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

Figure 9.

Figure 9

VIGS of WRKY40 in N. benthamiana affects stomatal closure in response to a MAMP stimulus. A, Verification of NbWRKY40 downregulation in pTRV2-NbWRKY40 (NbWRKY40 silencing) compared with pTRV2 (EV, silencing control) in VIGS N. benthamiana plants. Two weeks after infiltrating N. benthamiana plants with the silencing constructs, total RNA was isolated from excised leaves treated with 5 mM SA for 4 h. The mRNA level of NbWRKY40 in pTRV2-NbWRKY40 was measured by RT-qPCR and compared to NbWRKY40 expression in pTRV2 control plants. Actin was used as a reference gene. Bars represent the mean of n = 4 biological replicates ± sd and asterisks (**P < 0.01) mark significant differences according to Student’s t test. B, Exclusion of potential off target effects during downregulation of NbWRKY40 in VIGS N. benthamiana plants. The mRNA levels of NbWRKY40a and NbWRKY40e in pTRV2-NbWRKY40 plants were measured by RT-qPCR and compared with NbWRKY40a and NbWRKY40e expression in pTRV2 control plants. Actin was used as a reference gene. Bars represent the mean of n = 5 biological replicates± sd. ns according to Student’s t test. The experiment was carried out twice with similar results. C, Stomatal aperture measurement in pTRV2-NbWRKY40 and pTRV2 VIGS N. benthamiana plants transiently expressing either GFP alone or XopS-GFP. 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 = 4 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. D, Verification of NbWRKY40 downregulation in pTRV2-NbWRKY40 VIGS N. benthamiana plants. Two weeks after infiltrating N. benthamiana plants with the silencing constructs, total RNA was isolated from excised leaves treated with 5mM SA for 4 h. The mRNA level of NbWRKY40 in pTRV2-NbWRKY40 was measured by RT-qPCR and compared to NbWRKY40 expression in pTRV2 control plants. Actin was used as a reference gene. Bars represent the mean of n = 4 biological replicates ± SD and asterisks (***P < 0.001) mark significant differences according to Student’s t test. E, Stomatal aperture measurement in pTRV2-NbWRKY40 and pTRV2 VIGS N. benthamiana plants transiently expressing either GFP alone or HopX1-GFP. 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 = 4 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.