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. Author manuscript; available in PMC: 2018 Jul 15.
Published in final edited form as: Nat Genet. 2018 Jan 15;50(2):186–192. doi: 10.1038/s41588-017-0032-5

Figure 3. miR845b-dependent easiRNA biogenesis from transgenes and transposons.

Figure 3

(a) GFP sensor including 3′UTR with a miR845b target site and driven by the UBIQUITIN10 (UBQ10) promoter in wild type and dcl1-5/+ heterozygous background. GFP expression was restored in dcl1 pollen, allowing FACS-purification of wild type, dcl1, dcl2/4 and dcl1/2/4 pollen grains. Scale bars represent 30 μm. (b) Loss of GFP siRNA was detected in dcl1, dcl2/4 and dcl1/2/4 pollen grains, indicating that miR845b triggers DCL2/4-dependent secondary siRNA from the GFP transgene. (c) Small RNA sequencing from wild type and mutant FACS-sorted pollen revealed that 21- and 22-nt TE siRNA were lost in the dcl2/4 mutants, while miRNAs were depleted in dcl1. (d) miR845a and miR845b were depleted in dcl1 mutant and Ler-0 pollen, but miR845b was restored in transgenic Ler-0 plants expressing Col-MIR845b (Ler:MIR845b). (e) 21- and 22-nt TE-derived siRNA levels were also depleted in wild-type Ler-0 pollen, but restored in transgenic Ler:MIR845b. RPM, reads per million.