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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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PSTVd accumulation in the WT scion on a CoYMV:hpPSTVd-ΔTLE stock.(A) Illustration of the experimental procedure. At 13 dpi, leaf disks were harvested from the most newly expanded leaf. Then, sampling was done every 3 days. Seven grafted plants per line were tested. Accumulation level of the PSTVd RNA was analyzed by dot-blot hybridization (Figure S4). (B) Levels of PSTVd accumulation in the WT scion presented in the form of a heat map. The intensity of the hybridization signals was normalized relative to the 5.8S RNA signal, and sorted out from 0 no signal to 7 maximal. (C) Changes in the accumulation levels of PSTVd. Total scores for 7 grafts in each line are represented.
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pone-0057736-g004: PSTVd accumulation in the WT scion on a CoYMV:hpPSTVd-ΔTLE stock.(A) Illustration of the experimental procedure. At 13 dpi, leaf disks were harvested from the most newly expanded leaf. Then, sampling was done every 3 days. Seven grafted plants per line were tested. Accumulation level of the PSTVd RNA was analyzed by dot-blot hybridization (Figure S4). (B) Levels of PSTVd accumulation in the WT scion presented in the form of a heat map. The intensity of the hybridization signals was normalized relative to the 5.8S RNA signal, and sorted out from 0 no signal to 7 maximal. (C) Changes in the accumulation levels of PSTVd. Total scores for 7 grafts in each line are represented.

Mentions: Finally, to clarify whether PSTVd accumulation can be attenuated by a RNA silencing signals transferred from hpPSTVd-ΔTLE stock to the scion, we performed grafting experiments using wild-type N. benthamiana as the scion and CoYMV:hpPSTVd-ΔTLE lines as the stock. As shown in Figure 4A, to enhance the movement of the RNA silencing signals from the stock to the scion, the leaves of scion plants were removed except for one leaf, which was inoculated with PSTVd. At 12, 16, 19 and 25 dpi, the most newly expanded leaf at each time point was sampled for RNA extraction. All hybridization data were normalized by the 5.8S rRNA concentrations as the internal control (Figure S4), and then the levels of PSTVd accumulation in each plant were converted to a heat map (Figure 4B). At 12 dpi it was evident that 12 out of 21 wild type (WT) scions on the three lines of CoYMV:hpPSTVd-ΔTLE stocks gave no accumulation signs of PSTVd infection. Furthermore, even at 16 dpi, five out of a total 21 plants in the three lines of wild type WT/CoYMV:hpPSTVd-ΔTLE combinations continued to be negative for PSTVd, whereas all the seven plants in WT/Empty combination turned PSTVd-positive by 16 dpi. Furthermore, although all the plants showed PSTVd-positive at 25 dpi, the positive signals were somewhat dense in WT/Empty combination plants (Figure 4B). The changes in the levels of PSTVd accumulation from 12–25 dpi were quantified by using the sum of sores in the seven representative plants. As the result, it was apparent that all the three WT/CoYMV:hpPSTVd-ΔTLE combination plants attenuate PSTVd accumulation in the scions (Figure 4C). Interestingly, line 141, in which PSTVd-specific siRNAs accumulation was not evident (Figure 1), also attenuated PSTVd accumulation. As shown in Figure 3 and Figure S3, some of line 141 plants exhibited the attenuating efficacy of PSTVd accumulation. Therefore, line 141 is thought to have small amount level of the siRNA which was no detectable by northern blot analysis. Despite the lack of very clear efficacy of the siRNA transporting from stock to the scion, our observations have indicated that further research into the use of the siRNA producing stock as a mean of viroid disease resistant scion production is promising.


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 WT scion on a CoYMV:hpPSTVd-ΔTLE stock.(A) Illustration of the experimental procedure. At 13 dpi, leaf disks were harvested from the most newly expanded leaf. Then, sampling was done every 3 days. Seven grafted plants per line were tested. Accumulation level of the PSTVd RNA was analyzed by dot-blot hybridization (Figure S4). (B) Levels of PSTVd accumulation in the WT scion presented in the form of a heat map. The intensity of the hybridization signals was normalized relative to the 5.8S RNA signal, and sorted out from 0 no signal to 7 maximal. (C) Changes in the accumulation levels of PSTVd. Total scores for 7 grafts in each line are represented.
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Related In: Results  -  Collection

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pone-0057736-g004: PSTVd accumulation in the WT scion on a CoYMV:hpPSTVd-ΔTLE stock.(A) Illustration of the experimental procedure. At 13 dpi, leaf disks were harvested from the most newly expanded leaf. Then, sampling was done every 3 days. Seven grafted plants per line were tested. Accumulation level of the PSTVd RNA was analyzed by dot-blot hybridization (Figure S4). (B) Levels of PSTVd accumulation in the WT scion presented in the form of a heat map. The intensity of the hybridization signals was normalized relative to the 5.8S RNA signal, and sorted out from 0 no signal to 7 maximal. (C) Changes in the accumulation levels of PSTVd. Total scores for 7 grafts in each line are represented.
Mentions: Finally, to clarify whether PSTVd accumulation can be attenuated by a RNA silencing signals transferred from hpPSTVd-ΔTLE stock to the scion, we performed grafting experiments using wild-type N. benthamiana as the scion and CoYMV:hpPSTVd-ΔTLE lines as the stock. As shown in Figure 4A, to enhance the movement of the RNA silencing signals from the stock to the scion, the leaves of scion plants were removed except for one leaf, which was inoculated with PSTVd. At 12, 16, 19 and 25 dpi, the most newly expanded leaf at each time point was sampled for RNA extraction. All hybridization data were normalized by the 5.8S rRNA concentrations as the internal control (Figure S4), and then the levels of PSTVd accumulation in each plant were converted to a heat map (Figure 4B). At 12 dpi it was evident that 12 out of 21 wild type (WT) scions on the three lines of CoYMV:hpPSTVd-ΔTLE stocks gave no accumulation signs of PSTVd infection. Furthermore, even at 16 dpi, five out of a total 21 plants in the three lines of wild type WT/CoYMV:hpPSTVd-ΔTLE combinations continued to be negative for PSTVd, whereas all the seven plants in WT/Empty combination turned PSTVd-positive by 16 dpi. Furthermore, although all the plants showed PSTVd-positive at 25 dpi, the positive signals were somewhat dense in WT/Empty combination plants (Figure 4B). The changes in the levels of PSTVd accumulation from 12–25 dpi were quantified by using the sum of sores in the seven representative plants. As the result, it was apparent that all the three WT/CoYMV:hpPSTVd-ΔTLE combination plants attenuate PSTVd accumulation in the scions (Figure 4C). Interestingly, line 141, in which PSTVd-specific siRNAs accumulation was not evident (Figure 1), also attenuated PSTVd accumulation. As shown in Figure 3 and Figure S3, some of line 141 plants exhibited the attenuating efficacy of PSTVd accumulation. Therefore, line 141 is thought to have small amount level of the siRNA which was no detectable by northern blot analysis. Despite the lack of very clear efficacy of the siRNA transporting from stock to the scion, our observations have indicated that further research into the use of the siRNA producing stock as a mean of viroid disease resistant scion production is promising.

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