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. 2015 Feb;167(2):307-22.
doi: 10.1104/pp.114.253898. Epub 2014 Dec 9.

Pepper heat shock protein 70a interacts with the type III effector AvrBsT and triggers plant cell death and immunity

Nak Hyun Kim  1 , Byung Kook Hwang  2
Affiliations

Pepper heat shock protein 70a interacts with the type III effector AvrBsT and triggers plant cell death and immunity

Nak Hyun Kim et al. Plant Physiol. 2015 Feb.

Abstract

Heat shock proteins (HSPs) function as molecular chaperones and are essential for the maintenance and/or restoration of protein homeostasis. The genus Xanthomonas type III effector protein AvrBsT induces hypersensitive cell death in pepper (Capsicum annuum). Here, we report the identification of the pepper CaHSP70a as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirm the specific interaction between CaHSP70a and AvrBsT in planta. The CaHSP70a peptide-binding domain is essential for its interaction with AvrBsT. Heat stress (37°C) and Xanthomonas campestris pv vesicatoria (Xcv) infection distinctly induce CaHSP70a in pepper leaves. Cytoplasmic CaHSP70a proteins significantly accumulate in pepper leaves to induce the hypersensitive cell death response by Xcv (avrBsT) infection. Transient CaHSP70a overexpression induces hypersensitive cell death under heat stress, which is accompanied by strong induction of defense- and cell death-related genes. The CaHSP70a peptide-binding domain and ATPase-binding domain are required to trigger cell death under heat stress. Transient coexpression of CaHSP70a and avrBsT leads to cytoplasmic localization of the CaHSP70a-AvrBsT complex and significantly enhances avrBsT-triggered cell death in Nicotiana benthamiana. CaHSP70a silencing in pepper enhances Xcv growth but disrupts the reactive oxygen species burst and cell death response during Xcv infection. Expression of some defense marker genes is significantly reduced in CaHSP70a-silenced leaves, with lower levels of the defense hormones salicylic acid and jasmonic acid. Together, these results suggest that CaHSP70a interacts with the type III effector AvrBsT and is required for cell death and immunity in plants.

