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. 2019 Jul 15;9:10230. doi: 10.1038/s41598-019-46695-7

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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CymMV and ORSV viral RNA and siRNA accumulation in WT and transgenic plants inoculated with C+O. (A) Illustrations of sampling tissues in WT and transgenic line pH7W-COCP line3 (pH7W-3) after Leaf 3 (L3) and leaf 4 (L4) co-inoculated with C+O. L3, L4, Stem between L4 and L5 (S4), Stem between L5 and L6 (S5) and systemic leaf 5 (L5) were harvested for tissue blotting (B) and RT-PCR (C) at 14 dpi. (B) The tissue blots prepared from various tissues of WT and transgenic line were hybridized with DIG-labeled CymMV- and ORSV-specific CP probes. (C) Accumulation of CymMV and ORSV viral RNA in L2, S4, S5 and L5 of WT and transgenic line by RT-PCR at 14 dpi. CymMV RNA was detected by primer pair (CymMV RdRp-F and CymMV RdRp-R) of expected size 152 bp and ORSV RNA by primer pair (ORSV RdRp-F and ORSV RdRp-R) of 151 bp. Actin was used for loading controls. (D) Detection of the vsiRNAs in mock and C+O infected IL of WT and transgenic pH7W-3 at 10 dpi by [γ-³²P] CTP-labeled CymMV or ORSV CP probes. The lower panel shows hybridization of U6 probes as equal loading control. The positions of 21 nt RNA is indicated by arrows. M: marker; mock: water inoculation; C+O: inoculated with CymMV and ORSV.