Over-expression of lariat41 caused reduced miRNA accumulation.

(A) Strategy to over-express lariat41 in Arabidopsis. Full length genomic sequence (indicated as lariat41-OE/gDNA-OE) or full length coding sequence (indicated as local41-OE/CDS-OE) was driven by the 35S promoter and fused by an YFP tag, and introduced into plants. Grey boxes indicate exons, and lines indicate the intron. (B) Morphological phenotypes of transgenic plants over-expressing lariat 41 (lariat41-OE) and the corresponding gene of lariat41 (local41-OE). Curly leaves, late flowering, altered phyllotaxy, and reduced fertility were shown in lariat41-OE. More than 10 independent transgenic lines were obtained for each. (C) RT-PCR and western blot analysis to detect expression of the corresponding gene of lariat41. Total RNA from inflorescences of a representative T3 transgenic line for each was used for cDNA synthesis, and At5g37720 was amplified to indicate the mRNA level of the corresponding gene, UBQ5 as the loading control. In similar, YFP was detected using total protein from inflorescences by western blot, Hsc70 as the loading control. Two additional biological replicates were performed, and similar results were obtained. (D) RT-PCR to detect expression of lariats. Total RNA from inflorescences of a representative T3 transgenic line for each was used for cDNA synthesis, and lariat41 was amplified, lariat28 was used as the negative control, and UBQ5 as the loading control. Two additional biological replicates were performed, and similar results were obtained. (E) miR159 and miR167 northern blot analysis in Col-0, local41-OE, and lariat41-OE. U6 was used as a loading control. Another biological replicate was performed, and similar results were obtained.