Figure 4.

Analysis of amiRNAs targeting GDU1 in stably transformed Arabidopsis and transiently transformed N. benthamiana. (A) amiRNAs targeting GDU1 (GDU1 miA, miB, miC, and miD) were stably introduced into an Arabidopsis line expressing GDU1-cMyc under the control of the GDU1 promoter. About four progenies of each transformant line were analyzed. GDU1 protein levels in stems were determined by western blot using an anti-cMyc antibody, band intensities are expressed relative to the maximum (line 294A). GDU1 relative mRNA levels were measured by qRT-PCR (expressed relative to the GDU1-cMyc line). Error bars represent standard error processed by qbase^PLUS. (B) Relative GDU1 mRNA levels in transiently transformed N. benthamiana. GDU1 was co-expressed with SUC2 or amiRNA^GDU1. GDU1 mRNA level were estimated by qRT-PCR and expressed relative to levels in leaves transformed with GDU1 and SUC2. Errors bars represent standard error of at least two biological replicates. (C) Western blot showing effects of amiRNA^GDU1 on GDU1 protein accumulation in transiently transformed N. benthamiana, band intensities are expressed relative to the maximum (sample LUL1). amiRNA^SnRK1.1 (SnRK miC) was used as a negative control, and co-expression of LUL1 (a paralog from LOG2; Pratelli et al., 2012) was used as co-expression control. Numbers on the right indicate molecular weight in kDa.