Limits...
A truncated NLR protein, TIR-NBS2, is required for activated defense responses in the exo70B1 mutant.

Zhao T, Rui L, Li J, Nishimura MT, Vogel JP, Liu N, Liu S, Zhao Y, Dangl JL, Tang D - PLoS Genet. (2015)

Bottom Line: Our study thus provides a link between the exocyst complex and the function of a 'TIR-NBS only' immune receptor like protein.Our data are consistent with a speculative model wherein pathogen effectors could evolve to target EXO70B1 to manipulate plant secretion machinery.TN2 could monitor EXO70B1 integrity as part of an immune receptor complex.

View Article: PubMed Central - PubMed

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

ABSTRACT
During exocytosis, the evolutionarily conserved exocyst complex tethers Golgi-derived vesicles to the target plasma membrane, a critical function for secretory pathways. Here we show that exo70B1 loss-of-function mutants express activated defense responses upon infection and express enhanced resistance to fungal, oomycete and bacterial pathogens. In a screen for mutants that suppress exo70B1 resistance, we identified nine alleles of TIR-NBS2 (TN2), suggesting that loss-of-function of EXO70B1 leads to activation of this nucleotide binding domain and leucine-rich repeat-containing (NLR)-like disease resistance protein. This NLR-like protein is atypical because it lacks the LRR domain common in typical NLR receptors. In addition, we show that TN2 interacts with EXO70B1 in yeast and in planta. Our study thus provides a link between the exocyst complex and the function of a 'TIR-NBS only' immune receptor like protein. Our data are consistent with a speculative model wherein pathogen effectors could evolve to target EXO70B1 to manipulate plant secretion machinery. TN2 could monitor EXO70B1 integrity as part of an immune receptor complex.

Show MeSH

Related in: MedlinePlus

EXO70B1 associates with SNAP33.(A) EXO70B1 interacted with SNAP33 in yeast two-hybrid assays. Overnight culture from a single colony was diluted with sterile water to OD = 0.5, and serial dilutions 1:30, 1:900, 1:27,000 were prepared and dropped onto SD-Ade-His-Leu-Trp plates at 28°C, respectively. Each drop was 10 μL. The photograph was taken at day 5 after plating. PEN1Δ: PEN1 without transmembrane domain. (B) EXO70B1 and SNAP33 interaction was examined with BiFC assays in N. benthamiana. EXO70B1 was fused to the N-terminal fragment of YFP (nYFP); SNAP33, PEN1 and VAMP721 were fused to the C-terminal fragment of YFP (cYFP). Cauliflower mosaic virus 35S promoter (35Spro) was used in this assay. YFP fluorescence was observed only with the transiently expressed EXO70B1-nYFP and SNAP33-cYFP, but not EXO70B1-nYFP and VAMP721-cYFP nor EXO70B1-nYFP and PEN1-cYFP in N. benthamiana. The combination of PEN1-nYFP and SNAP33-cYFP was used as the positive control. Bar = 50 μm. (C) EXO70B1 interacted with SNAP33 in a Co-IP assay in N. benthamiana. EXO70B1pro:EXO70B1-GFP was co-expressed with 35Spro:SNAP33-Myc in N. benthamiana leaves. Total protein was extracted and subjected to immunoprecipitation of SNAP33 protein by anti-Myc antibody. Proteins were analyzed in an immunoblot using antibodies as indicated. (D) Co-IP of EXO70B1 and SNAP33 using transgenic Arabidopsis plants. The exo70B1-3 transgenic plants expressing EXO70B1pro:EXO70B1-GFP and SNAP33pro:SNAP33-HA were used in the Co-IP assay. Total protein was extracted from 3-week-old transgenic plants that express both EXO70B1-GFP and SNAP33-HA, or EXO70B1-GFP alone (negative control). The SNAP33 protein was immunoprecipitated by anti-HA antibody, followed by immunoblot analysis with the GFP antibody to detect the presence of EXO70B1-GFP in the precipitate.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4305288&req=5

