Sense- and antisense-mediated gene silencing in tobacco is inhibited by the same viral suppressors and is associated with accumulation of small RNAs - PubMed (original) (raw)
Sense- and antisense-mediated gene silencing in tobacco is inhibited by the same viral suppressors and is associated with accumulation of small RNAs
F Di Serio et al. Proc Natl Acad Sci U S A. 2001.
Abstract
Antisense-mediated gene silencing (ASGS) and posttranscriptional gene silencing (PTGS) with sense transgenes markedly reduce the steady-state mRNA levels of endogenous genes similar in transcribed sequence. RNase protection assays established that silencing in tobacco plants transformed with plant-defense-related class I sense and antisense chitinase (CHN) transgenes is at the posttranscriptional level. Infection of tobacco plants with cucumber mosaic virus strain FN and a necrotizing strain of potato virus Y, but not with potato virus X, effectively suppressed PTGS and ASGS of both the transgenes and homologous endogenes. This suggests that ASGS and PTGS share components associated with initiation and maintenance of the silent state. Small, ca. 25-nt RNAs (smRNA) of both polarities were associated with PTGS and ASGS in CHN transformants as reported for PTGS in other transgenic plants and for RNA interference in Drosophila. Similar results were obtained with an antisense class I beta-1,3-glucanase transformant showing that viral suppression and smRNAs are a more general feature of ASGS. Several current models hold that diverse signals lead to production of double-stranded RNAs, which are processed to smRNAs that then trigger PTGS. Our results provide direct evidence for mechanistic links between ASGS and PTGS and suggest that ASGS could join a common PTGS pathway at the double-stranded RNA step.
Figures
Figure 1
Pre-mRNA accumulation induced by ethylene treatment is unaffected by PTGS and ASGS. RNase protection assays of total RNA (25 μg) extracted from leaves of untransformed tobacco (Wt), TAC, TAG, sibling silent (STSC), and high-expressing (HTSC) TSC plants before (−) and after (+) treatment with 20 ppm ethylene for 2 days. Intron/exon RNA probes for_CHN50_ (A and C) and_Gla_ (B) mixed with a control U2 RNA probe were used. The positions of the RNA-species protected and probes before (−RNase) and after (+RNase) RNase digestion protected with tRNA are indicated.
Figure 2
CMV and PVY, but not PVX, suppress PTGS in TSC. RNA blot hybridization with a CHN cDNA probe and immunoblot analyses of leaves from sibling STSC plants (STSC1–STSC4). Tissues were harvested from the inoculated leaf just before inoculation (0), from leaves showing systemic virus infection 10–15 DPI (V), and from leaves showing systemic virus infection 2 days later after treatment of plants with 20-ppm ethylene (+ET). Total RNA (10 μg) was hybridized with a_CHN48_ cDNA probe, which detects CHN48 and_CHN50_ RNA (CHN RNA). The double bands detected with the probe have been described earlier and may result from alternative polyadenylation (26). Equal loading was confirmed by rehybridization with a probe for 18S rRNA. Immunoblot analyses of equal volumes of protein extracts of the same tissues used for RNA analyses are shown at the bottom. The positions of the class I chitinases CHN48, its truncated form ΔCHN48 and CHN50, and the class II chitinases (CHN II) are indicated.
Figure 3
CMV and PVY, but not PVX, suppress accumulation of transgene-encoded antisense transcripts in TAC and TAG. Tissues were harvested from the inoculated leaf just before inoculation (0) and from leaves showing systemic virus infection 10–15 DPI of sibling TAC (TAC1–TAC4) and TAG (TAG1–TAG4) plants. RNA blot hybridization of total RNA (10 μg) by using RNA probes to detect antisense CHN RNA (A) or antisense GLU RNA (B). Equal loading was confirmed by rehybridization with a probe for 18S rRNA.
Figure 4
CMV and PVY, but not PVX, restore ethylene-induction of endogenous-gene expression in antisense transformants. RNA blot and immunoblot analyses of leaf tissues harvested as indicated in Fig. 2 from sibling TAC plants (A) and TAG (B) plants. Total RNA (10 μg) was hybridized with RNA probes for detecting sense CHN RNA (A) or sense GLU RNA (B). Equal loading was confirmed by rehybridization with a probe for 18S rRNA. Protein extracts representing equal amounts of the same tissues were immunoblotted by using probes for CHN antigens and GLU antigens indicated in Fig. 2. Purified GLU I protein was used as size marker (B, GLU I). Positions of GLU I and the class II (GLU II) and class III (GLU III) β-1,3-glucanases are indicated. Note that induction of GLU I antigen is suppressed in mock- and PVX-infected plants, but not in CMV- and PVY-infected plants.
Figure 5
Small, ca. 25-nt sense- and antisense-RNAs are associated with both PTGS and ASGS. RNA blot hybridization of 20 μg of the low-molecular-weight RNA fraction prepared from total RNA of untreated plants. Membranes were hybridized with RNA probes for sense and antisense CHN (A) and GLU (B) RNA. The position of the ca. 25-nt RNAs was determined by using single-stranded DNA primers as size markers. Unspecific cross-hybridization with tRNAs (top of each gel) was used as the control for equal loading. Note that an additional ca. 30-nt RNA was detected with sense and antisense GLU probes. This RNA was present at roughly the same abundance in wild type and transformed lines and was not correlated with ASGS.
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