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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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Manipulation of MLA10 subcellular localization and the relevance to MLA10 cell death signaling and disease resistance.(A) Confocal images of barley leaf epidermal cells expressing MLA10 fusion proteins. Indicated fusion proteins were expressed in barley leaf epidermal cells upon biolistic delivery, the confocal images were taken at 36 hrs post bombardment. Upper panel: A representative barley cell coexpressing MLA10-YFP-NLS and a nucleus marker CFP-WRKY2 (2D z plane). Lower panel: a cell expressing MLA10-YFP-nls alone (2D z plane). NLS: nuclear localization signal; nls: mutated nuclear localization signal. Arrowheads mark the nucleus and scale bar is 50 µm. (B) Analyses of disease resistance activity of MLA10 fusions or mutant variants. Relative single cell resistance/susceptibility is shown by fungal haustorium index upon biolistic delivery of plasmids expressing indicated protein and a GUS reporter into the barley leaf epidermal cells of a susceptible barley line (Golden promise). Bombarded leaves were inoculated with B. graminis fungal spores expressing AVRA10, and the fungal haustorium index was microscopically scored at 36 hrs post spore inoculation. Histogram bar represents average of three independent experiments and error bar represents SD. In the case of MLA10(F99E) expression, the number of cells expressing GUS reporter were extremely low. (C) Analysis of cell death triggering activity of MLA10 fusion proteins. Indicated MLA10 fusion proteins were expressed in N. benthamiana leaves by Agro-infiltration, and cell-death triggered by each fusion protein was scored by trypan blue staining at 40 hpi. NES: nuclear exclusion signal; nes: mutated nuclear exclusion signal. (D) Protein expression of indicated MLA10 fusions. Proteins were extracted at 23 hpi and MLA was detected by immunoblotting using an anti-MLA27 monoclonal antibody. Asterisk indicates non-specific signals. (E) Quantification of cell-death inducing activity of MLA10 fusion proteins. Upon expression of indicated MLA10 fusion proteins by Agro-infiltration in N. benthamiana, ion leakage was measured each hour from 23 to 34 hpi. Error bars (SE) were calculated from three replicates per time point and construct. Experiment was done at least twice with similar result. Letters (a–c) represent groups with significant differences [p<0.05, Tukey's honest significant difference (HSD) test].
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ppat-1002752-g005: Manipulation of MLA10 subcellular localization and the relevance to MLA10 cell death signaling and disease resistance.(A) Confocal images of barley leaf epidermal cells expressing MLA10 fusion proteins. Indicated fusion proteins were expressed in barley leaf epidermal cells upon biolistic delivery, the confocal images were taken at 36 hrs post bombardment. Upper panel: A representative barley cell coexpressing MLA10-YFP-NLS and a nucleus marker CFP-WRKY2 (2D z plane). Lower panel: a cell expressing MLA10-YFP-nls alone (2D z plane). NLS: nuclear localization signal; nls: mutated nuclear localization signal. Arrowheads mark the nucleus and scale bar is 50 µm. (B) Analyses of disease resistance activity of MLA10 fusions or mutant variants. Relative single cell resistance/susceptibility is shown by fungal haustorium index upon biolistic delivery of plasmids expressing indicated protein and a GUS reporter into the barley leaf epidermal cells of a susceptible barley line (Golden promise). Bombarded leaves were inoculated with B. graminis fungal spores expressing AVRA10, and the fungal haustorium index was microscopically scored at 36 hrs post spore inoculation. Histogram bar represents average of three independent experiments and error bar represents SD. In the case of MLA10(F99E) expression, the number of cells expressing GUS reporter were extremely low. (C) Analysis of cell death triggering activity of MLA10 fusion proteins. Indicated MLA10 fusion proteins were expressed in N. benthamiana leaves by Agro-infiltration, and cell-death triggered by each fusion protein was scored by trypan blue staining at 40 hpi. NES: nuclear exclusion signal; nes: mutated nuclear exclusion signal. (D) Protein expression of indicated MLA10 fusions. Proteins were extracted at 23 hpi and MLA was detected by immunoblotting using an anti-MLA27 monoclonal antibody. Asterisk indicates non-specific signals. (E) Quantification of cell-death inducing activity of MLA10 fusion proteins. Upon expression of indicated MLA10 fusion proteins by Agro-infiltration in N. benthamiana, ion leakage was measured each hour from 23 to 34 hpi. Error bars (SE) were calculated from three replicates per time point and construct. Experiment was done at least twice with similar result. Letters (a–c) represent groups with significant differences [p<0.05, Tukey's honest significant difference (HSD) test].