pgen.1004945.g005: EXO70B1 associates with SNAP33.(A) EXO70B1 interacted with SNAP33 in yeast two-hybrid assays. Overnight culture from a single colony was diluted with sterile water to OD = 0.5, and serial dilutions 1:30, 1:900, 1:27,000 were prepared and dropped onto SD-Ade-His-Leu-Trp plates at 28°C, respectively. Each drop was 10 μL. The photograph was taken at day 5 after plating. PEN1Δ: PEN1 without transmembrane domain. (B) EXO70B1 and SNAP33 interaction was examined with BiFC assays in N. benthamiana. EXO70B1 was fused to the N-terminal fragment of YFP (nYFP); SNAP33, PEN1 and VAMP721 were fused to the C-terminal fragment of YFP (cYFP). Cauliflower mosaic virus 35S promoter (35Spro) was used in this assay. YFP fluorescence was observed only with the transiently expressed EXO70B1-nYFP and SNAP33-cYFP, but not EXO70B1-nYFP and VAMP721-cYFP nor EXO70B1-nYFP and PEN1-cYFP in N. benthamiana. The combination of PEN1-nYFP and SNAP33-cYFP was used as the positive control. Bar = 50 μm. (C) EXO70B1 interacted with SNAP33 in a Co-IP assay in N. benthamiana. EXO70B1pro:EXO70B1-GFP was co-expressed with 35Spro:SNAP33-Myc in N. benthamiana leaves. Total protein was extracted and subjected to immunoprecipitation of SNAP33 protein by anti-Myc antibody. Proteins were analyzed in an immunoblot using antibodies as indicated. (D) Co-IP of EXO70B1 and SNAP33 using transgenic Arabidopsis plants. The exo70B1-3 transgenic plants expressing EXO70B1pro:EXO70B1-GFP and SNAP33pro:SNAP33-HA were used in the Co-IP assay. Total protein was extracted from 3-week-old transgenic plants that express both EXO70B1-GFP and SNAP33-HA, or EXO70B1-GFP alone (negative control). The SNAP33 protein was immunoprecipitated by anti-HA antibody, followed by immunoblot analysis with the GFP antibody to detect the presence of EXO70B1-GFP in the precipitate.

Mentions: The ternary SNARE complex PEN1-SNAP33-VAMP721/VAMP722 affects powdery mildew resistance [56]. To examine whether EXO70B1 functions in the PEN1-SNAP33-VAMP721/VAMP722 pathway, we first used yeast two-hybrid assays to test whether EXO70B1 interacts with the PEN1, SNAP33 and VAMP721. As shown in Fig. 5A, EXO70B1 interacted with SNAP33, but not PEN1 or PEN1 without its transmembrane domain. To confirm the interaction between EXO70B1 and SNAP33, we used bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana [57] to examine whether EXO70B1 and SNAP33 can interact. EXO70B1 and SNAP33 were fused to the N-terminal and C-terminal fragments of YFP, respectively. In N. benthamiana leaves co-transformed with the EXO70B1-YFPN and SNAP33-YFPC or PEN1-YFPN and SNAP33-YFPC constructs, we observed YFP fluorescence (Fig. 5B and S9 Fig.). However, we observed no YFP fluorescence in leaves co-transformed with EXO70B1-YFPN and VAMP721-YFPC or PEN1-YFPC, suggesting that EXO70B1 interacts with SNAP33 but not PEN1 in N. benthamiana. These results were consistent with the observations from the yeast two-hybrid assays.


A truncated NLR protein, TIR-NBS2, is required for activated defense responses in the exo70B1 mutant.

Zhao T, Rui L, Li J, Nishimura MT, Vogel JP, Liu N, Liu S, Zhao Y, Dangl JL, Tang D - PLoS Genet. (2015)

EXO70B1 associates with SNAP33.(A) EXO70B1 interacted with SNAP33 in yeast two-hybrid assays. Overnight culture from a single colony was diluted with sterile water to OD = 0.5, and serial dilutions 1:30, 1:900, 1:27,000 were prepared and dropped onto SD-Ade-His-Leu-Trp plates at 28°C, respectively. Each drop was 10 μL. The photograph was taken at day 5 after plating. PEN1Δ: PEN1 without transmembrane domain. (B) EXO70B1 and SNAP33 interaction was examined with BiFC assays in N. benthamiana. EXO70B1 was fused to the N-terminal fragment of YFP (nYFP); SNAP33, PEN1 and VAMP721 were fused to the C-terminal fragment of YFP (cYFP). Cauliflower mosaic virus 35S promoter (35Spro) was used in this assay. YFP fluorescence was observed only with the transiently expressed EXO70B1-nYFP and SNAP33-cYFP, but not EXO70B1-nYFP and VAMP721-cYFP nor EXO70B1-nYFP and PEN1-cYFP in N. benthamiana. The combination of PEN1-nYFP and SNAP33-cYFP was used as the positive control. Bar = 50 μm. (C) EXO70B1 interacted with SNAP33 in a Co-IP assay in N. benthamiana. EXO70B1pro:EXO70B1-GFP was co-expressed with 35Spro:SNAP33-Myc in N. benthamiana leaves. Total protein was extracted and subjected to immunoprecipitation of SNAP33 protein by anti-Myc antibody. Proteins were analyzed in an immunoblot using antibodies as indicated. (D) Co-IP of EXO70B1 and SNAP33 using transgenic Arabidopsis plants. The exo70B1-3 transgenic plants expressing EXO70B1pro:EXO70B1-GFP and SNAP33pro:SNAP33-HA were used in the Co-IP assay. Total protein was extracted from 3-week-old transgenic plants that express both EXO70B1-GFP and SNAP33-HA, or EXO70B1-GFP alone (negative control). The SNAP33 protein was immunoprecipitated by anti-HA antibody, followed by immunoblot analysis with the GFP antibody to detect the presence of EXO70B1-GFP in the precipitate.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4305288&req=5