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Figures

Figure 1.
Figure 1.
CaHSP70a interacts with AvrBsT in yeast and plants. A, Yeast two-hybrid analysis of CaHSP70a and AvrBsT. CaHSP70a and AvrBsT fused with the AD or BD domain of GAL4 were cointroduced into Saccharomyces cerevisiae strain AH109, and reporter gene activation was monitored on SD-adenine-His-Leu-Trp (AHLT) medium containing 5-bromo-4-chloro-3-indolyl alpha-d-galactopyranoside (X-α-gal). Combinations of Lam and p53 with SV40-T were used as negative and positive controls, respectively. Lam, Laminin; p53, tumor protein p53; SV40-T, simian virus 40 large T antigen. B, Coimmunoprecipitation analyses of transiently expressed CaHSP70a:HA and AvrBsT:cMyc in N. benthamiana leaves. Extracted proteins were immunoprecipitated (IP) with anti-HA or anti-cMyc beads and immunoblotted with anti-HA and anti-cMyc antibodies.
Figure 2.
Figure 2.
Transient CaHSP70a expression during heat stress triggers cell death in pepper leaves. A, Cell death phenotypes and quantification. Pepper plants were exposed to 37°C for varying times and photographed 2 d after infiltration with A. tumefaciens carrying binary vector constructs (OD600 = 0.5). Cell death levels were rated based on a 0 to 3 scale: 0, no cell death (less than 10%); 1, weak cell death (10%–30%); 2, partial cell death (30%–80%); and 3, full cell death (80%–100%). B, Trypan Blue staining of leaves infiltrated with A. tumefaciens. C, Electrolyte leakage from leaf discs infiltrated with A. tumefaciens carrying the indicated constructs. Data represent means ± sd from three independent experiments. Asterisks indicate statistically significant differences (Student’s t test; P < 0.05). D, Immunoblot analyses of transient CaHSP70a expression. Protein loading was visualized by Coomassie Brilliant Blue (CBB) staining.
Figure 3.
Figure 3.
Deletion analysis of the actin-like ATPase- and peptide-binding domains of CaHSP70a in pepper leaves. A, Schematic diagrams of CaHSP70a deletion mutants. aa, Amino acids. B, Immunoblot analyses of CaHSP70a-deletion mutant transient expression. Protein loading was visualized by Coomassie Brilliant Blue (CBB) staining. C, Cell death phenotypes. Pepper plants were exposed to 37°C for varying times and photographed 2 d after infiltration with A. tumefaciens carrying binary vector constructs (OD600 = 0.5). Cell death levels were rated based on a 0 to 3 scale: 0, no cell death (less than 10%); 1, weak cell death (10%–30%); 2, partial cell death (30%–80%); and 3, full cell death (80%–100%). WT, Wild type. D, Electrolyte leakage from leaf discs infiltrated with A. tumefaciens carrying the indicated constructs. Data represent means ± sd from three independent experiments. Different letters indicate statistically significant differences (lsd, P < 0.05).
Figure 4.
Figure 4.
Subcellular localization of NLS or NES fusion AvrBsT and CaHSP70a constructs in N. benthamiana cells. A, Confocal images show transient expression of AvrBsT:GFP, AvrBsT:NLS:GFP, AvrBsT:NES:GFP, CaHSP70a:GFP, CaHSP70a:NLS:GFP, and CaHSP70a:NES:GFP in N. benthamiana cells. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Bars = 50 μm. B, Immunoblot (IB) analysis of total, nuclear, and cytoplasmic fractions of AvrBsT:cMyc, AvrBsT:NLS:cMyc, AvrBsT:NES:cMyc, CaHSP70a:HA, CaHSP70a:NLS:HA, and CaHSP70a:NES:HA proteins transiently expressed in N. benthamiana leaves. Anti-H3 and anti-Hsc70 antibodies were used to detect nuclear histone and cytoplasmic Hsc70 proteins, respectively.
Figure 5.
Figure 5.
Cytoplasmic localization of CaHSP70a during heat stress triggers cell death in pepper leaves. A, Cell death phenotypes and quantification. Pepper plants were exposed to 37°C for varying times and photographed 2 d after infiltration with A. tumefaciens carrying binary vector constructs (OD600 = 0.5). Cell death levels were rated based on a 0 to 3 scale: 0, no cell death (less than 10%); 1, weak cell death (10%–30%); 2, partial cell death (30%–80%); and 3, full cell death (80%–100%). nd, Not detected. B, Trypan Blue staining. C, Electrolyte leakage from leaf discs infiltrated with A. tumefaciens carrying the indicated constructs. Data represent means ± sd from three independent experiments. Different letters indicate statistically significant differences (Student’s t test, P < 0.05).
Figure 6.
Figure 6.
Quantitative real-time RT-PCR analysis of expression of CaHSP70a and defense- and cell death-related genes in pepper leaves transiently expressing CaHSP70a. Expression values are normalized by the expression level of 18S rRNA.
Figure 7.
Figure 7.
