Figure 1.
IRE1 is involved in abiotic stresses.
A, The transcript accumulation of IRE1a and IRE1b and B, SRO2 and GLP1 in response to Tm treatment for 0, 2 and 5 hours in the listed genotypes measured by real-time RT-PCR. Induction of IRE1a, IRE1b, SRO2 and suppression of GLP1 can be visualized in the treated wild-type Col-0. IRE1a and IRE1b gene expression was analyzed to confirm the absence of mRNA in their respective T-DNA insertional mutants. Data represent the mean and SE of three technical replicates per treatment. Statistical analysis was performed using Student's t-test, *, p<0.05, **, p<0.01, ***, p≤0.001. Experiments with at least two independent biological replications demonstrate similar results. C, Abiotic-dependent UPR was induced in the wild-type and indicated mutant seedlings by growing them on MS medium containing 0.3 µg/mL Tm for three days. Percentage of recovery was plotted by calculating alive/dead seedlings recovered ten days post Tm treatment. Statistical analysis was performed using Student's t-test, *, p<0.05, ***, p≤0.001. Experiments were repeated at least three times with similar results.
Figure 2.
UPR-responsive genes and PR1 secretion is affected in ire1 mutants.
A, The expression of IRE1a and IRE1b and B, SRO2 and GLP1were quantified in response to SA and Psm ES4326(avrRpt2) for 4 hours in the indicated genotypes using real-time RT-PCR. Increased expression of IRE1a, IRE1b, SRO2 and reduced transcript of GLP1 can be observed in the treated wild-type Col-0. Data represent the mean and SE of three technical replicates per treatment. Statistical analysis was performed using Student's t-test, *, p<0.05, **, p<0.01, ***, p≤0.001. Experiments with at least two independent biological replications demonstrate similar results. C, PR1 protein accumulation in the ire1 mutants was compared with wild-type. Intercellular wash fluid (IWF) was collected from 20 leaves derived from 10 plants per indicated genotype treated with SA for 16 hours. Total protein was extracted from five leaves derived from three plants per indicated genotype treated with SA for 16 hours. Accumulation of PR1 was detected by Western blotting with anti-PR1 antibody in IWF and total leaf extract from the indicated genotypes. Ponceau S stain verifies equal loading. Experiments were repeated at least four times with similar results.
Figure 3.
IRE1 is required to mount effective systemic acquired resistance.
A, Bacterial growth (colony forming unit – cfu/leaf disc, expressed on a log scale) of leaves of the indicated genotypes infected with Psm ES4326 (OD = 0.0002). Bacterial growth was assessed at 3 dpi. Hypersusceptible npr1 mutant was used as control. Error bars: 95% confidence interval of the mean (n = 8). Bars connected by the same letter did not differ from each other at p<0.05 (Tukey's HSD tests). B, Chemical SAR was established by treating indicated genotypes with 1 mM SA, while uninduced plants were sprayed with water 16 hours prior to Psm ES4326 (OD = 0.001). Bacterial growth was monitored 3 days post infection. Hypersusceptible npr1 mutant was used as control. Error bars represent 95% confidence interval of the mean (n = 8). Bars within a class connected by the same letter (lowercase for water treatment; uppercase for SA treatment) did not differ from each other at p<0.05 (Tukey's HSD tests). All the experiments were performed at least three times with similar results.
Figure 4.
bZIP60 mRNA splicing is stimulated by chemicals that trigger the UPR.
A, Schematic representation of two approaches used to detect the bZIP60 mRNA spliced forms. Primers sets flanking the putative splicing regions (solid arrows) are indicated (Top) to amplify bZIP60u and bZIP60s forms using RT-PCR. Alternative, RT-PCR products are subsequently digested using Alw21I restriction enzyme (Bottom). The latter approach will highlight the length differences between bZIP60u and bZIP60s since the Alw21I restriction site is present in bZIP60u and absent in bZIP60s. bZIP60u and bZIP60s PCR products upon digestion are shown. B, Processing of bZIP60 mRNA was analyzed by gel electrophoresis in agarose (3.5% p/v). RT-PCR products (Top) or RT-PCR products digested with Alw21I (bottom) were obtained from RNA samples of Arabidopsis seedlings (6-day-old) treated for 2 hours with several chemicals that trigger the UPR (Tm 5 µg/mL; DTT 5 mM; CPA 100 µg/mL; Thapsigargin 500 nM). DMSO and water-treated samples served as mock controls for chemicals. Asterisk indicates a hybrid band formed by the bZIP60u and bZIP60s PCR products. Such hybrid band has been also observed and documented in RT-PCR analysis of XBP-1 processing [56]. Elongation factor 1 alpha (EF1α) expression served as a control. All the experiments were performed at least three times with similar results.
