Dataset Information

Rapid Detection of Tobacco Rattle Tobravirus in Viruliferous Paratrichodorus allius from Greenhouse and Field Specimens.

ABSTRACT: The stubby root nematode, Paratrichodorus allius, is important to the potato industry in the Pacific Northwest of USA, because it vectors Tobacco rattle virus (TRV), the causal agent of corky ringspot disease. The current method for determining if nematodes are viruliferous for TRV takes several weeks, requiring a glasshouse bioassay followed by a serological test. To overcome this drawback, a rapid and affordable molecular test was developed using reverse transcription polymerase chain reaction (RT-PCR) to identify viruliferous P. allius nematodes within 48 hours. Primers from the 16 kDa gene of TRV were used to detect TRV in both greenhouse-reared and field collected P. allius. TRV RNA can be detected consistently in nucleic acids equivalent to one quarter of a viruliferous adult nematode reared in the greenhouse. In order to reduce the time and expense of processing individual nematodes from field samples, viral RNA was consistently and affordably detected in extracts from 5 field-collected adult P. allius.

SUBMITTER: Riga E

PROVIDER: S-EPMC3365295 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Rapid Detection of Tobacco Rattle Tobravirus in Viruliferous Paratrichodorus allius from Greenhouse and Field Specimens.

Riga E E Larsen R R Eastwell K K Guerra N N Guerra L L Crosslin J M JM

Journal of nematology 20090301 1

The stubby root nematode, Paratrichodorus allius, is important to the potato industry in the Pacific Northwest of USA, because it vectors Tobacco rattle virus (TRV), the causal agent of corky ringspot disease. The current method for determining if nematodes are viruliferous for TRV takes several weeks, requiring a glasshouse bioassay followed by a serological test. To overcome this drawback, a rapid and affordable molecular test was developed using reverse transcription polymerase chain reaction ...[more]

PMID: 22661778

Similar Datasets

Project description:BackgroundRNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA) of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we developed a rapid, reliable and transient method. We used viral vectors to produce dsRNA in the host plant Nicotiana attenuata to transiently silence midgut genes of the plant's lepidopteran specialist herbivore, Manduca sexta. To compare the efficacy of longer, undiced dsRNA for insect gene silencing, we silenced N. attenuata's dicer genes (NaDCL1- 4) in all combinations in a plant stably transformed to express dsRNA targeting an insect gene.Methodology/principal findingsStable transgenic N. attenuata plants harboring a 312 bp fragment of MsCYP6B46 in an inverted repeat orientation (ir-CYP6B46) were generated to produce CYP6B46 dsRNA. After consuming these plants, transcripts of CYP6B46 were significantly reduced in M. sexta larval midguts. The same 312 bp cDNA was cloned in an antisense orientation into a TRV vector and Agro-infiltrated into N. attenuata plants. When larvae ingested these plants, similar reductions in CYP6B46 transcripts were observed without reducing transcripts of the most closely related MsCYP6B45. We used this transient method to rapidly silence the expression of two additional midgut-expressed MsCYPs. CYP6B46 transcripts were further reduced in midguts, when the larvae fed on ir-CYP6B46 plants transiently silenced for two combinations of NaDCLs (DCL1/3/4 and DCL2/3/4) and contained higher concentrations of longer, undiced CYP6B46 dsRNA.ConclusionsBoth stable and transient expression of CYP6B46 dsRNA in host plants provides a specific and robust means of silencing this gene in M. sexta larvae, but the transient system is better suited for high throughput analyses. Transiently silencing NaDCLs in ir-CYP6B46 plants increased the silencing of MsCYP6B46, suggested that insect's RNAi machinery is more efficient with longer lengths of ingested dsRNA.

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

Rapid Detection of Tobacco Rattle Tobravirus in Viruliferous Paratrichodorus allius from Greenhouse and Field Specimens.

Publications

Rapid Detection of Tobacco Rattle Tobravirus in Viruliferous Paratrichodorus allius from Greenhouse and Field Specimens.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets