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. Author manuscript; available in PMC: 2017 Mar 19.
Published in final edited form as: Nat Plants. 2016 Sep 19;2:16151. doi: 10.1038/nplants.2016.151

Figure 3. Environmental Bc-DCL1/2-sRNAs and -dsRNAs are taken into B. cinerea cells and where they silence fungal DCL genes; Bc-DCL1/2-sRNAs move from plants into fungal cells.

Figure 3

(a) Fluorescein-labeled Bc-DCL1/2-sRNAs and -dsRNAs, as well as YFP-sRNAs and -dsRNAs, were applied onto B. cinerea spores and fluorescent signals were detected in the B. cinerea cells at 12 h post culturing on agar (malt extract) ME medium. Fluorescence signals remained visible in the B. cinerea cells after MNase treatment. Fluorescein-UTP and water were used as controls. (b) Fluorescein-labeled Bc-DCL1/2-sRNAs and -dsRNAs, as well as YFP-sRNAs and -dsRNAs, were observed in B. cinerea protoplasts after MNase treatment. Fluorescent sRNAs or dsRNAs were applied onto germinated B. cinerea spores and protoplasts were isolated after culturing for 20 h. The fluorescent signals were detected within fungal protoplasts, and the MNase enzyme treatment did not reduce the fluorescent signal intensities. (c) Bc-DCL1 and Bc-DCL2 were down-regulated in Bc-DCL1/2-RNA-treated B. cinerea. (d) In vitro synthesized Bc-DCL1/2-dsRNAs were taken up and processed into sRNAs after co-culturing with the B cinerea WT strain but not with the dcl1 dcl2 mutant. (e) Bc-DCL1-sRNAs and Bc-DCL2-sRNAs were detected by stem-loop RT-PCR in B. cinerea dcl1 dcl2 protoplasts after infection on Arabidopsis Bc-DCL1/2-RNAi plants but not in mock-treated Arabidopsis Bc-DCL1/2-RNAi plants mixed with B. cinerea dcl1 dcl2 mycelium prior to protoplast formation. Similar results were obtained from two biological replicates.