Figure 2.

Post-transcriptional gene silencing was enhanced in scions by grafting to a strong silencing stock and self-grafting. (A) The transmission of strong silencing from sense ACO1 silencing stocks to antisense ACO1 silencing scions. ACO1 antisense line (AS) shoots were grafted onto ACO1 strong silencer V or T lines as AS/V or AS/T, with AS shoots grafted onto wild type (Ailsa Craig) as controls (AS/AC).⁵,¹⁷ Total RNAs were extracted from the scions two weeks after grafting and analyzed using Northern hybridization for AS transgene mRNA level. The AS transgene generates two AS transcripts due to an alternative polyadenylation site.⁷ The levels of the two AS mRNAs were reduced in the scions (AS/V, AS/T) grafted to V or T lines, in contrast to those grafted to the wild type AC (AS/AC). Additional AS/V or AS/T graftings were carried out and showed similar results (data not shown). (B) Self-grafting of ACO1 overexpressers led to decreased level of ACO1 mRNA in scions. The ACO1 overexpressing lines C3-1, C3-5 and C8-4 containing siRNAs were self-grafted by cutting the shoots and grafting back to the same source plants using the same method as for heterografting, C3-1/C3-1 (lane 2), C3-5/C3-5 (lane 5) and C8-4/C8-4 (lane 8). ACO1 overexpressers C3-1, C3-5 and C8-4 were also grafted to wild type (AC) stock as negative controls (C3-1/AC, lane 1; C3-5/AC, lane 4; C8-4/AC, lane 7) and onto ACO1 strong silencer V or T lines as positive controls (C3-1/V, lane 3; C3-5/T, lane 6).⁴,⁵ Samples were taken for total RNA extraction and Northern analysis at seven weeks and 10 weeks after grafting. Self-grafted scions (C3-1/C3-1, lane 2; C3-5/C3-5, lane 5; C8-4/C8-4, lane 8) showed further silencing compared to grafting to wild type, especially by week 10. (C) siRNA accumulation in self-grafted scions (C3-1/C3-1, lane 2; C3-5/C3-5, lane 5) and scions grafted to strong silencers (C3-1/V, lane 3; C3-5/T, lane 6) increased compared to the scions grafted onto wild type stocks (C3-1/AC, lane 1; C3-5/C3-5, lane 4). Predominant small RNA stained with ethidium bromide is shown as equal loading controls. (D) ACO1 AS plants were self-grafted as in (B). Reduction in the AS mRNA was observed with AS self-grafting (AS/AS, lane 2 and 5) as for those grafted onto V or T lines (AS/V, lane 3; AS/T, lane 6), but not when grafted to wild type (AC) (lane 1, lane 4). Ethidium bromide staining of rRNA is shown as a control for equal loading for all the Northern hybridizations.