Fig 1.
PSTVd secondary structure and conservation of the 5'-UUUUCA-3' stem-loop among Pospiviroid species.
(A) Secondary structure of PSTVd. Nucleotide coordinates are indicated and loops/bulges are numbered 1 to 27 (red). The inset highlights loop 27 and the Watson-Crick U-A base pair that closes the loop. (B) Conservation of the 5'-UUUUCA-3' stem-loop across eight Pospiviroid species. The number of occurrences observed in the total number of variants analyzed for each species is indicated. Numbers in red indicate that the loop 27 secondary structure is present in a majority of variants.
Fig 2.
Tertiary structure model of loop 27 and validation by chemical modification.
(A) Model of loop 27 obtained using the JAR3D program. Two different views are shown. The loop is closed by a Watson-Crick (WC) base pair between U177 and A182, and has two bases within the structure (U178 and U180). U180 stacks on the closing base pair. Two bases (U179 and C181) bulge out of the structure. (B) DMS modification. High reactivity of C181 (red arrow) indicates its WC edge is available for modification, whereas low reactivity (black arrow) indicates the WC edge of A182 is not. (C) SHAPE reactivity using BzCN. (D) SHAPE reactivity using NMIA. SHAPE modifies flexible (usually unpaired) bases. Low reactivity indicates a higher probability of base pairing. SHAPE reactivity is indicated by color: red = high (>0.85), orange = intermediate (0.4–0.85), black = low (0–0.40).
Table 1.
Summary of PSTVd loop 27 mutant phenotypes.
Fig 3.
Comparison of conserved protein binding sites in the histone 3' UTR mRNA stem-loop and loop 27 bases critical for PSTVd replication and trafficking.
(A) Residues in the histone stem-loop involved in SLBP binding are indicated by red boxes. Residues in lower case were added to stabilize the structure for in vitro binding assays. Exchanging the closing U-A pair with an A-U pair reduced but did not eliminate SLBP binding (Zanier et al., 2002) [35]. (B) Bases predicted to be involved in the PSTVd loop 27 3D structure and to contact an unknown plant binding protein(s) (black boxes). (C) Summary of loop 27 sites critical for replication and/or trafficking (blue boxes), as determined by single site mutagenesis. Note correlation between mutagenesis data and structure and binding site predictions.
Fig 4.
U178G/U179G replicates but fails to exit local leaves following rub-inoculation.
Total RNA was collected from: (A) inoculated leaves, (B) upper systemically infected leaves, or (C) petioles of inoculated leaves of 10 plants inoculated with U178G/U179G or wild type PSTVd (WT, one plant, positive control). Mock inoculation (M) was a negative control. (A) RNA blot assay indicates U178G/U179G replication in rub-inoculated leaves. (B) RNA blot assay indicates U178G/U179G is unable to traffic to upper leaves following rub inoculation. (C) RT-PCR indicates U178G/U179G is not present in petioles and fails to exit inoculated leaves. In A and B, the region of the blot corresponding to circular progeny genomes is shown. Loading controls were ribosomal RNA (rRNA) (A and B) and RT-PCR of actin mRNA (C), detected by ethidium bromide staining. Images are representative of 10 (A and B) and three (C) independent experiments.
Fig 5.
Stability of U178G/U179G is similar to wild type PSTVd.
(A) RNA blot of in vitro degradation assays performed at 28°C in buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM phenylmethylsulfonyl fluoride), or uninfected N. benthamiana leaf extract prepared with the same buffer. (B) Percentage of remaining PSTVd wild type (WT) and U178G/U179G RNA over time was determined using Quantity One software. Data are representative of three independent experiments.
Fig 6.
U178G/U179G replicates and spreads in rub-inoculated leaves.
(A) Whole-mount in situ hybridization was used to monitor infection in leaves rub-inoculated with wild type PSTVd (WT), U178G/U179G, and replication defective A271G/C273G (negative control) at 8, 10, and 12 days post-inoculation (dpi). Mock inoculation was another negative control. Purple dots (red arrows) are viroid hybridization signals in nuclei. Bars = 100 μm. Images for PSTVd WT and U178G/U179G are representative of more than 200 visual fields. (B) Mean numbers of infected cells per visual field after U178G/U179G (blue) and WT (red) inoculation. Asterisks indicate significant differences (p < 0.05) as determined by Student's t test. Bars indicate standard error of the mean.
Fig 7.
U178G/U179G fails to exit epidermal cells in rub-inoculated leaves.
Transverse section (12 μm) in situ hybridization of (A) mock inoculated leaves (negative control), (B) leaves inoculated with replication defective A271G/C273G (negative control), (C) wild type PSTVd (positive control), and (D-F) U178G/U179G. Images for PSTVd WT and U178G/U179G are representative of more than 200 sections. Purple dots (red arrows) are viroid hybridization signals in nuclei. uEp, upper epidermis; Pm, palisade mesophyll; Sm, spongy mesophyll; lEp, lower epidermis. Bars = 100 μm. (G and H) Number of infected cells per leaf section (~1 x 0.15 mm) in the upper epidermis (uEp) or adjacent palisade mesophyll (Pm) of plants inoculated with WT PSTVd (red) or U178G/U179G (blue) at 12 dpi (G) and 18–20 dpi (H). Data were compiled from 40 sections obtained from 20 infected plants. Asterisks indicate significant differences (p < 0.05*; p < 0.01**) as determined by Student's t test. Bars indicate standard error of the mean.
Table 2.
Sequences of progeny from plants inoculated with U178G/U179G by needle puncture.
Fig 8.
Needle puncture inoculation of stems and petioles allows trafficking of U178G/U179G to all cell types in upper leaves.
Transverse section (12 μm) in situ hybridization of samples from upper leaves of plants that were: (A) mock inoculated (negative control), or needle puncture inoculated with (B) replication defective A271G/C273G (negative control), (C) wild type PSTVd (positive control), or (D) U178G/U179G. Image in (D) is representative of more than 100 sections. Purple dots (red arrows) are viroid hybridization signals in nuclei. uEp, upper epidermis; Pm, palisade mesophyll; Sm, spongy mesophyll; lEp, lower epidermis. Bars = 100 μm. (E and F) RNA blot analysis. Total RNA was collected from systemically infected leaves of 10 plants inoculated by needle puncture with (E) wild type PSTVd (WT) or (F) U178G/U179G. RNA from mock inoculated plants (M) was a negative control, and linear PSTVd RNA (L) was a hybridization control. Linear PSTVd RNA migrates faster than the circular form that predominates in infected plants. Ribosomal RNA (rRNA) was a loading control. Blots are from Table 2, Experiment 2.
Fig 9.
Comparison of loop 27 structural models for wild type PSTVd and the U178G/U179G mutant.
(A) Structural model of wild type loop 27 (5'-UUUUCA-3', positions 177–182, loop bases underlined), shown from a different perspective than in Fig 2A. U179 and C181 bulge out of the structure, while U180 (yellow box) stacks on the closing WC base pair between U177 and A182. (B) The predicted structure of the U178G/U179G mutant (5'-UGGUCA-3') is compatible with structural model (HL_4OOG_001; 5'-GAGUCC-3'). The HL_4OOG_001 model structure shown has bulges at positions corresponding to 178, 179, and 181. U180 (yellow box) stacks on the closing WC base pair between G177 and C182.