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. 2016 May 19;171(4):2620–2632. doi: 10.1104/pp.16.00231

Figure 4.

Figure 4.

A Cre-Lox allele of AGO1 to study the effect of slicing on miRNA regulation. A, Schematic representation of the T-DNA containing AGO1P:3xHA-AGO1-AGO1T in Cre-LoxP DNA-excision system used to construct conditional loss of AGO1 function in the ago1-3 background (adapted from Zuo et al., 2001). Expression of the XVE chimeric transcription factor is controlled by the constitutive G10-90 promoter. XVE binding to β-estradiol leads to transcriptional activation of CRE recombinase from the OLexA-46 promoter. CRE excises the DNA segment placed between the Lox P sites from the host genome, leading to loss of 3xHA-AGO1 and to fusion of the G10-90 promoter with the downstream GFP. B, Phenotype of 20-d-old ago1Cre-Lox seedlings germinated on MS-agar media with or without 10 μm β-estradiol. GFP fluorescence in root tips of induced plants is shown below. C, Western-blot analyses of protein extracted from the aerial part of seedlings germinated on ±10 μm estradiol and harvested 15 d after germination. All lines analyzed contain the pX6 3xHA-AGO1 transgene in the different backgrounds indicated. Coomassie staining is used as loading control. Note efficient disappearance of 3xHA-AGO1 protein upon CRE induction only in lines with an additional wild-type copy of AGO1.