The expression of both constructs was driven by a 35S promoter. Upon Agro-infiltration in N. benthamiana, we compared the fluorescence signal as well as the cell death phenotype of CC-NB-YFP with MLA10-YFP. Confocal imaging revealed that both fusion proteins localized to the nucleus and the cytoplasm but the CC-NB variant had a brighter YFP fluorescence signal and a clearer nuclear localization than full-length MLA10, possibly due to differences in protein folding and/or protein turnover (Figure S6). Since both fusions triggered cell death, we decided to use CC-NB-YFP instead of MLA10-YFP in the following experiments. We fused the C-terminus of CC-NB-YFP to either an NES or NLS, and upon expression confocal imaging showed that in most cells the CC-NB-YFP-NES proteins localized to the cytoplasm, whilst the CC-NB-YFP-NLS proteins exclusively localized in the nuclei (Figure 6A, upper panel). Significantly, in cell death staining assays, CC-NB-YFP-NES induced a much stronger cell death phenotype as compared to the CC-NB-YFP fusion, whereas CC-NB-YFP-NLS did not trigger any cell death (Figure 6A, lower panel). All these fusion proteins accumulated to similar levels (Figure S7). These results are consistent with those obtained with the MLA10 full-length protein tagged with NES/NLS or nes/nls (compare to Figure 5C).


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)

MLA10 CC-NB or full-length trigger cell death signaling in the cytoplasm.(A) Analysis of cell death inducing activity of MLA10 CC-NB fusion proteins. MLA10 CC-NB fusion proteins, CC-NB-YFP, CC-NB-YFP-NES and CC-NB-YFP-NLS, were expressed in N. benthamiana leaves by Agro-infiltration; confocal images were taken at ∼22 hpi (upper panel) and cell-death triggered by each fusion protein was scored by trypan blue staining at ∼48 hpi (lower panel). Scale bar is 50 µm. (B) Analysis of cell death phenotype upon expression of MLA10 CC-NB-YFP-GR or co-expression of MLA10 CC-NB-YFP-NLS and CC-NB-YFP-GR fusions before and after Dex treatment. The indicated fusion(s) were expressed in N. benthamiana leaves by Agro-infiltration. Buffer with/or without Dex was sprayed onto N. benthamiana leafs 20 hpi before the confocal images were taken (upper panel). The cell-death phenotype of each fusion protein was scored by trypan blue staining at ∼48 hrs post treatment with/or without Dex (lower panel). GR: Steroid binding domain of the mammalian glucocorticoid receptor. Scale bar is 50 µm. (C) Analysis of cell death activity of GR fusions of MLA10 WT and autoactive variants. Indicated MLA10-GR fusion, and the C-terminal YFP-GR fusions of MLA10 WT and two autoactive mutant variants were expressed in N. benthamiana leaves by Agro-infiltration. The cell-death phenotype was revealed by trypan blue staining at ∼24 hrs post infiltration without Dex induction.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369952&req=5

ppat-1002752-g006: MLA10 CC-NB or full-length trigger cell death signaling in the cytoplasm.(A) Analysis of cell death inducing activity of MLA10 CC-NB fusion proteins. MLA10 CC-NB fusion proteins, CC-NB-YFP, CC-NB-YFP-NES and CC-NB-YFP-NLS, were expressed in N. benthamiana leaves by Agro-infiltration; confocal images were taken at ∼22 hpi (upper panel) and cell-death triggered by each fusion protein was scored by trypan blue staining at ∼48 hpi (lower panel). Scale bar is 50 µm. (B) Analysis of cell death phenotype upon expression of MLA10 CC-NB-YFP-GR or co-expression of MLA10 CC-NB-YFP-NLS and CC-NB-YFP-GR fusions before and after Dex treatment. The indicated fusion(s) were expressed in N. benthamiana leaves by Agro-infiltration. Buffer with/or without Dex was sprayed onto N. benthamiana leafs 20 hpi before the confocal images were taken (upper panel). The cell-death phenotype of each fusion protein was scored by trypan blue staining at ∼48 hrs post treatment with/or without Dex (lower panel). GR: Steroid binding domain of the mammalian glucocorticoid receptor. Scale bar is 50 µm. (C) Analysis of cell death activity of GR fusions of MLA10 WT and autoactive variants. Indicated MLA10-GR fusion, and the C-terminal YFP-GR fusions of MLA10 WT and two autoactive mutant variants were expressed in N. benthamiana leaves by Agro-infiltration. The cell-death phenotype was revealed by trypan blue staining at ∼24 hrs post infiltration without Dex induction.
Mentions: To further study the subcellular localization of MLA10 in N. benthamiana, we constructed MLA10 CC-NB-YFP and MLA10-YFP fusions with a C-terminal YFP sequence. The expression of both constructs was driven by a 35S promoter. Upon Agro-infiltration in N. benthamiana, we compared the fluorescence signal as well as the cell death phenotype of CC-NB-YFP with MLA10-YFP. Confocal imaging revealed that both fusion proteins localized to the nucleus and the cytoplasm but the CC-NB variant had a brighter YFP fluorescence signal and a clearer nuclear localization than full-length MLA10, possibly due to differences in protein folding and/or protein turnover (Figure S6). Since both fusions triggered cell death, we decided to use CC-NB-YFP instead of MLA10-YFP in the following experiments. We fused the C-terminus of CC-NB-YFP to either an NES or NLS, and upon expression confocal imaging showed that in most cells the CC-NB-YFP-NES proteins localized to the cytoplasm, whilst the CC-NB-YFP-NLS proteins exclusively localized in the nuclei (Figure 6A, upper panel). Significantly, in cell death staining assays, CC-NB-YFP-NES induced a much stronger cell death phenotype as compared to the CC-NB-YFP fusion, whereas CC-NB-YFP-NLS did not trigger any cell death (Figure 6A, lower panel). All these fusion proteins accumulated to similar levels (Figure S7). These results are consistent with those obtained with the MLA10 full-length protein tagged with NES/NLS or nes/nls (compare to Figure 5C).

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
Related in: MedlinePlus