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Characterizing virus-induced gene silencing at the cellular level with in situ multimodal imaging.

ABSTRACT: Background:Reverse genetic strategies, such as virus-induced gene silencing, are powerful techniques to study gene function. Currently, there are few tools to study the spatial dependence of the consequences of gene silencing at the cellular level. Results:We report the use of multimodal Raman and mass spectrometry imaging to study the cellular-level biochemical changes that occur from silencing the phytoene desaturase (pds) gene using a Foxtail mosaic virus (FoMV) vector in maize leaves. The multimodal imaging method allows the localized carotenoid distribution to be measured and reveals differences lost in the spatial average when analyzing a carotenoid extraction of the whole leaf. The nature of the Raman and mass spectrometry signals are complementary: silencing pds reduces the downstream carotenoid Raman signal and increases the phytoene mass spectrometry signal. Conclusions:Both Raman and mass spectrometry imaging show that the biochemical changes from FoMV-pds silencing occur with a mosaic spatial pattern at the cellular level, and the Raman images show carotenoid expression was reduced at discrete locations but not eliminated. The data indicate the multimodal imaging method has great utility to study the biochemical changes that result from gene silencing at the cellular spatial level of expression in many plant tissues including the stem and leaf. Our demonstrated method is the first to spatially characterize the biochemical changes as a result of VIGS at the cellular level using commonly available instrumentation.

SUBMITTER: Burkhow SJ

PROVIDER: S-EPMC5968576 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Characterizing virus-induced gene silencing at the cellular level with in situ multimodal imaging.

Burkhow Sadie J SJ Stephens Nicole M NM Mei Yu Y Dueñas Maria Emilia ME Freppon Daniel J DJ Ding Geng G Smith Shea C SC Lee Young-Jin YJ Nikolau Basil J BJ Whitham Steven A SA Smith Emily A EA

Plant methods 20180525

<h4>Background</h4>Reverse genetic strategies, such as virus-induced gene silencing, are powerful techniques to study gene function. Currently, there are few tools to study the spatial dependence of the consequences of gene silencing at the cellular level.<h4>Results</h4>We report the use of multimodal Raman and mass spectrometry imaging to study the cellular-level biochemical changes that occur from silencing the <i>phytoene desaturase</i> (<i>pds</i>) gene using a <i>Foxtail mosaic virus</i> ( ...[more]

PMID: 29849743

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Project description:BackgroundVirus-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).ResultsNbPDS 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.ConclusionBased 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.

Dataset Information

Characterizing virus-induced gene silencing at the cellular level with in situ multimodal imaging.

Publications

Characterizing virus-induced gene silencing at the cellular level with in situ multimodal imaging.

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