(A), Ten translationally silent nucleotide
changes (shown in red) were introduced to the CHLI sequence to disrupt
the binding between Y-Sat siRNAs and the CHLI mRNA. The amino acid
sequence is given above. (B), Schematic
diagrams of wild-type (wt) and mutant (mt) CHLI overexpression
constructs plus the RNAi construct used for tobacco transformation.
35S-P, cauliflower mosaic virus 35S promoter; SSU-P, tobacco rubisco
small subunit promoter; Ocs-T, Agrobacterium
tumefaciens octopine synthase 3′ terminator region.
Empty boxes represent introns of the CHLI genomic sequence; red lines
indicate the sequence-modified region. The RNAi construct contains a
partial (571 bp) sense (sCHLI) and antisense (asCHLI) CHLI sequence
spanning the Y-Sat target site. (C),
Tobacco plants transformed with wild-type CHLI constructs develop the
yellowing symptoms upon CMV Y-Sat infection, but those transformed with
mutant CHLI constructs do not show the symptoms. The picture was taken
20 days post-inoculation. Some of the CMV Y-Sat-infected wtCHLI plants
showed a delayed onset of the yellowing symptoms, presumably because the
CHLI transgene provided additional CHLI mRNA to the endogenous
transcript, causing a reduction in the rate of CHLI silencing.
(D), An example of reciprocal
grafting between CMV Y-Sat-infected mtCHLI plants and wtCHLI plants.
Only the wtCHLI scion or root stock developed the yellowing symptoms.
(E), Northern blot hybridization shows that the CHLI
mRNA expressed from the mtCHLI construct is resistant to Y-Sat-induced
silencing. The ‘+’ symbol indicates samples from plants
infected with CMV Y-Sat, while the ‘−’ symbol
identifies samples extracted from plants prior to CMV Y-Sat infection.
wt1, wt2, mt3 and mt6 are line numbers for the 35S-gwtCHLI and
35S-gmtCHLI transformants. Gel lane numbers are given at the bottom.