Article Figures & Data
Figures
- Figure 1.
Methylation Associated with VIGS of GFP.
(A) Structure of the GFP transgene, including the 35S promoter (35S; open box); the chitinase endoplasmic reticulum targeting signal sequence (C); 5′ 453 bp of GFP (GF; solid box); 3′ 359 bp of GFP (P; hatched box); the nopaline synthase terminator (tnos); sites for the restriction endonucleases AluI (A), HaeIII (H), and Sau96I (S); and sizes of expected digestion products in kilobases. Restriction sites marked by asterisks contain cytosines in a symmetrical (CpG or Cp-NpG) configuration.
(B) and (C) DNA gel blot analysis of samples from nonrecombinant PVX-infected, nonsilenced leaves (lanes 1, 5, and 9) and PVX-GFP–silenced (lanes 2, 6, and 10), PVX-GF–silenced (lanes 3, 7, and 11), and PVX-P–silenced (lanes 4, 8, and 12) leaves of 16c plants. Results are representative of at least three separate experiments. DNA samples were digested with AluI, Sau96I, or HaeIII as indicated. The blot was probed with a full-length GFP-specific probe (B) followed by reprobing with a N. benthamiana hsp70–specific probe (C). Results for lines 16c (data shown) and 8a (data not shown) were identical. Sizes (in kilobases) of relevant DNA fragments are indicated.
- Figure 2.
VIGS of 35S:GFP by PVX-35S.
(A) Genomic organization of the PVX vector carrying the 35S promoter sequence. The PVX open reading frames are shown as RdRp (RNA-dependent RNA polymerase), 25K (25-kD protein), 12K (12-kD protein), 8K (8-kD protein), and CP (coat protein). The 35S insert was a 347-bp fragment carrying the 35S promoter sequence inserted downstream of a duplicated coat protein promoter (dark boxes).
(B) Upper leaves of 16c plants that were either mock-inoculated (leaf 1) or inoculated with PVX-GFP (leaf 2) or PVX-35S (leaf 3). It is clear that silencing exhibited by PVX-35S plants is not as complete as that exhibited by PVX-GFP plants. The leaves were photographed at 30 DPI.
(C) GFP mRNA levels in mock-inoculated (M; lane 1) or PVX-35S–inoculated (lanes 2 to 7) 16c plants. Lanes 2 to 4 contain samples prepared from tissue of infected plants that was not exhibiting GFP silencing (non-sil). Lanes 5 to 7 are samples from tissue that was exhibiting GFP silencing (sil). Five micrograms of total RNA was run per lane, and a probe specific for GFP was used for detection.
(D) PVX-35S RNA levels in systemically infected leaves. Samples were prepared from tissue of infected 16c plants that was not (nonsil; lanes 1 to 4) or was (sil; lanes 5 to 8) exhibiting silencing of the GFP transgene, and from infected nontransformed N. benthamiana (NT; lane 9). One microgram of total RNA was loaded per lane, and a probe specific for the 35S promoter was used for detection. The genomic (g) and major subgenomic (sg) RNA species are labeled.
- Figure 3.
Differences in Methylation Associated with VIGS of 35S:GFP Induced by PVX-35S and PVX-GFP.
(A) Structure of the GFP transgene. Features shown are the 35S promoter (35S; open box), chitinase signal sequence (C), 5′ 453 bp of GFP (GF; solid box), 3′ 359 bp of GFP (P; hatched box), nopaline synthase terminator (tnos), restriction sites for Sau3A (filled triangles) and Sau96I (filled circles), and expected digestion products. Also illustrated are the largest methylated products for each enzyme detected by either the 35S (open boxes) or P (hatched boxes) probes, when silencing is induced by PVX-35S or PVX-GFP.
(B) and (C) DNA gel blot analysis of samples from nonsilenced (lanes 1 and 4), PVX-35S–silenced (lanes 2 and 5), and PVX-GFP–silenced (lanes 3 and 6) 16c tissue. DNA samples were digested with Sau3A or Sau96I as indicated and probed with the 35S promoter sequence (B) or the P 3′ 359 bp of GFP (C). Sizes (in kilobases) of relevant DNA fragments are indicated.
- Figure 4.
VIGS Induced by PVX-rbcS.
(A) Genomic organization of the PVX vector carrying 500 nucleotides of the gene encoding the Rubisco small subunit (rbcS). The genome is labeled as given in the legend to Figure 2A.
(B) rbcS mRNA levels in mock-inoculated (m) or wild-type PVX and PVX-rbcS–(PVX:rbcS) and PVX-ABP–(PVX:ABP) infected plants. RNA was prepared from systemically infected leaves at 25 DPI, and detection was by RNase protection with a probe specific for endogenous rbcS transcripts.
(C) RNA gel blot analysis of mock-inoculated (m) and PVX, PVX-rbcS (PVX:rbcS), and PVX-ABP (PVX:ABP) levels in systemically infected leaves at 25 DPI. Probes specific for viral RNA (top) and ribosomal RNA (bottom) were used. Migration of PVX-rbcS RNA is slower than that of wild-type PVX, indicating that the accumulated RNA had not developed the ability to overcome VIGS due to loss of the rbcS-derived insert.
(D) DNA gel blot analysis of samples from N. benthamiana tissue systemically infected by PVX-rbcS (lanes 1 and 3) or PVX-GUS (lanes 2 and 4) at 25 DPI. DNA was digested with AluI or HaeIII as indicated. Filters were probed with the same fragment of rbcS as that carried in PVX-rbcS.
- Figure 5.
Methylation Associated with Systemic Silencing of GFP after Agrobacterium Infiltration of GFP Sequences.
(A) Mature leaves of 16c plants that were infiltrated with either MgCl2 (left) or Agrobacterium carrying 35S:GF (right). Silencing of GFP is visible as a patch of red fluorescence in leaves infiltrated with 35S:GF. Leaves were photographed at 6 DPI.
(B) RNA gel blot analysis of RNA samples prepared from tissue infiltrated with MgCl2 (lane 1) or 35S:GF (lane 2) and systemically silenced tissue, which was mature (lane 3) or undeveloped (lane 4) at the time of infiltration. Samples 1 and 2 were taken at 7 DPI, and samples 3 and 4 were taken at 35 DPI. Five micrograms of total RNA was loaded per lane, and a probe specific for the entire GFP sequence was used. Ethidium bromide staining of the electrophoresed gel shows the rRNA loading.
(C) and (D) DNA gel blot analysis of samples prepared from tissues described in (B). DNA was digested with Sau96I and probed with the 3′ 359 bp of GFP (C) followed by reprobing with the N. benthamiana hsp70 gene (D). Sizes (in kilobases) of relevant DNA fragments are indicated.
- Figure 6.
Suppression of GFP Silencing by PVY and CMV.
(A) RNA gel blot analysis of samples prepared from nonsilenced (NS) and silenced (S) 16c plants and silenced plants that were infected with either CMV (OL and NL) or PVY. For CMV infection, RNA was taken from old leaves (OL) that had emerged before systemic infection with CMV and from new leaves (NL) that had emerged after systemic infection. Five micrograms of total RNA was loaded per lane, and hybridization was to a probe specific for GFP.
(B) and (C) DNA gel blot analysis of samples prepared from tissue as described in (A): nonsilenced (lanes 1 and 6), silenced (lanes 2 and 7), CMV-infected old (lanes 3 and 8) and new (lanes 4 and 9), and PVY-infected (lanes 5 and 10) leaves. DNA was digested with AluI or HaeIII as indicated. The blot was probed with a full-length GFP-specific probe (B) followed by reprobing with the N. benthamiana hsp70 probe (C). Sizes (in kilobases) of relevant DNA fragments are indicated.
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