The pepper extracellular xyloglucan-specific endo-β-1,4-glucanase inhibitor protein gene, CaXEGIP1, is required for plant cell death and defense responses

Abstract

Plants produce various proteinaceous inhibitors to protect themselves against microbial pathogen attack. A xyloglucan-specific endo-β-1,4-glucanase inhibitor1 gene, CaXEGIP1, was isolated and functionally characterized in pepper (Capsicum annuum) plants. CaXEGIP1 was rapidly and strongly induced in pepper leaves infected with avirulent Xanthomonas campestris pv vesicatoria, and purified CaXEGIP1 protein significantly inhibited the hydrolytic activity of the glycoside hydrolase74 family xyloglucan-specific endo-β-1,4-glucanase from Clostridium thermocellum. Soluble-modified green fluorescent protein-tagged CaXEGIP1 proteins were mainly localized to the apoplast of onion (Allium cepa) epidermal cells. Agrobacterium tumefaciens-mediated overexpression of CaXEGIP1 triggered pathogen-independent, spontaneous cell death in pepper and Nicotiana benthamiana leaves. CaXEGIP1 silencing in pepper conferred enhanced susceptibility to virulent and avirulent X. campestris pv vesicatoria, accompanied by a compromised hypersensitive response and lowered expression of defense-related genes. Overexpression of dexamethasone:CaXEGIP1 in Arabidopsis (Arabidopsis thaliana) enhanced resistance to Hyaloperonospora arabidopsidis infection. Comparative histochemical and proteomic analyses revealed that CaXEGIP1 overexpression induced a spontaneous cell death response and also increased the expression of some defense-related proteins in transgenic Arabidopsis leaves. This response was also accompanied by cell wall thickening and darkening. Together, these results suggest that pathogen-inducible CaXEGIP1 positively regulates cell death-mediated defense responses in plants.

Figure 1.

Induction of CaXEGIP1 in pepper leaves by Xcv infection. Shown are RNA gel-blot (A) and immunoblot (B) analyses of CaXEGIP1 expression in pepper leaves at various time points after inoculation with the Xcv virulent strain Ds1 (compatible) and avirulent strain Bv5-4a (incompatible) at the six-leaf stage. H, Healthy leaves; Mock, leaves treated with 10 mm MgCl₂. Equal loading of total RNA (20 μg per lane) was verified by visualizing ribosomal RNA (rRNA) on a gel stained with ethidium bromide. Protein loading was verified by Coomassie Brilliant Blue (CBB) staining. [See online article for color version of this figure.]

Figure 2.

Inhibitory effect of the recombinant CaXEGIP1 protein on XEG activity. A, Expression and purification of recombinant CaXEGIP1 in E. coli BL21 (DE3) cells. Protein loading was verified by Coomassie Brilliant Blue staining. Lane 1, uninduced E. coli cell extracts; lane 2, crude protein extracts of E. coli cells expressing the His-tagged CaXEGIP1, as induced by 1 mm IPTG; lane 3, purified His-tagged CaXEGIP1. B, Inhibition of XEG activity by CaXEGIP1. The data represent means ± sd (n = 4) from three independent experiments. Different letters indicate significant differences, as determined by Fisher’s lsd test (P < 0.05, n = 4). [See online article for color version of this figure.]

Figure 3.

Subcellular localization of CaXEGIP1 protein by transient expression of the CaXEGIP1:smGFP construct in onion epidermal cells. The smGFP gene was fused to the CaXEGIP1 3′ region. Transient expression of smGFP or CaXEGIP1:smGFP was detected by confocal laser scanning microscopy 24 h after biolistic transformation. White arrows indicate the localization of CaXEGIP1 protein in the external and intercellular regions of the onion epidermal cells. Bars = 100 µm.

Figure 4.

Induction of the cell death response in pepper leaves by A. tumefaciens-mediated transient expression of CaXEGIP1. A and B, RT-PCR (A) and immunoblot (B) analyses of CaXEGIP1 expression in pepper leaves. Expression of pepper 18S rRNA served as a loading control. Protein loading was verified by Coomassie Brilliant Blue (CBB) staining. C, Induction of cell death in pepper leaves. The leaf areas between the lateral veins were infiltrated with A. tumefaciens at the indicated OD₆₀₀ concentrations. Photographs were taken 48 h after agroinfiltration: left, visible symptoms; right, UV exposure to visualize the deposition of fluorescent pigments. D and E, Trypan blue staining (D) and electrolyte leakage measurements (E) from the leaves infiltrated by A. tumefaciens (OD₆₀₀ = 0.5) strains carrying the indicated constructs. The data represent means ± sd from three independent experiments. Asterisks indicate significant differences in electrolyte leakage, as determined by the two-tailed Student’s t test (P < 0.05). Bars = 200 µm.

Figure 5.

Effect of A. tumefaciens-mediated transient expression of Pto, avrPto, Pto/avrPto, and CaXEGIP1 on the cell death response of N. benthamiana leaves. A and B, RT-PCR (A) and immunoblot (B) analyses of CaXEGIP1 expression in N. benthamiana leaves. Expression of the N. benthamiana Actin gene served as a control. Protein loading was verified by Coomassie Brilliant Blue (CBB) staining. C, Induction of cell death in N. benthamiana leaves. D, Measurements of electrolyte leakage from N. benthamiana leaves infiltrated with A. tumefaciens (OD₆₀₀ = 0.5) carrying the indicated constructs. The blue, yellow, and red circles indicate no cell death, intermediate cell death, and full cell death, respectively. The data represent means ± sd from three independent experiments. Different letters indicate statistically significant differences, as determined by Fisher’s lsd test (P < 0.05).

