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. 2020 Dec 14;16(12):e1008983. doi: 10.1371/journal.pgen.1008983

Fig 1. TRV-mediated guide RNA delivery in SunTag-VP64 plants causes transcriptional activation of FWA.

Fig 1

Reverse transcriptase-qPCR was performed on total RNA extracted from leaves of individual plants at different time points post TRV inoculation to examine the accumulation of FWA mRNAs by single guide RNAs (A) and multiple guide RNAs (C). Fold expression change is relative to control plants inoculated with TRV with no guide RNA. Left panel shows FWA expression in samples collected from 3 dpi inoculated leaves and right panel shows samples from 22 dpi upper uninoculated cauline leaves in (A) and (C). Experiment 1 (Expt.1) and experiment 2 (Expt.2) indicate two independent biological experiments, each using multiple plants. Individual samples are denoted as “1a”, “2a” and so on. The prefix 1 or 2 denotes the experiment, the alphabets “a”, “b” denotes individual plants. Error bars indicate standard deviations (n = 2 technical replicates for experiment 1, and n = 3 technical replicates for experiment 2). To simplify labelling, TRV with no guide RNA is denoted as-gRNA, TRV with guide 4 as g4, and TRV with multiple guides as g4.g10.g18. Agarose gel electrophoresis of reverse transcriptase PCR products amplified to detect guide RNA specific sequences in g4 (B) and g4.g10.g18 (D) inoculated plants from experiment 1. 100 bp band corresponds to guide 4 sequence (98 bp), while 273 bp likely corresponds to unprocessed guide 4 sequence containing RNAs. Numbers at the bottom of the gels indicate lanes with samples or DNA ladder. Samples run on the lane numbers that are grouped here in parentheses (1–4), (5–6), (7–10) in (B) and (1–4), (5–8) in (D) were run on the same gel. In addition, lane (5–6) in (B) and (1–4) in (D) were run on the same gel and so have a common marker. (E) Schematic representation of primer binding sites from (B) and (D) in the modified viral RNA2 containing three separate guide RNAs. Blue arrow indicates the forward primer, green indicates reverse primers. Red arrow downstream of Coat protein (CP) denotes the sgPeBV promoter. Expected sizes of PCR products are shown at the bottom.