Schematic diagrams of transgene constructs used in tobacco and/or tomato transformation. (Right) The predicted structure of RNA transcripts from these transgenes. RPVSat is a full-length (322-nt) sequence of the cereal yellow dwarf virus RPV satellite. The PSTVd sequence in GUS:PSTVd[+] and GUS:PSTVd[-] contains several minor sequence mutations (arising from cloning) compared with the 359-nt wild-type PSTVd-RG1 sequence (deletions of G₁₆₈ and C₃₁₈G₃₁₉ and single-nucleotide substitutions of C₁₈₅→T and C₂₃₁→G), but these mutations do not cause significant alteration to the predicted rod-like structure of the viroid RNA. The sense PSTVd sequence in hpPSTVd starts at nucleotide 16 and ends at nucleotide 355 of the RG1 strain, with one nucleotide substitution of G₂₈₁→A, and the antisense sequence is the same as that in the GUS:PSTVd fusion constructs. There are two versions (A and B) for the hpY-Sat and d/rY-Sat constructs; the Y-SatA sequence has the size of a wild-type Y-Sat RNA (369 nt) but with two single-nucleotide substitutions of A₂₉₃→G and A₃₀₈→G, and Y-SatB has a 5-nt deletion (nucleotides 192-196), which constitutes part of the yellow symptom domain, plus four single-nucleotide substitutions of G₈₀→A, G₁₂₈→ C, G₂₀₄→T, and A₂₁₄→T. The hpGUS sequence is the same as that described in ref. 18. 35S-P, cauliflower mosaic virus 35S promoter; Ocs-T, 3′ region of Agrobacterium octopine synthase gene; i/r, inverted-repeat sequences.

Expression of Hc-Pro in tobacco dramatically reduces the yellow mosaic symptom of Y-Sat. (A) Y-Sat induces severe chlorosis in Hc-Pro^- tissue but not in Hc-Pro⁺ tissue. The Hc-Pro⁺ and Hc-Pro^- plants were siblings of the same F₁ plant that was obtained by pollinating N. tobacum Wisconsin 38 with pollen from the previously described Hc-Pro plant (22). The presence or absence of the Hc-Pro transgene was confirmed by PCR analysis. (B) An Hc-Pro^- scion in a grafted plant showed severe Y-Sat symptom, whereas the shoot from the Hc-Pro⁺ stock exhibited very little yellowing. (C) Detection of plus (+) strand (Top) or minus (-) strand (Bottom) of Y-Sat RNA in Hc-Pro⁺ and Hc-Pro^- tobacco infected with Y-Sat. (Middle) RNA loading control. Twenty micrograms of total RNA was hybridized with either antisense (Top) or sense (Bottom) Y-Sat RNA probe. (D) Detection of siRNA from Hc-Pro⁺ (lanes 1 and 2) or Hc-Pro^- (lanes 3-6) tobacco infected with Y-Sat, or uninfected tobacco transformed with the hpY-Sat construct (lane 7). Total RNA (40 μg) was hybridized with the full-length antisense Y-Sat sequence. (Lower) An overexposure of Upper. Note that the infecting Y-Sat produced at least 100-fold more siRNA than the hpY-Sat transgene; hpY-SatA-3 is the same as that in Fig. 4A, where small RNA enriched from 120 μg of total RNA was loaded.

Expression of PSTVd hpRNA in tomato induces symptoms reminiscent of PSTVd infection. (A) Phenotypes of regenerated tomato shoots in tissue culture that were transformed with hpPSTVd or d/rPSTVd. (B) Phenotypes of hpPSTVd or d/rPSTVd transgenic tomato in the glasshouse. (C) T1 seedlings of hpPSTVd-4 and d/rPSTVd-3. (D) Analysis of hpPSTVd and d/rPSTVd transgenic tomato for the presence of siRNAs. Total RNA (30 μg) was hybridized with full-length antisense PSTVd RNA.

(A) Replicating Y-Sat RNA is resistant to hpRNA-induced RNA silencing. (Top) Northern blot hybridization analysis of (+) strand Y-Sat RNA in Y-Sat-infected d/rY-Sat or hpY-Sat tobacco. Total RNA (20 μg) isolated from leaf tissue collected from a pool of approximately two to five Y-Sat-infected T1 plants of each line was hybridized with antisense Y-Sat RNA probe. (Bottom) Detection of Y-Sat siRNA in uninfected d/rY-Sat or hpY-Sat tobacco. High-molecular weight RNA was removed from 120 μg of total RNA (isolated from a pool of several uninfected T1 plants of each line) by precipitation with 5% polyethylenglycol 8000 and 0.5 M NaCl, and the small RNAs were pelleted from the supernatant with 0.3 M NaOAc and 3 volumes of ethanol and used for the analysis. Hybridization was performed by using full-length antisense Y-Sat RNA as probe. (Middle) RNA loading control for Top. (B-E) Nonreplicating subviral RNA sequences are also resistant to RNA silencing. (B and D) Relative GUS activity of 5 μg of protein from tobacco transformed with GUS:PSTVd[+], GUS:PSTVd[-], or GUS:RPVSat or their supertransformants with hpGUS, hpPSTVd or hpRPVSat. The uppercase letter above each bar represents plants regenerated from the same primary transformant (for GUS:PSTVd[+] and GUS:PSTVd[+]) or T1 plant (for GUS:RPVSat), and the number above each bar indicates an independent supertransformant. (C) Detection of siRNA from a subset of plants in B by Northern blot hybridization using full-length sense PSTVd RNA as probe. (E) Northern blot hybridization analysis of duplicate GUS:RPVSat regenerants uninfected or infected with potato leaf roll virus that is required to support RPVSat replication. The blots, from 1 to 4, show total RNA hybridized with antisense GUS RNA (11); siRNA hybridized with antisense RPVSat sequence (11); total RNA hybridized with antisense RPVSat RNA, where the strong signals indicate the presence of replicating RPVSat RNA; and RNA loading control for blots 1 and 3.

A model for the role of RNA silencing in the pathogenicity and evolution of viroids and viral satellite. Replication of the subviral RNAs generates dsRNA intermediates, which are processed by Dicer into 21- to 25-nt siRNAs, and these siRNAs are then incorporated into siRNA-ribonuclease complexes (RISC). If significant sequence identity exists between a region in the subviral RNA genome and a region in host gene mRNA (shown in red), RISC will target the host gene for degradation leading to symptom development. RISC can also target the subviral genome for degradation, forcing the subviral RNA to evolve and to adopt and maintain RNA silencing-resistant secondary structure.