Figure 1.
Growth of Pst hrcC- (A) and Psp 1448a (B) in Col-0, tga1-1 tga4-1, npr1-1 and tga1-1 tga4-1 npr1-1 plants.
Four-week old leaves were syringe-infiltrated with a bacterial suspension of Pst hrcC- at 105 colony forming units (cfu) ml-1 or Psp 1448a at 106 cfu ml-1. The error bars represent the standard deviation of six replicates, each containing 8 leaf discs from one plant. An ANOVA of the log-transformed data was performed at α = 0.05; treatments with common letters over bars are not significantly different from each other. Post-hoc tests are presented in Table S2. Each experiment was repeated twice with similar results.
Figure 2.
Defence-related gene expression in Col-0 and tga1-1 tga4-1.
Four-week-old leaves were syringe-infiltrated with 108 cfu ml-1 of Pst hrcC- or Psp 1448a. Leaf tissues from three plants were collected and pooled as one sample for RNA isolation. Values were normalized to the expression of UBIQUITIN5. An asterisk indicates a statistically significant difference compared with Col-0 at the same time point (p<0.05, Student’s t-test), and two asterisks indicate p<0.01. The error bars represent the standard deviation of three biological samples.
Figure 3.
MAMPs- and pathogen-induced callose deposition in Col-0 and tga1-1 tga4-1 plants.
Four-week-old leaves were syringe-infiltrated with 108 cfu ml-1 of Pst hrcC- or Psp 1448a, 5µM flg22, and 10mM MgCl2 as control. Leaves were stained with Aniline blue and observed under a florescent microscope 12 h after treatment. Microscopic photographs of callose deposits are shown with the number of callose deposits indicated below each. Results are presented as means ± standard deviation. An asterisk indicates a statistically significant difference between Col-0 and tga1-1 tga4-1 (p<0.05, Student’s t-test), and two asterisks indicate p<0.01. Each experiment was repeated three times with similar results. Scale bar = 0.1mm, all photos are at the same magnification.
Figure 4.
MAMPs-induced oxidative burst in Col-0 and tga1-1tga4-1 plants.
Four-week-old leaf discs (3 per each sample) were treated with or without 2 µM flg22 in the presence of luminol, and the H2O2 generated was measured every 30 sec after treatment for 20 min. The error bars represent the standard deviation of six replicates. The experiment was repeated five times with similar results.
Figure 5.
PR-1 protein accumulation in Col-0 and tga1-1 tga4-1 after pathogen inoculation.
Four-week-old leaves were syringe-infiltrated with 108 cfu ml-1 of Pst hrcC- or Psp 1448a. Intercellular washing fluids (IWFs) and total protein were collected at 0, 1 and 2 dpi, separated on 16% Tricine-SDS-polyacrylamide gels and blotted with a PR-1 antibody. The Arabidopsis PR-1 protein has apredicted molecular weight of 16 kilodaltons (kDa) [74]. Arrows indicate the position of a 17-kDa molecular weight masker. The same gels were stained with Coomassie Brilliant Blue R250 (Sigma) as a loading control. These experiments were repeated three times with similar results.
Figure 6.
Tunicamycin sensitivity of Col-0 and the tga1-1 tga4-1 plants.
A, Five-day-old seedlings of Col-0 and the tga1-1 tga4-1 double mutant grown on Murashige and Skoog (MS) agar medium with different concentrations of TM were transplanted to TM-free MS agar and grown for a further 5 days prior to photography. This experiment was repeated three times with similar results.
B, Five-day-old seedlings of Col-0, tga1-1 tga4-1, and a line overexpressing TGA1 in the tga1-1 tga4-1 background (TGA1OE/tga1x4) grown on TM-free MS agar were submerged in MS liquid medium with or without 0.8 µg ml-1 TM for 6 h, and were allowed to recover for 5 days without TM prior to photography.
C, Fresh weight of seedlings in (B) was quantified. The fresh weight of TM-treated seedlings was divided by the average fresh weight of 5 untreated seedlings to generate percentage of control. The results are averages ± standard deviation (n=5). An ANOVA of data was performed at α = 0.05; treatments with common letters over the error bars are not significantly different from each other. This experiment was repeated twice with similar results.
Figure 7.
ER stress gene expression in Col-0 and the tga1-1 tga4-1 plants.
Ten-day-old seedlings were immersed with 5 µg ml-1 TM for the indicated time periods. Fifty mg samples were collected and pooled for RNA isolation. Values were normalized to the expression of UBIQUITIN5. An asterisk indicates a statistically significant difference compared with Col-0 at the same time point (p<0.05, Student’s t-test), and two asterisks indicate p<0.01. The error bars represent the standard deviation of four biological samples. The oligonucleotides used in (B) did not distinguish between BiP1 and BiP2. Accordlingly, the target is referred to as BiP1/2.