Expression analysis of MhAGO1 in response to B. dothidea infection. (A) Northern blot analysis of mature miR168 and MhAGO1 mRNA. LMW RNA was probed with DIG-labeled oligonucleotides complementary to miR168 and U6, while the total RNA was hybridized to the probe as indicated in the schematic of MhAGO1. FL, full length; 5′ CF, 5′ cleavage fragment. The experiment was repeated three times, and similar results were observed. (B) RT-qPCR analysis of uncleaved MhAGO1 mRNA and 5′ cleavage products. The white column indicates the accumulation of MhAGO1 uncleaved mRNA, which was amplified using a primer pair flanking the cleavage site. The black column represents the data obtained using a primer pair located upstream of the target site, which provided both uncleaved mRNA and 5′ cleavage products. (C) RT-qPCR analysis of the accumulation of 3′ cleavage products of MhAGO1. The total mRNA exacted from inoculated or mock-treated M. hupehensis leaves was ligated with the adaptor used in the 5′ RLM-RACE and subsequently reverse transcribed. A sense primer located in the adaptor region and a gene-specific antisense primer were used to amplify the 3′ cleavage products. (D and E) RT-qPCR analysis of GUS and AGO1 mRNA abundance in leaves transiently transformed with a pMhAGO1::GUS construct and treated with B. dothidea (D) and SA (E). EF-1α transcripts were used to normalize all expression values. The data represent the relative expression level compared with the corresponding mock-treated controls at each time point. Data represent the mean ± SD of three biological replicates (n = 3). RT-qPCR analysis included three technical replicates of each biological replicate. Significant differences between time points are indicated with asterisks in (B) and (C). Asterisks in (D) and (E) indicate significant differences between GUS and MhAGO1 (*P ≤ 0.05; **P ≤ 0.01).