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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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Related in: MedlinePlus

MLA10 CC is the cell death signaling domain whose activity is regulated by other domains.(A) Schematic diagram of the MLA10 domain structure and the derived fragments expressed in N. benthamiana. Individual domains of MLA10 are represented by colored boxes, and the relative positions of relevant amino acids and motifs (in parentheses) are indicated on top (upper panel). Lower panel: MLA10 fragments consisting only of the CC domain or the CC combined with other domains are drawn schematically in solid lines. All fragments were expressed as C-terminal -3×HA fusions in N. benthamiana leaves using Agrobacterium-mediated transient transformation (Agro-infiltration). (B) Analysis of cell death inducing activity of MLA10 fragments. MLA10 fragments or full-length(FL) protein fused with a C-terminal 3×HA tag were expressed by Agro-infiltration in N. benthamiana (left), and cell-death triggered by each protein was visualized by trypan blue staining at 42 hrs post Agro-infiltration (hpi) (right). Red circles indicate cell death; white circles indicate no obvious cell death in the infiltrated area. (C) Quantification of cell-death inducing activity of MLA10 fragments. Upon expression of the same fragment fusions as indicated in (B) by Agro-infiltration in N. benthamiana, electrolyte leakage was measured each hour from 28 to 34 hpi. Error bars were calculated from three replicates per time point and per construct. Experiments were done at least twice with similar results. Letters (a–d) represent groups with significant differences [p<0.05, Tukey's honest significant difference (HSD) test]. (D) Protein expression of MLA10 fragments. Total protein was extracted from N. benthamiana leaves at 40 hpi and MLA10-HA was detected by immunoblotting using an anti-HA antibody. Asterisk indicates non-specific signals throughout this article except specified. Ponceau staining of Rubisco small subunit was used to show equal loading throughout this article except specified. EV: empty vector.
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ppat-1002752-g001: MLA10 CC is the cell death signaling domain whose activity is regulated by other domains.(A) Schematic diagram of the MLA10 domain structure and the derived fragments expressed in N. benthamiana. Individual domains of MLA10 are represented by colored boxes, and the relative positions of relevant amino acids and motifs (in parentheses) are indicated on top (upper panel). Lower panel: MLA10 fragments consisting only of the CC domain or the CC combined with other domains are drawn schematically in solid lines. All fragments were expressed as C-terminal -3×HA fusions in N. benthamiana leaves using Agrobacterium-mediated transient transformation (Agro-infiltration). (B) Analysis of cell death inducing activity of MLA10 fragments. MLA10 fragments or full-length(FL) protein fused with a C-terminal 3×HA tag were expressed by Agro-infiltration in N. benthamiana (left), and cell-death triggered by each protein was visualized by trypan blue staining at 42 hrs post Agro-infiltration (hpi) (right). Red circles indicate cell death; white circles indicate no obvious cell death in the infiltrated area. (C) Quantification of cell-death inducing activity of MLA10 fragments. Upon expression of the same fragment fusions as indicated in (B) by Agro-infiltration in N. benthamiana, electrolyte leakage was measured each hour from 28 to 34 hpi. Error bars were calculated from three replicates per time point and per construct. Experiments were done at least twice with similar results. Letters (a–d) represent groups with significant differences [p<0.05, Tukey's honest significant difference (HSD) test]. (D) Protein expression of MLA10 fragments. Total protein was extracted from N. benthamiana leaves at 40 hpi and MLA10-HA was detected by immunoblotting using an anti-HA antibody. Asterisk indicates non-specific signals throughout this article except specified. Ponceau staining of Rubisco small subunit was used to show equal loading throughout this article except specified. EV: empty vector.

Mentions: The CC domain of MLA10 dimerizes in solution and defines the minimal functional unit required to trigger cell death [25]. To better understand the involvement of MLA10 domains in cell death induction a series of MLA10 fragments was generated. These fragments include the CC, NB, NB-ARC and NB-ARC-LRR alone, and the CC combined with one or multiple domains: CC-NB, CC-NB-ARC and CC-NB-ARC-LRR, all fused with 3×HA tag (Figure 1A). Upon expression of these fragments in N. benthamiana leaves and subsequent trypan blue staining or electrolyte leakage measurements, we observed a cell death phenotype by expression of the CC domain alone or CC domain-containing fragments (CC-NB, CC-NB-ARC and full-length [FL] MLA10). We never observed a cell death phenotype after expressing MLA10 fragments lacking the CC, i.e. NB, NB-ARC or NB-ARC-LRR (Figure 1B, 1C). All fusion proteins accumulated to detectable levels as revealed by immunoblotting (Figure 1D). To make sure that the C-terminal tags used here or in experiments below do not interfere with functions, we tested cell death activity of CC or CC-NB fused to different types of tag (Figure S1). Cell death activity of CC was not affected by small tags such as 3×HA or 3×Myc, whilst the function of CC-NB retained when fused to even larger tags, like YFP (Figure S1). These data indicate that only the CC domain can initiate the cell death response. When comparing the individual CC-containing fragments, we found that the CC-NB reproducibly triggered the strongest cell death response. The CC and CC-NB-ARC triggered comparatively weaker responses, suggesting that the NB positively regulates cell death whereas the ARC domain might exert a negatively regulatory effect (Figure 1B, 1C). Notably, MLA10 FL protein triggered an even stronger cell death response when compared to that of the CC-NB (Figure 1B, 1C), indicating that the FL protein is partially auto-active in the N. benthamiana heterologous expression system. In summary, the CC domain of MLA10 is necessary and sufficient for triggering cell death in N. benthamiana, and its activity is modulated by other MLA10 domains.