Figure 5.
T-DNA insertions in both IRE1 genes affectbZIP60 processing under ER stress conditions.
A, RT-PCR products derived from bZIP60 mRNA were digested with Alw21I and resolved by gel electrophoresis in agarose (3.5% p/v). RNA samples were obtained from wild-type or ire1b-4, ire1a-2 and ire1a-2 ire1b-4 mutant seedlings (6-day-old) treated with Tm for 2 hours. IRE1b and IRE1a gene expression was analyzed to confirm the absence of mRNA in their respective T-DNA insertional mutants. Elongation factor 1 alpha (EF1α) gene expression served as a control. B, Quantitative measurement of bZIP60 splicing activity. cDNA was made from the leaf tissue of 3-week-old plants of the indicated genotypes, untreated or infiltrated with 0.5 µg/mL Tm for 2 hours and 5 hours. Ratios of fold induction of spliced and unspliced bZIP60 are plotted, while setting ratio of Col-0 as 100%. Statistical analysis was performed using Student's t-test, *, p<0.05, ***, p≤0.001. All the experiments were performed at least three times with similar results.
Figure 6.
Salicylic acid stimulates bZIP60 processing.
A, RT-PCR products derived from bZIP60 mRNA were digested with Alw21I and resolved by gel electrophoresis in agarose (3.5% p/v). RNA samples were obtained from seedlings (6-days-old) of wild-type plants treated with salicylic acid (SA) for the indicated time. As a positive control we used a RNA sample obtained from seedlings treated with DTT (5 mM) for 2 hours. GRXC9 gene expression served as control for the action of SA at transcriptional level [29], [57]. Elongation factor 1 alpha (EF1α) gene expression served as a control. B, Pathogen infection and SA induce bZIP60 splicing in IRE1a-dependent manner. cDNA was made from the leaf tissue of 3-week-old plants of the indicated genotypes infected with Psm ES4326(avrRpt2) or sprayed with SA for 4 hours. Ratios of fold induction of spliced and unspliced bZIP60 in the listed genotypes are plotted, while ratio of Col-0 was set as 100%. Statistical analysis was performed using Student's t-test, **, p<0.01, ***, p≤0.001. All the experiments were performed at least three times with similar results.
Figure 7.
SA-induced up-regulation of UPR responding genes is altered in ire1 and bzip60 mutants.
Plants (Col-0, bzip60 and ire1a-2 ire1b-4) were treated with SA for 3 and 5 hours. RNA was extracted and quantitative PCR was performed for BiP1/2, calreticulin 2 (CRT2) and the UDP-glucose transporter (UTr1). The results were normalized against a housekeeping gene (putative clathrin adaptor). Experiment was performed at least three times with similar results.
Figure 8.
bZIP60 is involved in plant defense.
A, Col-0, bzip60, ire1a-3 ire1b-4 and hypersusceptible npr1 mutant were infected with Psm ES4326 (OD = 0.0002). Bacterial growth (colony forming units – cfu/leaf disc, expressed on a log scale) was quantified in the leaves of indicated genotypes at 3 dpi. Error bars represent 95% confidence intervals of the mean (n = 8). Bars connected by the same letter did not differ from each other at p<0.05 (Tukey's HSD tests). B, Chemical SAR was established by treating Col-0, bzip60, ire1a-3 ire1b-4 and hypersusceptible npr1 mutant with 1 mM SA or mock (water) 16 hours prior to Psm ES4326 (OD = 0.001) infection. Bacterial growth was monitored 3 days post inoculation. Error bars represent 95% confidence intervals of the mean (n = 8). Bars within a class connected by the same letter (lowercase for water treatment; uppercase for SA treatment) did not differ from each other at p<0.05 (Tukey's HSD tests). All the experiments were performed at least three times with similar results.