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. 2022 Feb 2;34(5):1684–1708. doi: 10.1093/plcell/koac032

Figure 2.

Figure 2

Inducible expression of XopS-GFP in transgenic Arabidopsis lines interferes with MTI and triggers JA signaling. A, Expression of XopS-GFP supports growth of Pst DC3000 ΔhrcC type-III defective bacteria. WT Col-0 and XopS-GFP transgenic Arabidopsis plants of indicated independent lines were treated with 50 µM β-estradiol and syringe-inoculated after 24 h with Pst DC3000 ΔhrcC bacteria at OD600 = 0.00002. CFU were determined 5 dpi. Bars represent the mean of at least n = 3 biological replicates and two technical replicates per biological replicate ± SD. Letters above bars represent a statistical significance determined by one-way ANOVA (P < 0.05). The experiment was carried out 3 times with similar results. B, Chlorotic phenotypes of indicated independent transgenic Arabidopsis lines expressing XopS-GFP under the control of the β-estradiol inducible promoter compared to the untransformed Col-0 control. Pictures were taken 5 days after spraying 50 µM β-estradiol. C, Gene expression analysis of JA marker genes in one representative XopS-GFP transgenic Arabidopsis line. Samples were taken 4 h after β-Estradiol induction. Total RNA was extracted and the mRNA levels of indicated marker genes was measured by RT-qPCR and compared to mock treated leaves. UBIQUITIN CONJUGATING ENZYME 9 was used as a reference gene. Bars represents the mean of n = 5 biological replicates ± SD and asterisks (**P < 0.01) mark significant differences according to Student’s t test. The experiment was carried out twice with similar results. D, JA levels in one representative XopS-GFP transgenic Arabidopsis line 24 h after induction of protein expression by spraying 50 µM β-estradiol, compared to Col-0 plants. - indicates plants that were sprayed with the mock control. DW, Dry weight. Bars represent the mean of n = 5 pools of four independent plants each ±SD. Letters above bars represent the statistical significance as determined by one-way ANOVA (P < 0.05). E, Stomatal aperture measurement in Col-0 and transgenic Arabidopsis XopS-GFP line 5 upon induction of protein expression with 50 µM β-estradiol. Leaf discs were floated on water (control) or on water supplemented with 25 µM flg22 for 2 h prior to the measurement of stomatal aperture under a microscope. Approximately 100 apertures from n = 3 independent plants were measured per individual treatment and are represented as width/length ratio. Bars represent the mean ± se. Letters above bars represent the statistical significance determined by one-way ANOVA (P < 0.05). The experiment was carried out twice with similar results. F, Translocation of XopS-HA supports growth of COR−Pst DC3000 bacteria. Arabidopsis Col-0 plants were surface-inoculated with Pst DC3000, Pst DC3000 COR− complemented with EV or the complementation strain Pst DC3000 COR− (XopS-HA) at OD600 = 0.2. CFU were determined 5 dpi. Bars represent the mean of n = 8 biological replicates (and two technical replicates per biological replicate) ±sd. Significant differences are marked by asterisks (****P < 0.0001; **P < 0.01) according to one-way ANOVA. The experiment was carried out 3 times with similar results. G, Stomatal aperture measurement in Arabidopsis Col-0 leaf discs floated on water (mock control), Pst DC3000, Pst DC3000 COR− complemented with EV or the complementation strain Pst DC3000 CO-− (XopS-HA) at OD600 = 0.2. The measurement of stomatal aperture under a microscope was performed 3 h posttreatment. Approximately 100 apertures from n = 4 different plants were measured per individual treatment and are represented as width/length ratio. Bars represent the mean ± se and letters above bars represent statistical significance determined by one-way ANOVA (P < 0.05). The experiment was carried out twice with similar results.