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Scion on a stock producing siRNAs of potato spindle tuber viroid (PSTVd) attenuates accumulation of the viroid.

Kasai A, Sano T, Harada T - PLoS ONE (2013)

Bottom Line: To test whether this is indeed the case, we prepared transgenic tobacco (Nicotiana benthamiana) expressing a hairpin RNA (hpRNA) of Potato spindle tuber viroid (PSTVd) in companion cells by using a strong companion cell-specific promoter.A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants.These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Japan.

ABSTRACT
Plants can attenuate the replication of plant viruses and viroids by RNA silencing induced by virus and viroid infection. In higher plants, silencing signals such as small interfering RNAs (siRNAs) produced by RNA silencing can be transported systemically through phloem, so it is anticipated that antiviral siRNA signals produced in a stock would have the potential to attenuate propagation of viruses or viroids in the scion. To test whether this is indeed the case, we prepared transgenic tobacco (Nicotiana benthamiana) expressing a hairpin RNA (hpRNA) of Potato spindle tuber viroid (PSTVd) in companion cells by using a strong companion cell-specific promoter. A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants. These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock.

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CoYMV:hpPSTVd-ΔTLE N. benthamiana plants.(A) Schematic diagrams of the CoYMV:hpPSTVd-ΔTLE construct. Arrows indicate PSTVd sequence used. pCoYMV; commelina yellow mottle virus promoter, Tn; nopaline synthase terminator, TL; terminal left, P; pathogenicity, CCR; conserved central region, V; variable region, TR; terminal right. (B) Northern blot analysis of PSTVd siRNAs in the transgenic plants. Small RNA enriched nucleic acid (10 µg) was analyzed in 15% polyacrylamide gel and probed with PSTVd negative (top) and positive (middle) strand RNA. 5.8S rRNA hybridization was used as a loading control.
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pone-0057736-g001: CoYMV:hpPSTVd-ΔTLE N. benthamiana plants.(A) Schematic diagrams of the CoYMV:hpPSTVd-ΔTLE construct. Arrows indicate PSTVd sequence used. pCoYMV; commelina yellow mottle virus promoter, Tn; nopaline synthase terminator, TL; terminal left, P; pathogenicity, CCR; conserved central region, V; variable region, TR; terminal right. (B) Northern blot analysis of PSTVd siRNAs in the transgenic plants. Small RNA enriched nucleic acid (10 µg) was analyzed in 15% polyacrylamide gel and probed with PSTVd negative (top) and positive (middle) strand RNA. 5.8S rRNA hybridization was used as a loading control.

Mentions: The expression of a truncated, near full-length, hpPSTVd-ΔTLE was controlled by a companion cell-specific promoter, pCoYMV [25] (Figure 1A), to increase the potential siRNAs level in the phloem. The usefulness of pCoYMV for enhancing siRNAs signal in phloem transport had been confirmed in our previous studies [24], [26]. Since the promoter sequence was linked to near full-length PSTVd cDNA lacking 12 nucleotides at the left terminal end of the genome, the resulting construct (Figure 1A) facilitated the production of non-infectious form of hpPSTVd-ΔTLE sequence in the transgenic N. benthamiana companion cells.


Scion on a stock producing siRNAs of potato spindle tuber viroid (PSTVd) attenuates accumulation of the viroid.

Kasai A, Sano T, Harada T - PLoS ONE (2013)

CoYMV:hpPSTVd-ΔTLE N. benthamiana plants.(A) Schematic diagrams of the CoYMV:hpPSTVd-ΔTLE construct. Arrows indicate PSTVd sequence used. pCoYMV; commelina yellow mottle virus promoter, Tn; nopaline synthase terminator, TL; terminal left, P; pathogenicity, CCR; conserved central region, V; variable region, TR; terminal right. (B) Northern blot analysis of PSTVd siRNAs in the transgenic plants. Small RNA enriched nucleic acid (10 µg) was analyzed in 15% polyacrylamide gel and probed with PSTVd negative (top) and positive (middle) strand RNA. 5.8S rRNA hybridization was used as a loading control.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585205&req=5

pone-0057736-g001: CoYMV:hpPSTVd-ΔTLE N. benthamiana plants.(A) Schematic diagrams of the CoYMV:hpPSTVd-ΔTLE construct. Arrows indicate PSTVd sequence used. pCoYMV; commelina yellow mottle virus promoter, Tn; nopaline synthase terminator, TL; terminal left, P; pathogenicity, CCR; conserved central region, V; variable region, TR; terminal right. (B) Northern blot analysis of PSTVd siRNAs in the transgenic plants. Small RNA enriched nucleic acid (10 µg) was analyzed in 15% polyacrylamide gel and probed with PSTVd negative (top) and positive (middle) strand RNA. 5.8S rRNA hybridization was used as a loading control.
Mentions: The expression of a truncated, near full-length, hpPSTVd-ΔTLE was controlled by a companion cell-specific promoter, pCoYMV [25] (Figure 1A), to increase the potential siRNAs level in the phloem. The usefulness of pCoYMV for enhancing siRNAs signal in phloem transport had been confirmed in our previous studies [24], [26]. Since the promoter sequence was linked to near full-length PSTVd cDNA lacking 12 nucleotides at the left terminal end of the genome, the resulting construct (Figure 1A) facilitated the production of non-infectious form of hpPSTVd-ΔTLE sequence in the transgenic N. benthamiana companion cells.

Bottom Line: To test whether this is indeed the case, we prepared transgenic tobacco (Nicotiana benthamiana) expressing a hairpin RNA (hpRNA) of Potato spindle tuber viroid (PSTVd) in companion cells by using a strong companion cell-specific promoter.A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants.These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Japan.

ABSTRACT
Plants can attenuate the replication of plant viruses and viroids by RNA silencing induced by virus and viroid infection. In higher plants, silencing signals such as small interfering RNAs (siRNAs) produced by RNA silencing can be transported systemically through phloem, so it is anticipated that antiviral siRNA signals produced in a stock would have the potential to attenuate propagation of viruses or viroids in the scion. To test whether this is indeed the case, we prepared transgenic tobacco (Nicotiana benthamiana) expressing a hairpin RNA (hpRNA) of Potato spindle tuber viroid (PSTVd) in companion cells by using a strong companion cell-specific promoter. A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants. These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock.

Show MeSH
Related in: MedlinePlus