Figure 6.

Enhanced susceptibility of CaXEGIP1-silenced pepper plants to infection with the Xcv virulent strain Ds1 and the avirulent strain Bv5-4a. A, Enhanced growth of CaXEGIP1-silenced pepper plants. B, RT-PCR analysis of the expression of CaXEGIP1 and defense-related genes in empty vector control (TRV:00) and gene-silenced (TRV:CaXEGIP1) pepper plants 12 h after inoculation with Xcv. Expression of the pepper 18S rRNA gene served as a control. H, Uninoculated healthy leaves; C, compatible; I, incompatible. C, Bacterial growth in leaves. Asterisks indicate significant differences, as determined by the two-tailed Student’s t test (P < 0.05). D and E, Trypan blue staining (D) and electrolyte leakage measurements (E) from leaves inoculated with avirulent Xcv. The data represent means ± sd from three independent experiments. Asterisks indicate significant differences, as determined by the two-tailed t test (P < 0.05). Bars = 200 µm. [See online article for color version of this figure.]

Figure 7.

CaXEGIP1-induced cell death and abnormal growth of DEX:CaXEGIP1 transgenic Arabidopsis plants after DEX treatment. A, Immunoblot analysis of CaXEGIP1 expression in leaves of transgenic plants at different time intervals after mock or 30 μm DEX treatment. Protein loading was verified by Coomassie Brilliant Blue (CBB) staining. B, CaXEGIP1-induced cell death phenotype in the leaves of DEX:CaXEGIP1 transgenic Arabidopsis plants. The visible cell death phenotype is mainly seen in the marginal region of DEX-treated leaves but not in that of mock-treated leaves, as indicated by white arrows. Photographs were taken 2 d after treatment. C, Trypan blue staining of leaves 24 h after mock or DEX treatment. Bars = 500 μm. D, Root-waving phenotype of transgenic Arabidopsis plants grown on MS medium with or without 3 μm DEX. E, Transverse sections from the roots and hypocotyls of wild-type (WT) and transgenic seedling plants (#3) grown on MS medium with or without DEX. Thin cross-sections of the roots and hypocotyls were stained with 0.1% Calcofluor (Fluorescent Brightener 28; Sigma) in water. Samples were observed with a microscope equipped with a UV lamp. Arrows indicate abnormal growth of root and hypocotyl cells of DEX:CaXEGIP1 seedling plants. [See online article for color version of this figure.]

Figure 8.

Cell death phenotype and bacterial growth in DEX:CaXEGIP1 transgenic Arabidopsis plants infiltrated with Pst DC3000. A and B, Disease symptoms developed on the leaves of DEX:CaXEGIP1 transgenic Arabidopsis 8 d after inoculation with virulent Pst DC3000 (A) and avirulent Pst DC3000 (avrRpm1) (B; 10⁷ cfu mL⁻¹). Arabidopsis plants were inoculated with Pst DC3000 24 h after mock treatment or 30 μm DEX treatment. C, Bacterial growth in DEX:CaXEGIP1 transgenic Arabidopsis leaves 0 and 3 d after inoculation with virulent Pst DC3000 and avirulent Pst DC3000 (avrRpm1) strains (10⁵ cfu mL⁻¹). The data represent means ± sd from three independent experiments. [See online article for color version of this figure.]

Figure 9.

Enhanced resistance of DEX:CaXEGIP1 transgenic Arabidopsis plants to infection with Hpa isolate Noco2. A, Trypan blue staining of Hpa-infected cotyledons of untreated and DEX-treated transgenic plants 3 d after inoculation. Arabidopsis seedlings were inoculated with Hpa 24 h after mock treatment or 30 μm DEX treatment. Bars = 200 µm. B, Reduced growth of Hpa in DEX-treated transgenic Arabidopsis plants. The disease severity was rated on a 0 to 3 scale after trypan blue staining to observe fungal structure development (0, no hyphal growth; 1, minor hyphal growth; 2, severe hyphal growth but no sporangiophore formation; 3, severe hyphal growth and sporangiophore formation). S, Sporangiophore. C, Disease symptoms on the cotyledons of untreated and DEX-treated transgenic plants 5 d after spray inoculation with Hpa (5 × 10⁴ spores mL⁻¹). Arrows indicate sporangiophores. Trypan blue-stained fungal structures are shown at higher magnification (bottom panels). ha, Haustorium; O, oospore. Bars = 500 µm (middle row) and 50 µm (bottom row). D, The number of sporangiophores per cotyledon of untreated or DEX-treated transgenic plants 7 d after inoculation with Hpa. Asterisks indicate significant differences in disease severity, as determined by the two-tailed Student’s t test (P < 0.05).

Figure 10.

Real-time RT-PCR analyses of the expression of Arabidopsis genes that are newly induced in DEX:CaXEGIP1 transgenic plants. RNA was extracted from the leaves of 4-week-old transgenic Arabidopsis plants 24 and 48 h after mock or DEX treatment. The relative amount of the gene transcript was normalized against the expression level of the UBQ gene. PR5, Pathogenesis-related gene5 (AT1G75040); BGL2, β-1,3-glucanase2 (AT3G57260); GSR1, Gln synthetase (AT5G37600); GSR2, copper-ion binding/Glu-ammonia ligase (AT1G66200). Asterisks indicate significant differences, as determined by the two-tailed Student’s t test (P < 0.05).