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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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Glutamate substitution analyses in the MLA10 CC domain identify the F99E autoactive mutation.(A) Analysis of cell death inducing activity of MLA10 CC mutant variants. MLA10 CC wild-type or mutant variants harboring indicated amino acid substitution were expressed in N. benthamiana leaves, and cell death induced by each protein was visualized by trypan blue staining at 42 hpi (upper panel). (B) Protein expression levels of each CC variant shown by Western blotting. Proteins were extracted from N. benthamiana leaves at 40 hpi and detection was done by immunoblotting with anti-HA antibody. (C) Comparison of cell death inducing activity of MLA10 FL and the FL(F99E) variant. FL and FL(F99E) were expressed on the same N. benthamiana leaf, and the amount of cell death induced by each protein was shown by Trypan blue staining at 24 hpi (upper panel); protein expression levels of FL and FL(F99E) were shown by protein immunoblotting analysis using anti-HA antibody (bottom panel), protein extracts were obtained at ∼22 hpi. (D) Quantification of cell-death inducing activity of FL and FL(F99E). Upon expression of FL or FL(F99E) by Agro-infiltration in N. benthamiana, electrolyte leakage was measured each hour from 22 to 30 hpi, and then every two hours from 30 to 34 hpi; empty vector (EV) was included as a negative control. Error bars representing standard error (SE) were calculated from three replicates per time point and per construct. Similar experiments were repeated at least twice with similar results. Letters (a–c) represent significant differences [p<0.05, Tukey's honest significant difference (HSD) test].
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ppat-1002752-g002: Glutamate substitution analyses in the MLA10 CC domain identify the F99E autoactive mutation.(A) Analysis of cell death inducing activity of MLA10 CC mutant variants. MLA10 CC wild-type or mutant variants harboring indicated amino acid substitution were expressed in N. benthamiana leaves, and cell death induced by each protein was visualized by trypan blue staining at 42 hpi (upper panel). (B) Protein expression levels of each CC variant shown by Western blotting. Proteins were extracted from N. benthamiana leaves at 40 hpi and detection was done by immunoblotting with anti-HA antibody. (C) Comparison of cell death inducing activity of MLA10 FL and the FL(F99E) variant. FL and FL(F99E) were expressed on the same N. benthamiana leaf, and the amount of cell death induced by each protein was shown by Trypan blue staining at 24 hpi (upper panel); protein expression levels of FL and FL(F99E) were shown by protein immunoblotting analysis using anti-HA antibody (bottom panel), protein extracts were obtained at ∼22 hpi. (D) Quantification of cell-death inducing activity of FL and FL(F99E). Upon expression of FL or FL(F99E) by Agro-infiltration in N. benthamiana, electrolyte leakage was measured each hour from 22 to 30 hpi, and then every two hours from 30 to 34 hpi; empty vector (EV) was included as a negative control. Error bars representing standard error (SE) were calculated from three replicates per time point and per construct. Similar experiments were repeated at least twice with similar results. Letters (a–c) represent significant differences [p<0.05, Tukey's honest significant difference (HSD) test].

Mentions: Previously, seventeen amino acid residues lining the interior between the protomers of the MLA10 CC dimer were chosen for Glutamate substitutions to assess their contributions to dimer stability [25]. Substitutions of most of these 17 residues were shown to reduce MLA10 self-interaction. In addition, all of these substitutions, except L18E and F99E, compromised MLA10-mediated disease resistance [25]. Here we tested whether the same 17 substitutions affect the cell death-inducing activity of the MLA10 CC domain. We created CC variants each containing a single substitution, i.e. L11E, L15E, L18E, L19E, F23E, L25E, V29E, I33E, L36E, M43E, V69E, L72E, I76E, F83E, F99E, M103E, and L110E. These CC variants, fused to a 3×HA tag, were transiently expressed in N. benthamiana. Significantly, except for F99E, all substitutions diminished the cell death-inducing activity of the CC domain (Figure 2A). Most variants accumulated to similar levels (Figure 2B) although some showed reduced levels (M43E, V69 and F99E). Three variants showed a reduced mobility in the polyacrylamide gel (L72E, I76E and F83E) for unknown reasons (Figure 2B, lower panel). The F99E substitution is interesting because it is the only mutation that retained CC cell death signaling activity. It is also noteworthy that the L18E substitution abrogated the CC activity leading to cell death, whilst full-length MLA10 containing the same mutation was shown to retain wild-type-like disease resistance activity against Bgh[25]. To exclude that the loss of cell death activity of CC(L18E) might be due to altered subcellular protein localization, we constructed C-terminal YFP fusions of CC(L18E) and FL(L18E), whose expression were driven by 35S or Ubiquitin promoter for transient expression in N. benthamiana or barley leaf cells, respectively (Figure S2). As controls we generated CC(F83E)-YFP and FL(K207R)-YFP fusions, the F83E and the P-loop K207R mutation rendering MLA10 inactive (Figure 2A, [25], and below Figure 3). Upon transient expression and confocal imaging we detected for CC(L18E)-YFP or FL(L18E)-YFP in N. benthamiana or barley cells similar nucleocytoplasmic distribution patterns compared to the respective controls (Figure S2). Our data are consistent with previously proposed sequence constraints acting on the CC domain for MLA disease resistance [25], [52] and assign a critical role of the invariant CC sequence to its cell death-inducing activity. Because the tested substitutions are also needed for efficient CC dimer formation [25], our new data substantiate a previous suggestion that only the CC homodimer, which has a characteristic surface charge segregation, is capable to initiate the cell death response.


