Silencing of NbCA1 accelerates N-immune receptor-mediated HR−PCD. (A) TMV-GFP-induced HR-PCD in VIGS-Vector control plants (upper panel) and VIGS-NbCA1 plants (lower panel) rub inoculated with equal amount of TMV-GFP extract. Images of TMV-GFP-infected leaves (panels 1) and magnified leaf area (panels 2–3) were captured under white light (panels 1 and 2) and UV light (panels 3) at 5 days post-infection (dpi). Experiments were repeated for four times, and four leaves from two individual plants were used in each experiment. (B) TMV-GFP accumulation at 0–5 dpi in TMV-GFP-infected leaves of VIGS-Vector plants and VIGS-NbCA1 plants. Total protein extracted from TMV-GFP-infected leaves was analysed by western blot using GFP antibody. Coomassie staining of Rubisco bands show total protein loading amount. Numbers above the panels indicate days post-infiltration. (C) TMV-p50-induced HR-PCD in VIGS-Vector plants (upper panel) and in VIGS-NbCA1 plants (lower panel) agro-infiltrated with TMV-p50 of OD₆₀₀=1.0. Numbers above the panels indicate days post-infiltration. Experiments were repeated for four times, and four leaves from two individual plants were used in each experiment. (D) Expression level of NbCA1 in NbCA1-RNAi plants (1) and control plants (2). Transcription level of NbCA1 was analysed by Northern blot using NbCA1 probe. Ethidium bromide (EtBr)-stained ribosomal RNA bands serve as loading control. (E) TMV-p50-induced HR-PCD in control plants (top panel) and in NbCA1-RNAi plants (bottom panel) agro-infiltrated with TMV-p50 of OD₆₀₀=1.0. hpi, hours post-infiltration. (F) Expression of PR1, PR2 and SGT1 in control plants and NbCA1-RNAi plants in response to TMV-p50. Transcription levels were analysed by Northern blots using PR1, PR2 and SGT1 probes at 0, 16, 24, 30 and 40 h post-infection (hpi). Ethidium bromide (EtBr)-stained ribosomal RNA bands serve as loading control.

Silencing of NbCA1 induces accelerated HR-PCD even in the absence of Rar1, HSP90 and SGT1. (A, B) TMV-GFP-induced HR-PCD in RAR1- (A) and HSP90-silenced (B) control plants (1–2) and NbCA1-RNAi plants (3–4) agro-infiltrated with TMV-GFP of OD₆₀₀=1.0. All pictures were taken at 6 dpi under white light (1 and 3) and the UV light (2 and 4). (C) TMV-GFP-induced HR-PCD in SGT1-silenced control plants (top row, panels 1 and 2) and NbCA1-RNAi plants (top row, panels 3 and 4). TMV-GFP moved to the upper uninoculated leaves of SGT1-silenced control plants (middle and bottom rows, panels 1 and 2) and NbCA1-RNAi plants (middle and bottom rows, panels 3 and 4). All pictures were taken at 6 dpi (top and middle row) and 8 dpi (bottom row) under white light (1 and 3) and the UV light (2 and 4).

NbCA1 is localized to ER. (A) Confocal images show that citrine expressed under NbCA1 promoter is localized to the cytoplasm (panel 1) and NbCA1 fused to citrine is expressed in the ER (panel 2) of epidermal (upper panels) and mesophyll (lower panel) cells. Image in the panel 3 is the magnified area of epidermal cell image shown in panel 2. The scale bar represents 20 μm. (B) NbCA1-citrine co-localizes with ER maker TagCFP-HDEL. Chloroplasts are illuminated by red autofluorescence. Merged image shows the overlay pattern of NbCA1 and TagCFP. The scale bar represents 20 μm.

Effect of silencing NbCAM1, NbrbohB and NbTPC on HR-PCD in NbCA1-RNAi plants. (A) TMV-p50-induced HR-PCD in the NbCaM1- (1), NbrbohB (2), and NbTPC- (3) silenced and Vector-alone (4) control plants (upper panel) and NbCA1-RNAi plants (lower panel) agro-infiltrated with TMV-p50 of OD₆₀₀=1.0. Pictures were taken at 3 days post-infiltration. (B) Avr9, AvrPto and Pst DC3000-induced cell death in NbrbohB-silenced and Vector-alone control plants (upper panels) and NbCA1−RNAi plants (lower panels) infiltrated with Pst DC3000 and agro-infiltrated with Cf9 plus Avr9, and Pto plus AvrPto. Pictures were taken at 3 days post-infiltration.

Silencing of NbCA1 accelerates Cf9 immune receptor as well as Pst DC3000- and cryptogein elicitor-induced cell death. Control plants (upper panel) and NbCA1-RNAi plants (lower panel) were co-infiltrated with a mixture of Agrobacterium with Cf9 and Avr9 (1), Pto and AvrPto (2) at a 1:1 ratio of OD₆₀₀=1.0, 10⁵ cfu of Pseudomonas syringae pv. tomato DC3000 (3) and 100 μg/ml of cryptogein (4). Photographs of the infiltrated leaves were taken at 3 days post-infiltration. Experiments were repeated four times, and four leaves from two individual plants were used in each experiment.

Expression of NbCA1 is induced during N-mediated HR-PCD. Total RNA was extracted from temperature-shifted TMV-infected wild-type and N-containing plants at time points indicated (hours post-temperature shift, hps) and analysed by RT–PCR using NbCA1 and EF1α gene-specific primers. EF1α was used as an internal control. Pictures are taken from the agarose gels loaded with 25 cycle PCR products. M, DNA marker size ladder.

Downregulation of NbCA1 alters calcium signature triggered by cryptogein elicitor. (A–E) Calcium oscillation triggered by cryptogein. Nuclear calcium oscillation of 5 μg/ml (A) and 10 μg/ml (B) cryptogein-treated control plant cells. Nuclear calcium oscillation of 5 μg/ml (C), 10 μg/ml (D) and 25 μg/ml (E) cryptogein-treated NbCA1-RNAi plant cells. Red, blue, black, yellow and green colour lines represent different individual cells. (F) Amplitude, duration and frequency of calcium oscillation in control and NbCA-RNAi plant cells treated with different concentration of cryptogein. (G) Amplitude of nuclear (upper panel) and cytosolic (lower panel) calcium spikes of 10 and 25 μg/ml treated control plant cells and 5 μg/ml cryptogein-treated NbCA1-RNAi plant cells. The individual bar represents amplitude of individual calcium spike from 14 independent control plant cells and 11 independent NbCA1-RNAi plant cells. (H) HR-PCD is induced by 25–500 μg/ml cryptogein in the control plants (upper panel) and NbCA1-RNAi plants (lower panel). Number 1–5 represent 25, 50, 100, 250 and 500 μg/ml cryptogein, respectively.