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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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Related in: MedlinePlus

PSTVd accumulation in the CoYMV:hpPSTVd-ΔTLE lines at 14 dpi.The data were obtained by dot-blot hybridization (Figure S3) using 8 individual plants of each line.
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pone-0057736-g003: PSTVd accumulation in the CoYMV:hpPSTVd-ΔTLE lines at 14 dpi.The data were obtained by dot-blot hybridization (Figure S3) using 8 individual plants of each line.

Mentions: To examine whether PSTVd accumulation can be attenuated in CoYMV:hpPSTVd-ΔTLE lines, we performed northern blot analyses to detect the mature PSTVd positive strand molecules in line 24 and 32 which accumulated detectable levels of the PSTVd siRNA by gel blot analysis (Figure 2). At 14 dpi, plants of all lines showed PSTVd accumulation. However, the signal intensities in lines 24 and 32 were weaker than those in the lines Empty control (Figure 2A and B). That is, PSTVd accumulation was suppressed in the two CoYMV:hpPSTVd-ΔTLE lines, in agreement with a previous report indicating that PSTVd resistance is correlated with the level of hpPSTVd siRNAs accumulation [11]. Although the period of attenuation was not prolonged and differences in the signal intensity between the lines were not observed at 22 dpi anymore, PSTVd accumulation was evidently restrained at 14 dpi. To confirm the attenuating effect of hpPSTVd-ΔTLE small RNAs on PSTVd accumulation, we performed another experiment using eight individual plants of CoYMV:hpPSTVd-ΔTLE lines including line 11 and 141 which did not show detectable levels of PSTVd-siRNAs (Figure S3). Dot-blot hybridization experiment was done as substitute for northern hybridization because of the concise analysis. As shown in Figure 3, although the levels of accumulation varied considerably within the same lines, all plants in line 24, and especially those in line 32, exhibited lower accumulation levels relative to the other three lines. Thus, it is appeared that PSTVd siRNAs production only in the companion cells was at least among the promising methods to attenuate PSTVd accumulation in the genetically modified plant.


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)

PSTVd accumulation in the CoYMV:hpPSTVd-ΔTLE lines at 14 dpi.The data were obtained by dot-blot hybridization (Figure S3) using 8 individual plants of each line.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057736-g003: PSTVd accumulation in the CoYMV:hpPSTVd-ΔTLE lines at 14 dpi.The data were obtained by dot-blot hybridization (Figure S3) using 8 individual plants of each line.
Mentions: To examine whether PSTVd accumulation can be attenuated in CoYMV:hpPSTVd-ΔTLE lines, we performed northern blot analyses to detect the mature PSTVd positive strand molecules in line 24 and 32 which accumulated detectable levels of the PSTVd siRNA by gel blot analysis (Figure 2). At 14 dpi, plants of all lines showed PSTVd accumulation. However, the signal intensities in lines 24 and 32 were weaker than those in the lines Empty control (Figure 2A and B). That is, PSTVd accumulation was suppressed in the two CoYMV:hpPSTVd-ΔTLE lines, in agreement with a previous report indicating that PSTVd resistance is correlated with the level of hpPSTVd siRNAs accumulation [11]. Although the period of attenuation was not prolonged and differences in the signal intensity between the lines were not observed at 22 dpi anymore, PSTVd accumulation was evidently restrained at 14 dpi. To confirm the attenuating effect of hpPSTVd-ΔTLE small RNAs on PSTVd accumulation, we performed another experiment using eight individual plants of CoYMV:hpPSTVd-ΔTLE lines including line 11 and 141 which did not show detectable levels of PSTVd-siRNAs (Figure S3). Dot-blot hybridization experiment was done as substitute for northern hybridization because of the concise analysis. As shown in Figure 3, although the levels of accumulation varied considerably within the same lines, all plants in line 24, and especially those in line 32, exhibited lower accumulation levels relative to the other three lines. Thus, it is appeared that PSTVd siRNAs production only in the companion cells was at least among the promising methods to attenuate PSTVd accumulation in the genetically modified plant.

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