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. 2014 Dec 3;66(5):1191–1203. doi: 10.1093/jxb/eru467

Fig. 5.

Fig. 5.

RNAi for the HvTIP3:1 gene interferes with ABA-mediated inhibition of PSV coalescence. (A) Diagram of the constructs used in transient expression assays. The C-terminal coding region or 167bp of the highly gene-specific 3’UTR of HvTIP3;1 was used to construct RNAi vectors to repress expression of HvTIP3;1 in aleurone cells. Expression of RNAi constructs and the GFP gene was under the control of the maize ubiquitin promoter, which works effectively as a constitutive and strong promoter in monocot grain. (B) Five stages of vacuolation in barley aleurone cells. The five stages indicated were used as an index of vacuolation in aleurone cells. (C) Transient expression assay of RNAi-TIP3;1 for PSV coalescence. Barley aleurone protoplasts were co-transfected with RNAi-TIP3;1/Ubi::GFP or empty vector/Ubi::GFP and incubated in medium containing ABA (10 μM) for up to 30h. At the indicated times, protoplasts from each transfection were examined for the status of vacuolation using the five categories indicated in (B). Bars indicate the number of protoplasts of each stage ± standard deviation after incubation with ABA for the indicated time. (D) Transient expression assay of RNAi-TIP3;1 3’UTR for PSV coalescence. The co-transfected protoplasts were assayed after 30h of incubation with ABA (10 μM) as described above.