Figure 1.
The effect of local infection with avirulent Pst DC3000 (AvrRps4) on phenotype and chlorophyll fluorescence parameter.
A. Pst DC3000 (AvrRps4) induced HR lesions are contained in 4-week-old wild-type (Col-0). Representative images of disease symptoms were photographed at 0, 1, 2, 3, 4, 5, 6 and 8 dpi after local infection with avirulent Pst DC3000 (AvrRps4). Region I:At the site of pathogen infection, in the red dashed-line areas. Region II: Regions adjacent to the site of infection, in the yellow dashed-line areas and outside the red dashed-line areas. Region III: Uninfected systemic tissues, outside the yellow dashed-line areas. This experiment was repeated three times with similar results. B. False color images and quantitative analyses of the changes of chlorophyll fluorescence parameters Fv/Fm (Region 1, 2 and 3) induced by avirulent Pst DC3000 (AvrRps4) at 1, 2, 4 and 6 dpi, and the false color code depicted at the bottom of each image ranged from 0.000 (black) to 1.000 (purple). The statistical data of Fv/Fm support the results seen in the images.
Figure 2.
Stroma-targeted GFP bodies induction upon avirulent Pst DC3000 (AvrRps4) infection in concanamycin A-treated leaves.
A, B and C. Mesophyll cells of fresh leaves excised from the plant were infiltrated with 10 mM MgCl2 (A) or avirulent Pst DC3000 (AvrRps4) (OD600 = 0.1) (B and C) and incubated in 10 mM MES-NaOH (pH 5.5) (A and B) or in 10 mM MES-NaOH (pH 5.5) with the addition of 1 µM CA (C) at 23 °C for 12 h. D, E and F, Magnification of a mesophyll cell of leaves incubated in the conditions described for A, B and C, respectively. Chlorophyll fluorescence appears red, and CT-GFP (Stroma-targeted GFP) appears green. In merged images, the overlap of GFP and chlorophyll fluorescence appears yellow. Spherical bodies only having GFP (arrowheads) were observed. Scale bars represent 20 μm.
Figure 3.
Autophagy induction upon avirulent Pst DC3000 (AvrRps4) infection in concanamycin A-treated leaves.
A and B. Mesophyll cells of fresh leaves excised from the Arabidopsis expressing CT-GFP protein were infiltrated with 10 mM MgCl2 (A) or avirulent Pst DC3000 (AvrRps4) (B) and incubated in 10 mM MES-NaOH (pH 5.5) with the addition of 1 μM CA at 23 °C for 12 h, leaves were vacuum-infiltrated with 1 μM LTR and kept for an additional hour. C and D. Mesophyll cells of fresh leaves excised from the Arabidopsis expressing the GFP-ATG8a fusion protein were infiltrated with 10 mM MgCl2 (C) or avirulent Pst DC3000 (AvrRps4) (D) and incubated in 10 mM MES-NaOH (pH 5.5) with the addition of 1 μM CA at 23 °C for 15 h. A and B. LTR staining of autophagosomal-like structures appears red, and CT-GFP (Stroma-targeted GFP) appears green. In merged images, the overlap of GFP and LTR staining of autophagosomal-like structures appears yellow. C and D. Chlorophyll fluorescence appears red, and autophagic bodies with GFP-ATG8a fusion protein appears green. Scale bars represent 20 μm.
Figure 4.
Effect of autophagic deficiency on the behavior of chloroplast degradation in mesophyll cells.
A, B, C and D. Mesophyll cells of fresh leaves excised from the CT-GFP transgenic atg5-1 plant (A and B) or CT-GFP plant (C and D) were infiltrated with 10 mM MgCl2 (A) or avirulent Pst DC3000 (AvrRps4) (OD600 = 0.1) (B, C and D) and incubated in 10 mM MES-NaOH (pH 5.5) with the addition of 1 μM CA (A and B) or in 10 mM MES-NaOH (pH 5.5) with the addition of 1 μM CA and 10 μM 3-MA (C and D) at 23 °C for 12 h. D, Magnification of a mesophyll cell of leaves incubated in the conditions described for C, respectively. Chlorophyll fluorescence appears red, and CT-GFP appears green. In merged images, the overlap of GFP and chlorophyll fluorescence appears yellow. Spherical bodies only having GFP (arrows) and whole chloroplast degradative bodies (arrowheads) were observed. Scale bars represent 20 μm.
Figure 5.
Chloroplast degradation induction upon virulent Pst DC3000 infection in concanamycin A-treated leaves.
Mesophyll cells of fresh leaves excised from the plant were infected with virulent Pst DC3000 (OD600 = 0.1) and incubated in 10 mM MES-NaOH (pH 5.5) with the addition of 1 μM CA (C) at 23 °C for 12 h. Respectively, chlorophyll fluorescence appears red, and CT-GFP (Stroma-targeted GFP) appears green. In merged images, the overlap of GFP and chlorophyll fluorescence appears yellow. Whole chloroplast degradative bodies (arrowheads) were observed. Scale bars represent 20 μm.
Figure 6.
Expression pattern of related genes in wild-type (WT) and atg5-1 plants.
A. Expression of RPS4, EDS1, PAD4, ATG8a, NPR1, PR1 and RBCS in normal light (N) environment and low light (L) environment of wild-type and atg5-1 plants during the avirulent Pst DC3000 (AvrRps4) treatment. Total RNA was isolated from third and fourth leaves (about 0.1g, collected at 0, 1, 2, 3 and 4 days) of each plant and subjected to semiquantitative RT-PCR using gene-specific primers. 18s ribosomal RNA was used as an internal control. B. Q-PCR quantification of NPR1 and EDS1 mRNA levels in WT (gray), atg5-1 (white) 0 and 2 or 3 d after inoculation with avirulent Pst DC3000 (AvrRps4). Error bars represent SD of the mean and standard deviation of values obtained from three biological samples per genotype and time point. The asterisk indicates a significant difference from control (*, P < 0.05; **, P<0.01).
Figure 7.
Contribution of Chloroplast via Autophagy to Disease Resistance against Avirulent Pst DC3000 (AvrRps4).
A. Bacterial growth quantification of Pst DC3000 (AvrRps4) on wild-type and atg5-1, which grow in normal light (N) and low light (L) environment. 4-week-old plants were infiltrated with 1×105 cfu/ml-1 (OD600 = 0.0001) and the samples were collected at 0 (white bars) and 3 dpi (gray bars) for assay. Error bars represent SD of the mean of three samples. B. Enhanced electrolyte leakage in the wild-type and atg5-1 mutant, which grow in normal light (N) and low light (L) environment, following inoculation with avirulent Pst DC3000 (AvrRps4). The error bars display standard deviation (SD) from four technical replicates from two independent replicates.
Figure 8.
The accumulations of H2O2 induced by pathogens in WT, atg5-1, rbohD and atg5-1 × rbohD.
A. The plants (4 weeks old) were infiltrated with Pst DC3000 (AvrRps4) (OD600 = 0.2) or MgCl2 (control). DAB staining of leaves from WT, atg5-1, rbohD and atg5-1 × rbohD were taken after 24 hpi, respectively. Experiments were performed three times with similar results. B. Show are Arabidopsis leaves after infiltrating Pst DC3000 (AvrRps4) (OD600 = 0.2) or 10 mM MgCl2 (control) for 6 hpi. Then the changes of 525 nm peak values in fluorescence emission spectra were scanned for 120 min. Excitation wavelength: 488 nm; Excitation slit width: 10 nm; Emission slit width: 8.5 nm; Scanning speed: 200 nm/min; Scanning wavelength range: 510-550 nm.