Mentions: We have previously shown that MLA10 locates to both the cytoplasm and the nucleus, and importantly, the nuclear pool is required for its disease resistance [44]. In this study, we are interested in how MLA10 subcellular partitioning relates to its cell death-inducing activity and disease resistance function. First we generated two fusion constructs, MLA10-YFP-NLS and MLA10-YFP-nls, whose expression is driven by the Ubiquitin promoter (NLS is a nuclear localization sequence from SV40 virus, while ‘nls’ is a mutated NLS and serves as a negative control; [59]). Upon expression of MLA10-YFP-NLS in barley leaf epidermal cells, YFP-derived fluorescence was exclusively detected in the nucleus. The YFP signal overlapped with that of a nuclear marker protein, CFP-WRKY2 [44], confirming its nuclear localization. The fluorescence signal of the MLA10-YFP-nls variant clearly partitioned to both nucleus and cytoplasm, similar to that of MLA10-YFP (Figure 5A, and [44]). This experiment demonstrated that the NLS functions to localize MLA10-YFP into plant cell nuclei. We wondered whether the nuclear localized MLA10-YFP-NLS confers disease resistance against Bgh. To address this, we utilized the single-cell transient expression assay [44], in which we delivered gold particles coated with DNA plasmids coexpressing a GUS reporter and either MLA10-YFP-NLS or MLA10-NLS into barley epidermal cells. Subsequently, the frequency of fungal structures formed inside the transformed cells (haustorium index; [44]) was scored upon inoculation with a Bgh race carrying the cognate AVRA10 effector. Included in these experiments are control plasmids expressing MLA10-YFP and the MLA10(K207R) P-loop mutant, respectively (Figure 5B). As expected the MLA10-YFP fusion conferred resistance against Bgh infection, resulting in an haustorium index around 29%, similar to our previous study [44]. The K207R P-loop mutation reduced MLA10 resistance activity as indicated by a haustorium index as high as 65%. In contrast, MLA10-YFP-NLS and MLA10-NLS fusions mediated similar levels of Bgh resistance as the MLA10-YFP control, with haustorium indexes of 35% and 30% respectively (Figure 5B). These data indicate that MLA10, forced to localize into the nucleus, is capable of triggering resistance to Bgh. Previously, we observed a haustorium index of 52% for the MLA10-YFP-NES fusion, which was undetectable in the nucleus. Since a similar haustorium index was obtained for the empty vector control, this indicates its loss-of-function against Bgh[44]. Together, these data suggest that the MLA10 nuclear pool is required for and capable of mediating disease resistance against the Bgh fungus in barley.


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)

Manipulation of MLA10 subcellular localization and the relevance to MLA10 cell death signaling and disease resistance.(A) Confocal images of barley leaf epidermal cells expressing MLA10 fusion proteins. Indicated fusion proteins were expressed in barley leaf epidermal cells upon biolistic delivery, the confocal images were taken at 36 hrs post bombardment. Upper panel: A representative barley cell coexpressing MLA10-YFP-NLS and a nucleus marker CFP-WRKY2 (2D z plane). Lower panel: a cell expressing MLA10-YFP-nls alone (2D z plane). NLS: nuclear localization signal; nls: mutated nuclear localization signal. Arrowheads mark the nucleus and scale bar is 50 µm. (B) Analyses of disease resistance activity of MLA10 fusions or mutant variants. Relative single cell resistance/susceptibility is shown by fungal haustorium index upon biolistic delivery of plasmids expressing indicated protein and a GUS reporter into the barley leaf epidermal cells of a susceptible barley line (Golden promise). Bombarded leaves were inoculated with B. graminis fungal spores expressing AVRA10, and the fungal haustorium index was microscopically scored at 36 hrs post spore inoculation. Histogram bar represents average of three independent experiments and error bar represents SD. In the case of MLA10(F99E) expression, the number of cells expressing GUS reporter were extremely low. (C) Analysis of cell death triggering activity of MLA10 fusion proteins. Indicated MLA10 fusion proteins were expressed in N. benthamiana leaves by Agro-infiltration, and cell-death triggered by each fusion protein was scored by trypan blue staining at 40 hpi. NES: nuclear exclusion signal; nes: mutated nuclear exclusion signal. (D) Protein expression of indicated MLA10 fusions. Proteins were extracted at 23 hpi and MLA was detected by immunoblotting using an anti-MLA27 monoclonal antibody. Asterisk indicates non-specific signals. (E) Quantification of cell-death inducing activity of MLA10 fusion proteins. Upon expression of indicated MLA10 fusion proteins by Agro-infiltration in N. benthamiana, ion leakage was measured each hour from 23 to 34 hpi. Error bars (SE) were calculated from three replicates per time point and construct. Experiment was done at least twice with similar result. Letters (a–c) represent groups with significant differences [p<0.05, Tukey's honest significant difference (HSD) test].
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getmorefigures.php?uid=PMC3369952&req=5

