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. 2014 May;165(1):76-91.
doi: 10.1104/pp.114.238840. Epub 2014 Mar 31.

Pepper suppressor of the G2 allele of skp1 interacts with the receptor-like cytoplasmic kinase1 and type III effector AvrBsT and promotes the hypersensitive cell death response in a phosphorylation-dependent manner

Nak Hyun Kim  1 , Dae Sung Kim, Eui Hwan Chung, Byung Kook Hwang
Affiliations

Pepper suppressor of the G2 allele of skp1 interacts with the receptor-like cytoplasmic kinase1 and type III effector AvrBsT and promotes the hypersensitive cell death response in a phosphorylation-dependent manner

Nak Hyun Kim et al. Plant Physiol. 2014 May.

Abstract

Xanthomonas campestris pv vesicatoria type III effector protein, AvrBsT, triggers hypersensitive cell death in pepper (Capsicum annuum). Here, we have identified the pepper SGT1 (for suppressor of the G2 allele of skp1) as a host interactor of AvrBsT and also the pepper PIK1 (for receptor-like cytoplasmic kinase1). PIK1 specifically phosphorylates SGT1 and AvrBsT in vitro. AvrBsT specifically binds to the CHORD-containing protein and SGT1 domain of SGT1, resulting in the inhibition of PIK1-mediated SGT1 phosphorylation and subsequent nuclear transport of the SGT1-PIK1 complex. Liquid chromatography-tandem mass spectrometry of the proteolytic peptides of SGT1 identified the residues serine-98 and serine-279 of SGT1 as the major PIK1-mediated phosphorylation sites. Site-directed mutagenesis of SGT1 revealed that the identified SGT1 phosphorylation sites are responsible for the activation of AvrBsT-triggered cell death in planta. SGT1 forms a heterotrimeric complex with both AvrBsT and PIK1 exclusively in the cytoplasm. Agrobacterium tumefaciens-mediated coexpression of SGT1 and PIK1 with avrBsT promotes avrBsT-triggered cell death in Nicotiana benthamiana, dependent on PIK1. Virus-induced silencing of SGT1 and/or PIK1 compromises avrBsT-triggered cell death, hydrogen peroxide production, defense gene induction, and salicylic acid accumulation, leading to the enhanced bacterial pathogen growth in pepper. Together, these results suggest that SGT1 interacts with PIK1 and the bacterial effector protein AvrBsT and promotes the hypersensitive cell death associated with PIK1-mediated phosphorylation in plants.

