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. 2001 May 22;98(11):6516-21.
doi: 10.1073/pnas.111440998. Epub 2001 May 15.

An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense

Z Xie  1 , B Fan, C Chen, Z Chen
Affiliations

An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense

Z Xie et al. Proc Natl Acad Sci U S A. .

Abstract

Plants contain RNA-dependent RNA polymerase (RdRP) activities that synthesize short cRNAs by using cellular or viral RNAs as templates. During studies of salicylic acid (SA)-induced resistance to viral pathogens, we recently found that the activity of a tobacco RdRP was increased in virus-infected or SA-treated plants. Biologically active SA analogs capable of activating plant defense response also induced the RdRP activity, whereas biologically inactive analogs did not. A tobacco RdRP gene, NtRDRP1, was isolated and found to be induced both by virus infection and by treatment with SA or its biologically active analogs. Tobacco lines deficient in the inducible RDRP activity were obtained by expressing antisense RNA for the NtRDRP1 gene in transgenic plants. When infected by tobacco mosaic virus, these transgenic plants accumulated significantly higher levels of viral RNA and developed more severe disease symptoms than wild-type plants. After infection by a strain of potato virus X that does not spread in wild-type tobacco plants, the transgenic NtRDRP1 antisense plants accumulated virus and developed symptoms not only locally in inoculated leaves but also systemically in upper uninoculated leaves. These results strongly suggest that inducible RdRP activity plays an important role in plant antiviral defense.

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Figures

Figure 1
Figure 1
Induction of tobacco RdRP activity by SA and TMV infection. (A) Tobacco leaves were treated by floating on 1 mM SA and harvested at indicated times before preparation and assays of RdRP activity. (B) Tobacco leaves were treated with 1 mM SA, 5-chloroSA (5-CSA), acetylSA (ASA), 3-hydroxybenzoic acid (3HBA), or 4-hydroxybenzoic acid (4-HBA) for 48 h before preparation and assays of RdRP activity. (C) Tobacco plants were inoculated on a low leaf with TMV. Leaf tissues were harvested 15 days after inoculation from the upper systemically infected leaves for preparation and assays for RdRP activity. Migration positions of two RNA size markers (generated by in vitro transcription of the TMV genome and a 26-bp DNA fragment cloned in a pBluscript vector) are indicated.
Figure 2
Figure 2
Northern blot analysis of the NtRdRP1 gene expression. Total RNA was prepared from tobacco plants and probed with an 860-bp HindIII fragment of the NtRdRP1 cDNA. (A) Expression of NtRdRP1 in tobacco leaves treated with 1 mM SA for indicated times. (B) Expression of NtRdRP1 in tobacco leaves treated for 24 h with 1 mM SA, 5-chloroSA (5-CSA), acetylSA (ASA), 3-hydroxybenzoic acid (3HBA), or 4-hydroxybenzoic acid (4-HBA). (C) Expression of NtRdRP1 in mock- or TMV-inoculated tobacco plants. Chemical treatments and TMV infection were performed as described in Fig. 1. The ethidium bromide stain of rRNA is shown for each lane to allow assessment of equal loading.
Figure 3
Figure 3
Suppression of inducible RdRP activity in transgenic tobacco NtRdRP1 antisense plants. (A) RdRP activity from vector-transformed wild-type plants (wt) or transgenic NtRdRP1 antisense lines 14 (L14) and 18 (L18) after treatment with water (−) or 1 mM SA (+) for 48 h. (B) RdRP activity from wild-type plants (wt) or transgenic antisense lines in upper uninoculated leaves 15 days after mock (−) or TMV (+) infection on lower leaves. (C) Transcript levels of NtRdRP1 in wild-type or antisense lines after treatment with water (−) or 1 mM SA (+) for 48 h. (D) Transcript levels of NtRdRP1 in wild-type or antisense lines in upper systemically infected leaves 15 days after mock (−) or TMV (+) infection on lower leaves. Northern blots were hybridized with an antisense-strand RNA probe transcribed from an 860-bp HindIII fragment of the NtRdRP1 cDNA clone. The ethidium bromide stain of rRNA is shown for each lane.
Figure 4
Figure 4
TMV symptom development and viral RNA accumulation in detached leaf discs. (A) Leaf discs from wild-type or antisense lines 14 (L14) and 18 (L18) were inoculated with TMV. The inoculated leaves were incubated in Petri dishes for 14 days before photographing. The enhanced chlorotic symptoms typically began in the antisense lines 10 days after inoculation. (B) Total RNA was isolated from TMV-infected leaf discs at indicated days postinoculation (dpi), separated on an agarose (1.2%)-formaldehyde gel, and probed with a DNA fragment corresponding to the TMV coat protein subgenomic RNA. The blot for the 2-dpi time point was exposed twice longer to detect low levels of TMV RNAs. The ethidium bromide stain of rRNA is shown for each lane. Migration positions of size markers (generated from HindIII-digested phage λDNA fragments) are indicated.
Figure 5
Figure 5
TMV symptom development and viral RNA accumulation on whole plants. (A) TMV-susceptible wild-type (Left) or antisense lines 14 (Center) and 18 (Right) were inoculated on one of the lower leaves with TMV. The plants were photographed 15 days after inoculation. (B) The difference in symptom development between the wild-type (Left) and antisense lines 14 (Center) and 18 (Right) 35 days after TMV inoculation. (C) Total RNA was isolated 15 days after TMV inoculation from the inoculated leaves and the fifth leaves directly above the inoculated ones of the wild-type (wt), antisense lines 14 (L14) and 18 (L18) and probed with a DNA fragment corresponding to the TMV coat protein subgenomic RNA. The ethidium bromide stain of rRNA is shown for each lane. Migration positions of size markers are indicated. No significant hybridization signal was detected in uninoculated plants.
Figure 6
Figure 6
PVX symptom development and virus accumulation in transgenic NtRdRP1 antisense plants. (A) The wild-type (Left) or antisense lines 14 (Center) and 18 (Right) were inoculated on one of the lower leaves with PVX. The plants were photographed 15 days after inoculation. (B) Total RNA was isolated 15 days after PVX inoculation from the inoculated leaves and the fifth leaves directly above the inoculated ones of the wild-type (wt) and antisense lines 14 (L14) and 18 (L18) and probed with a DNA fragment corresponding to the coding region for the PVX coat protein. The ethidium bromide stain of rRNA is shown for each lane. Migration positions of size markers are indicated. (C) Total soluble proteins were isolated 15 days after PVX inoculation from the lower inoculated and upper systemically infected leaves of the wild-type (wt) and antisense lines 14 (L14) and 18 (L18) and probed with a PVX-specific polyclonal antibody.
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