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 is the cell death signaling domain whose activity is regulated by other domains.(A) Schematic diagram of the MLA10 domain structure and the derived fragments expressed in N. benthamiana. Individual domains of MLA10 are represented by colored boxes, and the relative positions of relevant amino acids and motifs (in parentheses) are indicated on top (upper panel). Lower panel: MLA10 fragments consisting only of the CC domain or the CC combined with other domains are drawn schematically in solid lines. All fragments were expressed as C-terminal -3×HA fusions in N. benthamiana leaves using Agrobacterium-mediated transient transformation (Agro-infiltration). (B) Analysis of cell death inducing activity of MLA10 fragments. MLA10 fragments or full-length(FL) protein fused with a C-terminal 3×HA tag were expressed by Agro-infiltration in N. benthamiana (left), and cell-death triggered by each protein was visualized by trypan blue staining at 42 hrs post Agro-infiltration (hpi) (right). Red circles indicate cell death; white circles indicate no obvious cell death in the infiltrated area. (C) Quantification of cell-death inducing activity of MLA10 fragments. Upon expression of the same fragment fusions as indicated in (B) by Agro-infiltration in N. benthamiana, electrolyte leakage was measured each hour from 28 to 34 hpi. Error bars were calculated from three replicates per time point and per construct. Experiments were done at least twice with similar results. Letters (a–d) represent groups with significant differences [p<0.05, Tukey's honest significant difference (HSD) test]. (D) Protein expression of MLA10 fragments. Total protein was extracted from N. benthamiana leaves at 40 hpi and MLA10-HA was detected by immunoblotting using an anti-HA antibody. Asterisk indicates non-specific signals throughout this article except specified. Ponceau staining of Rubisco small subunit was used to show equal loading throughout this article except specified. EV: empty vector.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002752-g001: MLA10 CC is the cell death signaling domain whose activity is regulated by other domains.(A) Schematic diagram of the MLA10 domain structure and the derived fragments expressed in N. benthamiana. Individual domains of MLA10 are represented by colored boxes, and the relative positions of relevant amino acids and motifs (in parentheses) are indicated on top (upper panel). Lower panel: MLA10 fragments consisting only of the CC domain or the CC combined with other domains are drawn schematically in solid lines. All fragments were expressed as C-terminal -3×HA fusions in N. benthamiana leaves using Agrobacterium-mediated transient transformation (Agro-infiltration). (B) Analysis of cell death inducing activity of MLA10 fragments. MLA10 fragments or full-length(FL) protein fused with a C-terminal 3×HA tag were expressed by Agro-infiltration in N. benthamiana (left), and cell-death triggered by each protein was visualized by trypan blue staining at 42 hrs post Agro-infiltration (hpi) (right). Red circles indicate cell death; white circles indicate no obvious cell death in the infiltrated area. (C) Quantification of cell-death inducing activity of MLA10 fragments. Upon expression of the same fragment fusions as indicated in (B) by Agro-infiltration in N. benthamiana, electrolyte leakage was measured each hour from 28 to 34 hpi. Error bars were calculated from three replicates per time point and per construct. Experiments were done at least twice with similar results. Letters (a–d) represent groups with significant differences [p<0.05, Tukey's honest significant difference (HSD) test]. (D) Protein expression of MLA10 fragments. Total protein was extracted from N. benthamiana leaves at 40 hpi and MLA10-HA was detected by immunoblotting using an anti-HA antibody. Asterisk indicates non-specific signals throughout this article except specified. Ponceau staining of Rubisco small subunit was used to show equal loading throughout this article except specified. EV: empty vector.
Mentions: The CC domain of MLA10 dimerizes in solution and defines the minimal functional unit required to trigger cell death [25]. To better understand the involvement of MLA10 domains in cell death induction a series of MLA10 fragments was generated. These fragments include the CC, NB, NB-ARC and NB-ARC-LRR alone, and the CC combined with one or multiple domains: CC-NB, CC-NB-ARC and CC-NB-ARC-LRR, all fused with 3×HA tag (Figure 1A). Upon expression of these fragments in N. benthamiana leaves and subsequent trypan blue staining or electrolyte leakage measurements, we observed a cell death phenotype by expression of the CC domain alone or CC domain-containing fragments (CC-NB, CC-NB-ARC and full-length [FL] MLA10). We never observed a cell death phenotype after expressing MLA10 fragments lacking the CC, i.e. NB, NB-ARC or NB-ARC-LRR (Figure 1B, 1C). All fusion proteins accumulated to detectable levels as revealed by immunoblotting (Figure 1D). To make sure that the C-terminal tags used here or in experiments below do not interfere with functions, we tested cell death activity of CC or CC-NB fused to different types of tag (Figure S1). Cell death activity of CC was not affected by small tags such as 3×HA or 3×Myc, whilst the function of CC-NB retained when fused to even larger tags, like YFP (Figure S1). These data indicate that only the CC domain can initiate the cell death response. When comparing the individual CC-containing fragments, we found that the CC-NB reproducibly triggered the strongest cell death response. The CC and CC-NB-ARC triggered comparatively weaker responses, suggesting that the NB positively regulates cell death whereas the ARC domain might exert a negatively regulatory effect (Figure 1B, 1C). Notably, MLA10 FL protein triggered an even stronger cell death response when compared to that of the CC-NB (Figure 1B, 1C), indicating that the FL protein is partially auto-active in the N. benthamiana heterologous expression system. In summary, the CC domain of MLA10 is necessary and sufficient for triggering cell death in N. benthamiana, and its activity is modulated by other MLA10 domains.

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