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Figures

Figure 1.
Figure 1.
AvrBsT and PIK1 interact with SGT1 in yeast. A, Interactions between AvrBsT and SGT1 proteins in yeast two-hybrid assays. B, Interactions between PIK1 and SGT1 proteins in yeast two-hybrid assays. Plasmids containing fusions to the GAL4 DNA-binding domain and transcriptional activation domain are indicated by BD and AD, respectively. Lam-SV40-T and p53-SV40-T combinations were used as negative and positive controls, respectively. KD, Kinase domain; SD, synthetic dropout agar medium; SD-LT, SD minus Leu (L) and Trp (T); SD-ALTH-X-α-gal, SD minus adenine (A), Leu (L), Trp, (T), and His (H) with 5-bromo-4-chloro-3-indoyl-α-d-galactoside (X-α-gal). Different letters indicate statistically significant differences (lsd; P < 0.05). The results represent mean values ± sd from three independent experiments.
Figure 2.
Figure 2.
AvrBsT, SGT1, and PIK1 form a complex in planta. A, Multicolor BiFC assay of interactions among AvrBsT, SGT1, and PIK1 in N. benthamiana leaves. The signals were visualized 30 h after agroinfiltration using a confocal laser scanning microscope. Fluorescence signals of VYNE/SCYCE (515 nm) and SCYNE/SCYCE (477 nm) channels were digitally colored in green and red, respectively. Bars = 50 µm. B, Coimmunoprecipitation and immunoblotting of AvrBsT and PIK1 with SGT1. The indicated proteins were transiently expressed in N. benthamiana leaves under the control of the 35S promoter. C, AvrBsT inhibits the binding of PIK1 to SGT1. D, SGT1 forms dimers in vivo, and phosphorylation by PIK1 negatively regulates dimer formation. E, AvrBsT inhibits the monomerization of SGT1. Protein extracts were incubated with anti-HA agarose beads, and coimmunoprecipitated proteins were analyzed by immunoblotting using the indicated antibodies.
Figure 3.
Figure 3.
SGT1 is phosphorylated by PIK1 in vitro. A, SGT1 is a specific target protein of PIK1 for phosphorylation. GST-tagged PIK1, His-tagged SGT1, MAL-tagged CaMBL1, MAL-tagged CaRING1, His-tagged CaPR4b, and MAL proteins were purified from E. coli and separated by SDS-PAGE. Proteins were incubated with [γ-32P]ATP, separated by SDS-PAGE, and visualized by autoradiography and Coomassie Brilliant Blue (CBB) staining. Arrows indicate His-tagged proteins separated on the Coomassie Brilliant Blue-stained gel. B, PIK1 mutant proteins with defective kinase activity do not phosphorylate SGT1. K130R and D228H mutants do not contain functional kinase activity. WT, Wild type. [See online article for color version of this figure.]
Figure 4.
Figure 4.
AvrBsT inhibits PIK1 autophosphorylation and phosphorylation of SGT1. A, Time courses of the inhibition of PIK1 and SGT1 phosphorylation by AvrBsT. B, Inhibitory effects of AvrBsT on PIK1 and SGT1 phosphorylation during their different in vitro interactions. Lanes 1 to 7, Each mixture was incubated with [γ-32P]ATP at 30°C for 1 h; lanes 8 and 9, after the first incubation of SGT1/PIK1 or SGT1/AvrBsT with [γ-32P]ATP at 30°C for 30 min, AvrBsT or PIK1 was added to the mixtures, respectively, followed by incubation for 30 min. C, Inhibition of the phosphorylation of SGT1 and PIK1 is independent of AvrBsT enzymatic activity. PIK1, AvrBsT, AvrBsT C222A, and SGT1 proteins were incubated with [γ-32P]ATP for 1 h. Proteins were separated by SDS-PAGE and visualized by autoradiography (top) and Coomassie Brilliant Blue (CBB) staining (bottom). AvrBsT low concentration was 10 μg, and AvrBsT high concentration was 20 μg. [See online article for color version of this figure.]
Figure 5.
Figure 5.
SGT1 is phosphorylated on Ser-98 and Ser-279 residues by PIK1. The in vitro-phosphorylated SGT1 protein was PAGE purified and in-gel digested with trypsin, and the resulting peptides were extracted and analyzed by LC-MS/MS using a capillary LC system directly connected to the LTQ linear ion-trap mass spectrometer. Each MS/MS spectrum is a collection of ions produced by collision-induced dissociation of the intact peptide. A and B, Amino acid sequences and electrospray ionization MS/MS spectra of tryptic peptides of SGT1. The predominant ion peaks of N-terminal fragments (b ions) and C-terminal fragments (y ions) are labeled accordingly, with the subscripts denoting their positions in the identified peptide and the + and ++ superscripts indicating singly and doubly protonated ions, respectively. Product ions eliciting neutral mass losses of H3PO4 and water (H2O) are also indicated. Identified phosphoseryl resides are denoted as pS. cC denotes carbamidomethylation of the Cys residue, and oM denotes oxidation of the Met residue. C, In vitro phosphorylation of the wild-type SGT1 (WT) and S98A, S161A, S279A, and S98A/S279A mutant proteins by PIK1. Phosphorylated proteins were detected using the anti-phosphoserine antibody (top). The SDS-PAGE gel was stained with Coomassie Brilliant Blue (CBB; bottom). [See online article for color version of this figure.]
