Destabilization of potato spindle tuber viroid by mutations in the left terminal loop.

Hu, Yan; Feldstein, Paul A.; Hammond, R. W.; Bottino, P.J.; Owens, Robert A.

doi:10.1099/0022-1317-78-6-1199

Cited by 36 publications

(36 citation statements)

References 27 publications

(39 reference statements)

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“…This rod-like structural model is well supported by microscopic and biophysical studies (Riesner et al, 1979), chemical/enzymatic mapping , and nuclear magnetic resonance spectroscopic studies (left terminal domain; Dingley et al, 2003). Mutational studies support the importance of the rod-like secondary structure of PSTVd for infection (Hammond and Owens, 1987;Hammond, 1994;Wassenegger et al, 1994;Hu et al, 1997;Owens and Thompson, 2005). Biophysical/chemical studies identified several premelting regions of low thermostability in the native structure of PSTVd, and denaturation of this structure initiated at these regions and subsequent refolding led to the formation of metastable hairpin (HP) structures via pairing of distant complementary nucleotide sequences (Riesner, 1987;Riesner et al, 1979).…”

Section: Introductionmentioning

confidence: 62%

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A Genomic Map of Viroid RNA Motifs Critical for Replication and Systemic Trafficking

Zhong¹,

Archual²,

Amin³

et al. 2008

The Plant Cell

160

162

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RNA replication and systemic trafficking play significant roles in developmental regulation and host-pathogen interactions. Viroids are the simplest noncoding eukaryotic RNA pathogens and genetic units that are capable of autonomous replication and systemic trafficking and offer excellent models to investigate the role of RNA structures in these processes. Like other RNAs, the predicted secondary structure of a viroid RNA contains many loops and bulges flanked by double-stranded helices, the biological functions of which are mostly unknown. Using Potato spindle tuber viroid infection of Nicotiana benthamiana as the experimental system, we tested the hypothesis that these loops/bulges are functional motifs that regulate replication in single cells or trafficking in a plant. Through a genome-wide mutational analysis, we identified multiple loops/bulges essential or important for each of these biological processes. Our results led to a genomic map of viroid RNA motifs that mediate single-cell replication and systemic trafficking, respectively. This map provides a framework to enable high-throughput studies on the tertiary structures and functional mechanisms of RNA motifs that regulate viroid replication and trafficking. Our model and approach should also be valuable for comprehensive investigations of the replication and trafficking motifs in other RNAs.

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Section: Introductionmentioning

confidence: 62%

“…PSTVd replicates efficiently in cultured cell protoplasts and easily develops systemic infection in this plant (Hu et al, 1997;Zhu et al, 2001). Through this analysis, we identified multiple loops/bulges as functional motifs that are critical for replication or trafficking.…”

Section: Introductionmentioning

confidence: 99%

A Genomic Map of Viroid RNA Motifs Critical for Replication and Systemic Trafficking

Zhong¹,

Archual²,

Amin³

et al. 2008

The Plant Cell

160

162

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show abstract

“…Furthermore, P1/HC-Pro can enhance miRNA accumulation (16,24,55). To test the effects of these suppressors on srPSTVd accumulation, we used Nicotiana benthamiana for the following reasons: (i) PSTVd actively replicates in N. benthamiana plants (38,104) as well as in protoplasts of cultured cells (67), and (ii) RNA silencing protocols, including assays of viral suppressor functions, have been well established in N. benthamiana plants (72) and cultured cells (68). Therefore, N. benthamiana provides a convenient experimental system to study RNA silencing and PSTVd biology.…”

Section: Resultsmentioning

confidence: 99%

A Structured Viroid RNA Serves as a Substrate for Dicer-Like Cleavage To Produce Biologically Active Small RNAs but Is Resistant to RNA-Induced Silencing Complex-Mediated Degradation

Itaya¹,

Zhong²,

Bundschuh

et al. 2007

J Virol

185

177

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RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21-to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.A major focus of current biology is to understand how a pathogen has evolved mechanisms to achieve a balance among several interrelated activities that are crucial to establish a full infection: evading or suppressing host defense, minimizing destructive interference of host metabolism, and maximizing utilization of host factors to support replication and systemic spread. A full understanding of these mechanisms is not only necessary to build a foundation for developing technologies to combat pathogen diseases but also can provide fundamental mechanistic insights into the regulation of basic cellular processes.Recent studies have discovered small RNA-mediated gene silencing as a powerful antiviral mechanism in plants and animals (6,22,25,47,49,50,72,77,84,(87)(88)(89)98). Furthermore, small RNA-mediated gene silencing plays essential roles in regulating a wide variety of growth and development processes (4-6, 11, 13, 17, 23, 28, 42). A key mediator of RNA silencing is several classes of 21-to 24-nucleotide (nt) small RNAs.MicroRNAs (miRNAs) are produced by cleavage of hairpin RNA precursors encoded by the genome of an organism. Short interfering RNAs (siRNAs) are generated by cleavage of double-stranded RNAs (dsRNAs) that may originate from several sources, including cellular genomes, viral replication intermediates, aberrant cellular RNAs, overexpresse...

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“…Plasmid pRZ6-2 containing the cDNA of PSTVd Int (pRZ6-2-Int) was constructed by Hu et al (1997) and was a gift from Robert Owens. All PSTVd mutants described in this article were generated by site-directed mutagenesis based on this template, as previously described (Zhong et al, 2008).…”

Section: Methods Cdna Construction Of Pstvd Loop 6 Mutantsmentioning

confidence: 99%

A Three-Dimensional RNA Motif inPotato spindle tuber viroidMediates Trafficking from Palisade Mesophyll to Spongy Mesophyll inNicotiana benthamiana

et al. 2011

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Cell-to-cell trafficking of RNA is an emerging biological principle that integrates systemic gene regulation, viral infection, antiviral response, and cell-to-cell communication. A key mechanistic question is how an RNA is specifically selected for trafficking from one type of cell into another type. Here, we report the identification of an RNA motif in Potato spindle tuber viroid (PSTVd) required for trafficking from palisade mesophyll to spongy mesophyll in Nicotiana benthamiana leaves. This motif, called loop 6, has the sequence 59-CGA-39...59-GAC-39 flanked on both sides by cis Watson-Crick G/C and G/U wobble base pairs. We present a three-dimensional (3D) structural model of loop 6 that specifies all non-Watson-Crick base pair interactions, derived by isostericity-based sequence comparisons with 3D RNA motifs from the RNA x-ray crystal structure database. The model is supported by available chemical modification patterns, natural sequence conservation/variations in PSTVd isolates and related species, and functional characterization of all possible mutants for each of the loop 6 base pairs. Our findings and approaches have broad implications for studying the 3D RNA structural motifs mediating trafficking of diverse RNA species across specific cellular boundaries and for studying the structure-function relationships of RNA motifs in other biological processes.

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Destabilization of potato spindle tuber viroid by mutations in the left terminal loop.

Cited by 36 publications

References 27 publications

A Genomic Map of Viroid RNA Motifs Critical for Replication and Systemic Trafficking

A Genomic Map of Viroid RNA Motifs Critical for Replication and Systemic Trafficking

A Structured Viroid RNA Serves as a Substrate for Dicer-Like Cleavage To Produce Biologically Active Small RNAs but Is Resistant to RNA-Induced Silencing Complex-Mediated Degradation

A Three-Dimensional RNA Motif inPotato spindle tuber viroidMediates Trafficking from Palisade Mesophyll to Spongy Mesophyll inNicotiana benthamiana

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