pgen.1004945.g005: EXO70B1 associates with SNAP33.(A) EXO70B1 interacted with SNAP33 in yeast two-hybrid assays. Overnight culture from a single colony was diluted with sterile water to OD = 0.5, and serial dilutions 1:30, 1:900, 1:27,000 were prepared and dropped onto SD-Ade-His-Leu-Trp plates at 28°C, respectively. Each drop was 10 μL. The photograph was taken at day 5 after plating. PEN1Δ: PEN1 without transmembrane domain. (B) EXO70B1 and SNAP33 interaction was examined with BiFC assays in N. benthamiana. EXO70B1 was fused to the N-terminal fragment of YFP (nYFP); SNAP33, PEN1 and VAMP721 were fused to the C-terminal fragment of YFP (cYFP). Cauliflower mosaic virus 35S promoter (35Spro) was used in this assay. YFP fluorescence was observed only with the transiently expressed EXO70B1-nYFP and SNAP33-cYFP, but not EXO70B1-nYFP and VAMP721-cYFP nor EXO70B1-nYFP and PEN1-cYFP in N. benthamiana. The combination of PEN1-nYFP and SNAP33-cYFP was used as the positive control. Bar = 50 μm. (C) EXO70B1 interacted with SNAP33 in a Co-IP assay in N. benthamiana. EXO70B1pro:EXO70B1-GFP was co-expressed with 35Spro:SNAP33-Myc in N. benthamiana leaves. Total protein was extracted and subjected to immunoprecipitation of SNAP33 protein by anti-Myc antibody. Proteins were analyzed in an immunoblot using antibodies as indicated. (D) Co-IP of EXO70B1 and SNAP33 using transgenic Arabidopsis plants. The exo70B1-3 transgenic plants expressing EXO70B1pro:EXO70B1-GFP and SNAP33pro:SNAP33-HA were used in the Co-IP assay. Total protein was extracted from 3-week-old transgenic plants that express both EXO70B1-GFP and SNAP33-HA, or EXO70B1-GFP alone (negative control). The SNAP33 protein was immunoprecipitated by anti-HA antibody, followed by immunoblot analysis with the GFP antibody to detect the presence of EXO70B1-GFP in the precipitate.
Mentions: The ternary SNARE complex PEN1-SNAP33-VAMP721/VAMP722 affects powdery mildew resistance [56]. To examine whether EXO70B1 functions in the PEN1-SNAP33-VAMP721/VAMP722 pathway, we first used yeast two-hybrid assays to test whether EXO70B1 interacts with the PEN1, SNAP33 and VAMP721. As shown in Fig. 5A, EXO70B1 interacted with SNAP33, but not PEN1 or PEN1 without its transmembrane domain. To confirm the interaction between EXO70B1 and SNAP33, we used bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana [57] to examine whether EXO70B1 and SNAP33 can interact. EXO70B1 and SNAP33 were fused to the N-terminal and C-terminal fragments of YFP, respectively. In N. benthamiana leaves co-transformed with the EXO70B1-YFPN and SNAP33-YFPC or PEN1-YFPN and SNAP33-YFPC constructs, we observed YFP fluorescence (Fig. 5B and S9 Fig.). However, we observed no YFP fluorescence in leaves co-transformed with EXO70B1-YFPN and VAMP721-YFPC or PEN1-YFPC, suggesting that EXO70B1 interacts with SNAP33 but not PEN1 in N. benthamiana. These results were consistent with the observations from the yeast two-hybrid assays.

Bottom Line: Our study thus provides a link between the exocyst complex and the function of a 'TIR-NBS only' immune receptor like protein.Our data are consistent with a speculative model wherein pathogen effectors could evolve to target EXO70B1 to manipulate plant secretion machinery.TN2 could monitor EXO70B1 integrity as part of an immune receptor complex.

View Article: PubMed Central - PubMed

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

ABSTRACT
During exocytosis, the evolutionarily conserved exocyst complex tethers Golgi-derived vesicles to the target plasma membrane, a critical function for secretory pathways. Here we show that exo70B1 loss-of-function mutants express activated defense responses upon infection and express enhanced resistance to fungal, oomycete and bacterial pathogens. In a screen for mutants that suppress exo70B1 resistance, we identified nine alleles of TIR-NBS2 (TN2), suggesting that loss-of-function of EXO70B1 leads to activation of this nucleotide binding domain and leucine-rich repeat-containing (NLR)-like disease resistance protein. This NLR-like protein is atypical because it lacks the LRR domain common in typical NLR receptors. In addition, we show that TN2 interacts with EXO70B1 in yeast and in planta. Our study thus provides a link between the exocyst complex and the function of a 'TIR-NBS only' immune receptor like protein. Our data are consistent with a speculative model wherein pathogen effectors could evolve to target EXO70B1 to manipulate plant secretion machinery. TN2 could monitor EXO70B1 integrity as part of an immune receptor complex.

Show MeSH
Related in: MedlinePlus