Figure 6.
Figure 6.
Transient coexpression of SGT1 and PIK1 with avrBsT enhances avrBsT-triggered hypersensitive cell death. A and C, Cell death phenotypes in N. benthamiana leaves 3 d after agroinfiltration (OD600 = 0.05). The infiltrated sites with no visible, partial, and full cell death phenotypes are circled in blue, yellow, and red, respectively. B and D, Quantification of electrolyte leakage from the N. benthamiana leaves after agroinfiltration (OD600 = 0.05). The data represent means ± sd from three independent experiments. Different letters indicate statistically significant differences (lsd; P < 0.05).
Figure 7.
Figure 7.
Transient coexpression of SGT1 phosphorylation-defective mutants with avrBsT does not enhance avrBsT-triggered hypersensitive cell death. A, Cell death phenotypes in N. benthamiana leaves 3 d after agroinfiltration (OD600 = 0.05). The infiltrated sites with no visible, partial, and severe cell death phenotypes are circled in blue, yellow, and orange, respectively. B, Quantification of electrolyte leakage from the N. benthamiana leaves after agroinfiltration (OD600 = 0.05). The data represent means ± sd from three independent experiments. Different letters indicate statistically significant differences (lsd; P < 0.05).
Figure 8.
Figure 8.
Nuclear localization of the SGT1-PIK1-AvrBsT complex reduces cell death phenotypes. A, BiFC analysis and DAPI counterstaining for the detection of nuclear localization. 35S:SGT1:SPYNE, 35:SGT1:SPYCE, and 35S:PIK1:SPYCE constructs were used for BiFC analysis. The 35S:avrBsT construct was also used to coexpress AvrBsT with SGT1:SPYNE and PIK1:SPYCE (SGT1/PIK1 + AvrBsT). Samples were counterstained with DAPI to visualize nuclei. Arrows indicate the nuclei colocalized with BiFC signals. Bars = 50 µm. B, Multicolor BiFC assay of the altered localizations of AvrBsT, SGT1, and PIK1. The NLS sequence was added to 35S:VYNE:avrBsT, 35S:SGT1:SCYCE, and 35S:SCYNE:PIK1 constructs. Arrowheads indicate the nuclei colocalized with SGT1/PIK1/AvrBsT. Fluorescence signals of VYNE/SCYCE (515 nm) or SPYNE/SPYCE (527 nm) and SCYNE/SCYCE (477 nm) or DAPI (470 nm) were digitally colored in green and red, respectively. Bar = 50 µm. C, Cell death scores in SGT1, PIK1, and AvrBsT localizations in N. benthamiana leaves. Cell death levels were rated based on a 1 to 3 scale: 1, no cell death (less than 10%); 2, partial cell death (10%–80%); and 3, full cell death (80%–100%). D, Quantification of electrolyte leakage from N. benthamiana leaves after agroinfiltration. E, Transient expression of AvrBsT:cMyc, SGT1:HA, and PIK1:FLAG after agroinfiltration. Total soluble proteins were resolved on 10% (w/v) SDS-PAGE gels, followed by immunoblotting with anti-cMyc, anti-HA, and anti-FLAG antibodies. CBB, Coomassie Brilliant Blue. For C and D, data are means ± sd from three independent experiments. Different letters indicate statistically significant differences (lsd; P < 0.05).
Figure 9.
Figure 9.
Silencing of SGT1, PIK1, and SGT1/PIK1 compromises the avrBsT-triggered hypersensitive cell death response. A, Bacterial growth in leaves of empty vector (EV) control (TRV:00) and silenced pepper plants infiltrated with Xcv (5 × 104 colony-forming units [cfu] mL−1). B, Cell death phenotypes developed on empty vector control (TRV:00) and silenced leaves 2 d after infiltration with Xcv (108 cfu mL−1). C, Quantification of electrolyte leakage from leaves infiltrated with Xcv (5 × 107 cfu mL−1). D, Quantification of H2O2 accumulation in leaves infiltrated with Xcv (5 × 107 cfu mL−1). Data are means ± sd from three independent experiments. Different letters indicate statistically significant differences (lsd; P < 0.05).
Figure 10.
Figure 10.
Silencing of SGT1, PIK1, and SGT1/PIK1 compromises defense-related gene expression and SA and JA accumulation. A, Quantitative real-time PCR analysis of the expression of SGT1, SGT1b, PIK1, and PR1 in pepper leaves infected by Xcv. The pepper 18S ribosomal RNA was used to normalize the mRNA abundance of the tested genes. B, Comparison of free SA and total SA (free SA plus Glc-conjugated SA) levels in leaves. Data are means ± sd from three independent experiments. Different letters indicate statistically significant differences (lsd; P < 0.05). EV, Empty vector; FW, fresh weight.
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