ppat-1002752-g005: Manipulation of MLA10 subcellular localization and the relevance to MLA10 cell death signaling and disease resistance.(A) Confocal images of barley leaf epidermal cells expressing MLA10 fusion proteins. Indicated fusion proteins were expressed in barley leaf epidermal cells upon biolistic delivery, the confocal images were taken at 36 hrs post bombardment. Upper panel: A representative barley cell coexpressing MLA10-YFP-NLS and a nucleus marker CFP-WRKY2 (2D z plane). Lower panel: a cell expressing MLA10-YFP-nls alone (2D z plane). NLS: nuclear localization signal; nls: mutated nuclear localization signal. Arrowheads mark the nucleus and scale bar is 50 µm. (B) Analyses of disease resistance activity of MLA10 fusions or mutant variants. Relative single cell resistance/susceptibility is shown by fungal haustorium index upon biolistic delivery of plasmids expressing indicated protein and a GUS reporter into the barley leaf epidermal cells of a susceptible barley line (Golden promise). Bombarded leaves were inoculated with B. graminis fungal spores expressing AVRA10, and the fungal haustorium index was microscopically scored at 36 hrs post spore inoculation. Histogram bar represents average of three independent experiments and error bar represents SD. In the case of MLA10(F99E) expression, the number of cells expressing GUS reporter were extremely low. (C) Analysis of cell death triggering activity of MLA10 fusion proteins. Indicated MLA10 fusion proteins were expressed in N. benthamiana leaves by Agro-infiltration, and cell-death triggered by each fusion protein was scored by trypan blue staining at 40 hpi. NES: nuclear exclusion signal; nes: mutated nuclear exclusion signal. (D) Protein expression of indicated MLA10 fusions. Proteins were extracted at 23 hpi and MLA was detected by immunoblotting using an anti-MLA27 monoclonal antibody. Asterisk indicates non-specific signals. (E) Quantification of cell-death inducing activity of MLA10 fusion proteins. Upon expression of indicated MLA10 fusion proteins by Agro-infiltration in N. benthamiana, ion leakage was measured each hour from 23 to 34 hpi. Error bars (SE) were calculated from three replicates per time point and construct. Experiment was done at least twice with similar result. Letters (a–c) represent groups with significant differences [p<0.05, Tukey's honest significant difference (HSD) test].
Mentions: We have previously shown that MLA10 locates to both the cytoplasm and the nucleus, and importantly, the nuclear pool is required for its disease resistance [44]. In this study, we are interested in how MLA10 subcellular partitioning relates to its cell death-inducing activity and disease resistance function. First we generated two fusion constructs, MLA10-YFP-NLS and MLA10-YFP-nls, whose expression is driven by the Ubiquitin promoter (NLS is a nuclear localization sequence from SV40 virus, while ‘nls’ is a mutated NLS and serves as a negative control; [59]). Upon expression of MLA10-YFP-NLS in barley leaf epidermal cells, YFP-derived fluorescence was exclusively detected in the nucleus. The YFP signal overlapped with that of a nuclear marker protein, CFP-WRKY2 [44], confirming its nuclear localization. The fluorescence signal of the MLA10-YFP-nls variant clearly partitioned to both nucleus and cytoplasm, similar to that of MLA10-YFP (Figure 5A, and [44]). This experiment demonstrated that the NLS functions to localize MLA10-YFP into plant cell nuclei. We wondered whether the nuclear localized MLA10-YFP-NLS confers disease resistance against Bgh. To address this, we utilized the single-cell transient expression assay [44], in which we delivered gold particles coated with DNA plasmids coexpressing a GUS reporter and either MLA10-YFP-NLS or MLA10-NLS into barley epidermal cells. Subsequently, the frequency of fungal structures formed inside the transformed cells (haustorium index; [44]) was scored upon inoculation with a Bgh race carrying the cognate AVRA10 effector. Included in these experiments are control plasmids expressing MLA10-YFP and the MLA10(K207R) P-loop mutant, respectively (Figure 5B). As expected the MLA10-YFP fusion conferred resistance against Bgh infection, resulting in an haustorium index around 29%, similar to our previous study [44]. The K207R P-loop mutation reduced MLA10 resistance activity as indicated by a haustorium index as high as 65%. In contrast, MLA10-YFP-NLS and MLA10-NLS fusions mediated similar levels of Bgh resistance as the MLA10-YFP control, with haustorium indexes of 35% and 30% respectively (Figure 5B). These data indicate that MLA10, forced to localize into the nucleus, is capable of triggering resistance to Bgh. Previously, we observed a haustorium index of 52% for the MLA10-YFP-NES fusion, which was undetectable in the nucleus. Since a similar haustorium index was obtained for the empty vector control, this indicates its loss-of-function against Bgh[44]. Together, these data suggest that the MLA10 nuclear pool is required for and capable of mediating disease resistance against the Bgh fungus in barley.

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