PR-2 | Pathogenesis-related protein 2

While growth retardation of siz1 is hardly rescued at 28°C, other hallmarks of auto-immunity fully recover at this temperature.(A) Picture of the rosettes of siz1 and SNC1-dependent auto-immune mutants (crp1-2, bon1, snc1-1, srfr1-4) grown for 5 weeks in SD conditions at 22°C or 28°C. The mutants are in the Col-0 background. (B) Box-plot (middle bar = median, box limit = upper and lower quartile, extremes = Min and Max values) depicting the rosette weight of the genotypes shown in (A). Plants were 5-week-old plants. Significant differences were detected using a two-way ANOVA with Tukey’s multiple comparisons test: Genotype p-value<0.0001 (29% of the variation), Temperature p-value<0.001 (37%); GxT interaction p-value<0.0001 (22%); letters indicate significantly different post hoc groups (n = 8–10). The experiment was repeated 3 times with similar result. (C, D) Similar to (A), plants were grown in parallel for 5-weeks at 22°C or 28°C. The top row depicts wild type Col-0 and the single mutants, while the bottom row depicts siz1 and siz1 crossed with the mutants of the top row. (E) Box-plot with the rosette weight of the plants of panels (C, D). The statistical test was similar to (B) with similar result (n = 8). siz pad4-1, siz1 eds1-2, and siz1 NahG show a small recovery at 22°C (C) without any additional effect at 28°C (D). The experiment was repeated three times with similar result. Left side, single mutants; right side, siz1 and the corresponding double mutants. (F) Spontaneous cell death is absent in siz1 at 28°C, but also 22°C when EDS1/PAD4 are mutated or SA accumulation is compromised (NahG). Fully elongated leaves of 5-week-old plants were stained with Trypan blue and examined under the microscope. (G) Accumulation of PR1/PR2 in siz1 double mutants grown at 22°C or 28°C. srfr1 and bon1 are shown as control for PR accumulation. Total protein was extracted from 5-week-old plants. PR proteins were detected with polyclonal antibodies. Blots were stained with Ponceau S to confirm equal protein loading (? = Rubisco). The blots shown are a single exposure on one film of gels run/blotted in parallel with the samples taken in parallel as well. The apparent Mw of marker proteins is shown on the left.

The siz1 auto-immune phenotype is partially rescued by loss of SNC1.(A) Picture of the rosettes of siz1 and the siz1 snc1-11 double at 22°C/28°C. At 22°C growth retardation of siz1 is partially recovered in the snc1-11 background, while at 28°C siz1 snc1-11 does not show any additional recovery to siz1. Plants were 5-weeks-old (SD). (B) Box plot showing the rosette weight of the plants in (A). Significant differences were determined using a two-ay ANOVA followed by a Tukey post-hoc test: Genotype p-value<0.0001 (75% of the variation), Temperature p-value = 0.0033 (2.1%); GxT interaction p-value<0.0001 (8.8%). The letters indicate statistically different post hoc groups (n = 8) (C) Spontaneous cell death in siz1 requires SNC1 function. Fully elongated leaves of 5-week-old plants were stained with Trypan blue. (D) Accumulation of PR1 and PR2 is partially suppressed in siz1 snc1-11. As control for thermosensitive accumulation of PR proteins, srf1-4, snc1-1, bon1 and cpr1-2 are shown. Total protein was extracted from 5-week-old plants. The blots were prepared in parallel and ECL detection was done on one film, except for the ‘long exposure’ to reveal PR2 accumulation. Blots were stained with Ponceau S to confirm equal protein loading. Asterisks mark enhanced PR2 accumulation in siz1-2 and siz1 snc1-11 at 28°C. (E) Normalized gene expression of PR1, PR2 and SNC1 (mean ± SE) in 5-week-old plants (fold change; Col-0 at 22°C = 1). PR1 and PR2 expression are still elevated in siz1 snc1-11 at 22°C. At 28°C PR1 expression is gone in siz1 snc1-11, while PR2 expression remains up regulated (7-fold up).