Transient CaHSP70a expression promotes avrBsT-triggered cell death in N. benthamiana leaves. A, Cell death phenotypes and quantification in leaves 2 d after infiltration with A. tumefaciens carrying empty vector (EV), CaHSP10a, Bax, and avrBsT at different inoculum ratios. Cell death levels were rated based on a 0 to 3 scale: 0, no cell death (less than 10%); 1, weak cell death (10%–30%); 2, partial cell death (30%–80%); and 3, full cell death (80%–100%). B, Electrolyte leakage from leaf discs at different time points after agroinfiltration. C, Immunoblot analyses of the transient expression of CaHSP70a, Bax, and avrBsT. Protein loading was visualized by Coomassie Brilliant Blue (CBB) staining. Data represent means ± sd from three independent experiments. Different letters indicate statistically significant differences (lsd, P < 0.05).
Figure 8.
Figure 8.
Cytoplasmic interaction between CaHSP70a and AvrBsT promotes cell death in N. benthamiana leaves. A, BiFC images of CaHSP70a/AvrBsT, CaHSP70a:NLS/AvrBsT:NLS, CaHSP70a:NES/AvrBsT:NES, and AvrBsT in leaves infiltrated with A. tumefaciens. CaHSP70a interacts with AvrBsT in both the cytosol and nucleus. CaHSP70a fused with the C-terminal yellow fluorescent protein (YFP) fragment and AvrBsT fused with the N-terminal YFP fragment were coexpressed in N. benthamiana cells. YFP signals were visualized by confocal microscopy. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Bars = 50 μm. B, Cell death phenotypes and quantification in leaves 2 d after infiltration with A. tumefaciens carrying the indicated constructs. Cell death levels were rated based on a 0 to 3 scale: 0, no cell death (less than 10%); 1, weak cell death (10%–30%); 2, partial cell death (30%–80%); and 3, full cell death (80%–100%). Data represent means ± sd from three independent experiments. Different letters indicate statistically significant differences (lsd, P < 0.05). C, Electrolyte leakage from leaf discs infiltrated by A. tumefaciens carrying the indicated constructs. Data represent means ± sd from three independent experiments. Different letters indicate statistically significant differences (lsd, P < 0.05).
Figure 9.
Figure 9.
The actin-like ATPase- and peptide-binding domains of CaHSP70a are required to promote avrBsT-triggered cell death in N. benthamiana leaves. A, BiFC images of interactions between CaHSP70a-deletion mutants and AvrBsT in N. benthamiana leaves. CaHSP70a-deletion mutants fused with the C-terminal yellow fluorescent protein (YFP) fragment and AvrBsT fused with the N-terminal YFP fragment were coexpressed in N. benthamiana cells. YFP signals were visualized by confocal microscopy. Cell nuclei were counterstained with 4′,6-diamidino-1-phenylindole (DAPI). Bars = 50 μm. B, Immunoblot and coimmunoprecipitation analyses of the transient expression of CaHSP70a-deletion mutants in N. benthamiana leaves. Protein loading was visualized by Coomassie Brilliant Blue (CBB) staining. C, Cell death phenotypes and quantification in N. benthamiana leaves 2 d after infiltration with A. tumefaciens carrying binary vector constructs. Cell death levels were rated based on a 0 to 3 scale: 0, no cell death (less than 10%); 1, weak cell death (10%–30%); 2, partial cell death (30%–80%); and 3, full cell death (80%–100%). D, Electrolyte leakage from leaf discs infiltrated with A. tumefaciens carrying the indicated constructs. Data represent means ± sd of three independent experiments. Different letters indicate statistically significant differences (lsd, P < 0.05).
Figure 10.
Figure 10.
Enhanced susceptibility of CaHSP70a-silenced pepper leaves to Xcv infection. A, Reduction of disease symptoms on CaHSP70a-silenced pepper leaves 2 d after inoculation with Xcv Ds1 (EV) and Ds1 (avrBsT) (107 cfu mL−1). Blue circles, No symptoms; yellow circles, symptoms visible only under UV light; orange circles, visible symptoms; red circles, complete cell death. B, Bacterial growth in Xcv-infected leaves (5 × 104 cfu mL−1). C, DAB staining and H2O2 quantification in Xcv-inoculated leaves (5 × 107 cfu mL−1). D, Trypan Blue staining and electrolyte leakage measurement in Xcv-inoculated leaves (5 × 107 cfu mL−1). Data represent means ± sd of three independent experiments. Asterisks indicate statistically significant differences (Student’s t test, P < 0.05).
Figure 11.
Figure 11.
Compromised induction of some defense-related genes in CaHSP70a-silenced pepper leaves inoculated with Xcv. Quantitative real-time PCR was performed for CaHSP70a, CaPR1, CaPR10, and CaDEF1. Expression levels of 18S rRNA were used for the normalization of defense-related gene expression levels. Data represent means ± sd of three independent experiments. Asterisks indicate statistically significant differences (Student’s t test, P < 0.05).
Figure 12.
Figure 12.
Reduced SA and JA accumulation in CaHSP70a-silenced pepper leaves inoculated with Xcv. A, Free SA and total SA (free SA plus Glc-conjugated SA [SAG]) levels in empty-vector control and silenced leaves. B, JA levels in empty-vector control and silenced leaves. Data represent means ± sd of three independent experiments. Asterisks indicate statistically significant differences (Student’s t test, P < 0.05). FW, Fresh weight.
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