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)

Glutamate substitution analyses in the MLA10 CC domain identify the F99E autoactive mutation.(A) Analysis of cell death inducing activity of MLA10 CC mutant variants. MLA10 CC wild-type or mutant variants harboring indicated amino acid substitution were expressed in N. benthamiana leaves, and cell death induced by each protein was visualized by trypan blue staining at 42 hpi (upper panel). (B) Protein expression levels of each CC variant shown by Western blotting. Proteins were extracted from N. benthamiana leaves at 40 hpi and detection was done by immunoblotting with anti-HA antibody. (C) Comparison of cell death inducing activity of MLA10 FL and the FL(F99E) variant. FL and FL(F99E) were expressed on the same N. benthamiana leaf, and the amount of cell death induced by each protein was shown by Trypan blue staining at 24 hpi (upper panel); protein expression levels of FL and FL(F99E) were shown by protein immunoblotting analysis using anti-HA antibody (bottom panel), protein extracts were obtained at ∼22 hpi. (D) Quantification of cell-death inducing activity of FL and FL(F99E). Upon expression of FL or FL(F99E) by Agro-infiltration in N. benthamiana, electrolyte leakage was measured each hour from 22 to 30 hpi, and then every two hours from 30 to 34 hpi; empty vector (EV) was included as a negative control. Error bars representing standard error (SE) were calculated from three replicates per time point and per construct. Similar experiments were repeated at least twice with similar results. Letters (a–c) represent significant differences [p<0.05, Tukey's honest significant difference (HSD) test].
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Related In: Results  -  Collection

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

ppat-1002752-g002: Glutamate substitution analyses in the MLA10 CC domain identify the F99E autoactive mutation.(A) Analysis of cell death inducing activity of MLA10 CC mutant variants. MLA10 CC wild-type or mutant variants harboring indicated amino acid substitution were expressed in N. benthamiana leaves, and cell death induced by each protein was visualized by trypan blue staining at 42 hpi (upper panel). (B) Protein expression levels of each CC variant shown by Western blotting. Proteins were extracted from N. benthamiana leaves at 40 hpi and detection was done by immunoblotting with anti-HA antibody. (C) Comparison of cell death inducing activity of MLA10 FL and the FL(F99E) variant. FL and FL(F99E) were expressed on the same N. benthamiana leaf, and the amount of cell death induced by each protein was shown by Trypan blue staining at 24 hpi (upper panel); protein expression levels of FL and FL(F99E) were shown by protein immunoblotting analysis using anti-HA antibody (bottom panel), protein extracts were obtained at ∼22 hpi. (D) Quantification of cell-death inducing activity of FL and FL(F99E). Upon expression of FL or FL(F99E) by Agro-infiltration in N. benthamiana, electrolyte leakage was measured each hour from 22 to 30 hpi, and then every two hours from 30 to 34 hpi; empty vector (EV) was included as a negative control. Error bars representing standard error (SE) were calculated from three replicates per time point and per construct. Similar experiments were repeated at least twice with similar results. Letters (a–c) represent significant differences [p<0.05, Tukey's honest significant difference (HSD) test].
Mentions: Previously, seventeen amino acid residues lining the interior between the protomers of the MLA10 CC dimer were chosen for Glutamate substitutions to assess their contributions to dimer stability [25]. Substitutions of most of these 17 residues were shown to reduce MLA10 self-interaction. In addition, all of these substitutions, except L18E and F99E, compromised MLA10-mediated disease resistance [25]. Here we tested whether the same 17 substitutions affect the cell death-inducing activity of the MLA10 CC domain. We created CC variants each containing a single substitution, i.e. L11E, L15E, L18E, L19E, F23E, L25E, V29E, I33E, L36E, M43E, V69E, L72E, I76E, F83E, F99E, M103E, and L110E. These CC variants, fused to a 3×HA tag, were transiently expressed in N. benthamiana. Significantly, except for F99E, all substitutions diminished the cell death-inducing activity of the CC domain (Figure 2A). Most variants accumulated to similar levels (Figure 2B) although some showed reduced levels (M43E, V69 and F99E). Three variants showed a reduced mobility in the polyacrylamide gel (L72E, I76E and F83E) for unknown reasons (Figure 2B, lower panel). The F99E substitution is interesting because it is the only mutation that retained CC cell death signaling activity. It is also noteworthy that the L18E substitution abrogated the CC activity leading to cell death, whilst full-length MLA10 containing the same mutation was shown to retain wild-type-like disease resistance activity against Bgh[25]. To exclude that the loss of cell death activity of CC(L18E) might be due to altered subcellular protein localization, we constructed C-terminal YFP fusions of CC(L18E) and FL(L18E), whose expression were driven by 35S or Ubiquitin promoter for transient expression in N. benthamiana or barley leaf cells, respectively (Figure S2). As controls we generated CC(F83E)-YFP and FL(K207R)-YFP fusions, the F83E and the P-loop K207R mutation rendering MLA10 inactive (Figure 2A, [25], and below Figure 3). Upon transient expression and confocal imaging we detected for CC(L18E)-YFP or FL(L18E)-YFP in N. benthamiana or barley cells similar nucleocytoplasmic distribution patterns compared to the respective controls (Figure S2). Our data are consistent with previously proposed sequence constraints acting on the CC domain for MLA disease resistance [25], [52] and assign a critical role of the invariant CC sequence to its cell death-inducing activity. Because the tested substitutions are also needed for efficient CC dimer formation [25], our new data substantiate a previous suggestion that only the CC homodimer, which has a characteristic surface charge segregation, is capable to initiate the cell death response.

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