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Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus.

Liu E, Page JE - Plant Methods (2008)

Bottom Line: Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested.Silencing efficiency was reduced by the inclusion of a 24 bp poly(A) or poly(G) homopolymeric region.Leaf nicotine levels were reduced by more than 90% in all plants infected with the NbPMT constructs.

View Article: PubMed Central - HTML - PubMed

Affiliation: NRC Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9 Canada. jon.page@nrc-cnrc.gc.ca.

ABSTRACT

Background: Virus-induced gene silencing (VIGS) has emerged as a method for performing rapid loss-of-function experiments in plants. Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested. In this study, we probed the optimal properties of cDNA fragments of the phytoene desaturase (PDS) gene for efficient VIGS in Nicotiana benthamiana using tobacco rattle virus (TRV).

Results: NbPDS inserts of between 192 bp and 1304 bp led to efficient silencing as determined by analysis of leaf chlorophyll a levels. The region of the NbPDS cDNA used for silencing had a small effect on silencing efficiency with 5' and 3' located inserts performing more poorly than those from the middle. Silencing efficiency was reduced by the inclusion of a 24 bp poly(A) or poly(G) homopolymeric region. We developed a method for constructing cDNA libraries for use as a source of VIGS-ready constructs. Library construction involved the synthesis of cDNA on a solid phase support, digestion with RsaI to yield short cDNA fragments lacking poly(A) tails and suppression subtractive hybridization to enrich for differentially expressed transcripts. We constructed two cDNA libraries from methyl-jasmonate treated N. benthamiana roots and obtained 2948 ESTs. Thirty percent of the cDNA inserts were 401-500 bp in length and 99.5% lacked poly(A) tails. To test the efficiency of constructs derived from the VIGS-cDNA libraries, we silenced the nicotine biosynthetic enzyme, putrescine N-methyltransferase (PMT), with ten different VIGS-NbPMT constructs ranging from 122 bp to 517 bp. Leaf nicotine levels were reduced by more than 90% in all plants infected with the NbPMT constructs.

Conclusion: Based on the silencing of NbPDS and NbPMT, we suggest the following design guidelines for constructs in TRV vectors: (1) Insert lengths should be in the range of ~200 bp to ~1300 bp, (2) they should be positioned in the middle of the cDNA and (3) homopolymeric regions (i.e. poly(A/T) tails) should not be included. Our VIGS-cDNA library method, which incorporates these guidelines to produce sequenced, VIGS-ready cDNAs, will be useful for both fast-forward and reverse genetics experiments in TRV vectors.

No MeSH data available.


Related in: MedlinePlus

Effect of cDNA insert position on the silencing of PDS in N. benthamiana leaves. (a) Position and length of NbPDS cDNA fragments used for silencing relative to the full-length NbPDS cDNA. All inserts were in the antisense orientation relative to the TRV coat protein. (b) Chlorophyll a levels in the aerial parts of plants infected with TRV-NbPDS constructs. Buffer control plants received infiltration buffer only and empty TRV plants were infected with TRV lacking a silencing insert. Chlorophyll a values for buffer, empty TRV and some constructs are also shown in Figure 1B. Bars represent mean ± SD (n = 3). (c) Photographs of representative leaves from TRV infected plants showing the extent of the photobleaching.
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Figure 2: Effect of cDNA insert position on the silencing of PDS in N. benthamiana leaves. (a) Position and length of NbPDS cDNA fragments used for silencing relative to the full-length NbPDS cDNA. All inserts were in the antisense orientation relative to the TRV coat protein. (b) Chlorophyll a levels in the aerial parts of plants infected with TRV-NbPDS constructs. Buffer control plants received infiltration buffer only and empty TRV plants were infected with TRV lacking a silencing insert. Chlorophyll a values for buffer, empty TRV and some constructs are also shown in Figure 1B. Bars represent mean ± SD (n = 3). (c) Photographs of representative leaves from TRV infected plants showing the extent of the photobleaching.

Mentions: We next tested if the position of the insert with respect to the full-length cDNA affected silencing. TRV constructs were synthesized using primers designed to amplify a non-overlapping series of short NbPDS fragments (Figure 2a). In this manner we synthesized constructs of TRV-NbPDS 1–385 (385 bp), TRV-NbPDS 386–742 (357 bp), TRV-NbPDS 743–1087 (345 bp), TRV-NbPDS 1088–1436 (349 bp) and TRV-NbPDS 1437–1789 (353 bp), which together with the NbPDS 1788–2046 (257 bp) construct, gave complete coverage of NbPDS. Plants infected with these constructs showed that all were effective in silencing but slightly more bleaching was produced by constructs located in the middle of the cDNA compared to the 5' and 3' ends (Figure 2b). This effect was apparent in the fine green mottling on the leaves of TRV-NbPDS 1–385 (385 bp) and TRV-NbPDS 1788–2046 (257 bp) infected plants while silencing inserts from the middle of the NbPDS cDNA gave more uniformly bleached leaves.


Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus.

Liu E, Page JE - Plant Methods (2008)

Effect of cDNA insert position on the silencing of PDS in N. benthamiana leaves. (a) Position and length of NbPDS cDNA fragments used for silencing relative to the full-length NbPDS cDNA. All inserts were in the antisense orientation relative to the TRV coat protein. (b) Chlorophyll a levels in the aerial parts of plants infected with TRV-NbPDS constructs. Buffer control plants received infiltration buffer only and empty TRV plants were infected with TRV lacking a silencing insert. Chlorophyll a values for buffer, empty TRV and some constructs are also shown in Figure 1B. Bars represent mean ± SD (n = 3). (c) Photographs of representative leaves from TRV infected plants showing the extent of the photobleaching.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254408&req=5

Figure 2: Effect of cDNA insert position on the silencing of PDS in N. benthamiana leaves. (a) Position and length of NbPDS cDNA fragments used for silencing relative to the full-length NbPDS cDNA. All inserts were in the antisense orientation relative to the TRV coat protein. (b) Chlorophyll a levels in the aerial parts of plants infected with TRV-NbPDS constructs. Buffer control plants received infiltration buffer only and empty TRV plants were infected with TRV lacking a silencing insert. Chlorophyll a values for buffer, empty TRV and some constructs are also shown in Figure 1B. Bars represent mean ± SD (n = 3). (c) Photographs of representative leaves from TRV infected plants showing the extent of the photobleaching.
Mentions: We next tested if the position of the insert with respect to the full-length cDNA affected silencing. TRV constructs were synthesized using primers designed to amplify a non-overlapping series of short NbPDS fragments (Figure 2a). In this manner we synthesized constructs of TRV-NbPDS 1–385 (385 bp), TRV-NbPDS 386–742 (357 bp), TRV-NbPDS 743–1087 (345 bp), TRV-NbPDS 1088–1436 (349 bp) and TRV-NbPDS 1437–1789 (353 bp), which together with the NbPDS 1788–2046 (257 bp) construct, gave complete coverage of NbPDS. Plants infected with these constructs showed that all were effective in silencing but slightly more bleaching was produced by constructs located in the middle of the cDNA compared to the 5' and 3' ends (Figure 2b). This effect was apparent in the fine green mottling on the leaves of TRV-NbPDS 1–385 (385 bp) and TRV-NbPDS 1788–2046 (257 bp) infected plants while silencing inserts from the middle of the NbPDS cDNA gave more uniformly bleached leaves.

Bottom Line: Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested.Silencing efficiency was reduced by the inclusion of a 24 bp poly(A) or poly(G) homopolymeric region.Leaf nicotine levels were reduced by more than 90% in all plants infected with the NbPMT constructs.

View Article: PubMed Central - HTML - PubMed

Affiliation: NRC Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9 Canada. jon.page@nrc-cnrc.gc.ca.

ABSTRACT

Background: Virus-induced gene silencing (VIGS) has emerged as a method for performing rapid loss-of-function experiments in plants. Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested. In this study, we probed the optimal properties of cDNA fragments of the phytoene desaturase (PDS) gene for efficient VIGS in Nicotiana benthamiana using tobacco rattle virus (TRV).

Results: NbPDS inserts of between 192 bp and 1304 bp led to efficient silencing as determined by analysis of leaf chlorophyll a levels. The region of the NbPDS cDNA used for silencing had a small effect on silencing efficiency with 5' and 3' located inserts performing more poorly than those from the middle. Silencing efficiency was reduced by the inclusion of a 24 bp poly(A) or poly(G) homopolymeric region. We developed a method for constructing cDNA libraries for use as a source of VIGS-ready constructs. Library construction involved the synthesis of cDNA on a solid phase support, digestion with RsaI to yield short cDNA fragments lacking poly(A) tails and suppression subtractive hybridization to enrich for differentially expressed transcripts. We constructed two cDNA libraries from methyl-jasmonate treated N. benthamiana roots and obtained 2948 ESTs. Thirty percent of the cDNA inserts were 401-500 bp in length and 99.5% lacked poly(A) tails. To test the efficiency of constructs derived from the VIGS-cDNA libraries, we silenced the nicotine biosynthetic enzyme, putrescine N-methyltransferase (PMT), with ten different VIGS-NbPMT constructs ranging from 122 bp to 517 bp. Leaf nicotine levels were reduced by more than 90% in all plants infected with the NbPMT constructs.

Conclusion: Based on the silencing of NbPDS and NbPMT, we suggest the following design guidelines for constructs in TRV vectors: (1) Insert lengths should be in the range of ~200 bp to ~1300 bp, (2) they should be positioned in the middle of the cDNA and (3) homopolymeric regions (i.e. poly(A/T) tails) should not be included. Our VIGS-cDNA library method, which incorporates these guidelines to produce sequenced, VIGS-ready cDNAs, will be useful for both fast-forward and reverse genetics experiments in TRV vectors.

No MeSH data available.


Related in: MedlinePlus