Figure 2—figure supplement 3. RNAi off-target controls and phenotypes associated with either overexpression of brush or a null BRUSH allele.(A) RT-qPCR of CNGC.IVA3, CNGC.IVA4, and CNGC.IVA5 in hairy roots transformed with an empty vector control (control) or an RNAi fragment targeting either the 5'UTR (5'RNAi) or 3'UTR (3'RNAi) of brush in the brush mutant. The normalized fold expression is shown relative to the average expression level in control roots transformed with the empty vector (n = 6 for all constructs). Transgenic root tissue was isolated 6 weeks after inoculation with rhizobia. (B) Hairy root formation efficiency of Gifu wild-type plants inoculated with A. tumefaciens carrying a T-DNA containing either UBQpro:BRUSHgenomic or UBQpro:brushgenomic or empty vector as a control. Numbers on the columns indicate the number of plants with GFP-positive hairy roots per total number of inoculated plants. (C) A BRUSH TILLING line (SL1484-1) with a G357A exchange leading to a stop codon (W119Stop) was isolated. Nodule formation in homozygous SL1484-1 plants (n = 3) was not impaired relative to wild-type (n = 8). Plants were cultivated at 26°C and inoculated with M. loti MAFF DsRed. Nodules were counted 2 weeks after inoculation. Letters in (A,C) indicate statistical groups. (A) ANOVA followed by Tukey’s HSD test; F(8, 45)=0.9405, p > 0.05, (C) t-test, p > 0.05.
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