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. 2012 Jun;159(2):558-64.
doi: 10.1104/pp.112.195214. Epub 2012 Apr 3.

Studies on subcellular compartmentalization of plant pathogenic noncoding RNAs give new insights into the intracellular RNA-traffic mechanisms

Gustavo Gómez  1 , Vicente Pallas
Affiliations

Studies on subcellular compartmentalization of plant pathogenic noncoding RNAs give new insights into the intracellular RNA-traffic mechanisms

Gustavo Gómez et al. Plant Physiol. 2012 Jun.
No abstract available

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Figures

Figure 1.
Figure 1.
Analysis of GFP expression in plants infected with PVX-derived transcripts. A, Physical map of the PVX-derived construct used in this work. PVX-based vector P2C2S and cDNAs (GFP, GFP/IV2, and GFP/IV2/ELVd) cloned under the control of the duplicated PVX-CP promoter are shown at top. The bottom shows representations of PVX-GFP, PVX-GFP/IV2, and PVX-GFP/IV2/ELVd(+) chimeric constructs. The constructs are not drawn to scale. B, GFP fluorescence stereomicroscopy images of N. benthamiana leaves inoculated with PVX (unmodified), PVX-GFP, PVX-GFP/IV2, and PVX-GFP/IV2/ELVd(+) transcripts at 7 and 10 d post inoculation. C, Serological detection of GFP. Total proteins were extracted from leaves, electrophoresed by 10% SDS-PAGE (top), and blotted for serological detection (bottom). GFP was clearly detected in both PVX-GFP and PVX-GFP/IV2/ELVd(+) systemically infected plants, confirming the correct processing of the chimeric GFP mRNA carrying the IV2/ELVd(+) insertion. D, RT-PCR amplification of processed and unprocessed GFP mRNAs. E, Confirmation (by RT-PCR) of systemic PVX infection in N. benthamiana plants inoculated with the construct used in this work.
Figure 2.
Figure 2.
Evidence for the stability of chimeric constructs in systemically infected leaves. RT-PCR amplification of IV2 and ELVd(+) cDNAs imbibed in a GFP open reading frame is shown. The sequences and positions of the specific primers (GFP-1 and GFP-2 and IV2-1 and IV2-2) used for the amplification are detailed in Supplemental Figure S2. The sizes in the top panel are not to scale. nt, Nucleotides.
Figure 3.
Figure 3.
Analysis of GFP expression in plants infected with PVX-derived transcripts carrying partial-length ELVd sequences. A, GFP fluorescence stereomicroscopy images of N. benthamiana leaves inoculated with PVX-GFP, PVX-GFP/IV2/L-ELVd, PVX-GFP/IV2/R-ELVd, and PVX-GFP/IV2/Up-ELVd transcripts at 6, 9, and 12 d post inoculation (dpi). B, Confirmation (by RT-PCR) of systemic PVX infection in N. benthamiana plants inoculated with the different constructs used in this assay. C, RT-PCR amplification of processed and unprocessed GFP mRNAs.
Figure 4.
Figure 4.
The full-length (plus strand) ELVd RNA mediates the trafficking of functional GFP-mRNA from the nucleus to chloroplasts. A, Physical map of the ELVd-5′UTR-GFP and unmodified GFP constructs used in this assay. The construct sizes are not to scale. B, ELVd-5′UTR/GFP and GFP were clearly detected and showed similar relative electrophoretic mobility in the western-blot assay, indicating that the full-length viroid sequence acts as a true untranslated RNA. Serial dilutions(1:5) are shown for each construct. C, Confocal microscopy observation of N. benthamiana leaves expressing unmodified GFP (top panels), ELVd-5′UTR-GFP (middle panels), or OE23/GFP (bottom panels). ELVd-5′UTR/GFP mimics the cellular localization of the OE23/GFP construct, which accumulates specifically in chloroplasts.
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