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A new BSMV-based vector with modified β molecule allows simultaneous and stable silencing of two genes

Abstract

Virus-induced gene silencing is an important tool for functional gene analysis and the vector based on Barley stripe mosaic virus (BSMV) is widely used for the purpose in monocots. Of the tripartite BSMV genome, currently the BSMV:γMCS molecule is used to clone a fragment of a target gene. As an alternative, the BSMV:β molecule was engineered with a unique BamHI site between the open reading frame of βc (ORF βc) and poly(A). The mixture of RNA particles α, βBamHI and γMCS was fully infectious. Barley phytoene desaturase and wheat phospholipase Dα fragments were cloned to βBamHI and γMCS. Delivery of the target gene fragment in γMCS induced stronger silencing, while delivery in βBamHI yielded more stable transcript reduction. A quantitative analysis (qRT-PCR) of the transcripts showed that the silencing induced with a fragment carried in both particles was stronger and more stable than that from a fragment placed in one particle. The modification of β enables simultaneous silencing of two genes. Quantifying the β and γ particles in virus-inoculated plants revealed a 2.5-fold higher level of γ than β, while the stability of the insert was higher in β compared with γ. The possible influence of the relative quantity of β and γ particles in virus-inoculated plants on insert stability and gene silencing efficiency is discussed.

Abbreviations

BMV:

Brome mosaic virus

BSMV:

Barley stripe mosaic virus

DAB:

3,3′-diaminobenzidine tetrahydrochloride

dpi:

days post inoculation

MCS:

multiple cloning site

PDS:

phytoene desaturase

PLD:

phospholipase Dα

VIGS:

virus-induced gene silencing

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Correspondence to Waclaw Orczyk.

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Kawalek, A., Dmochowska-Boguta, M., Nadolska-Orczyk, A. et al. A new BSMV-based vector with modified β molecule allows simultaneous and stable silencing of two genes. Cell Mol Biol Lett 17, 107–123 (2012). https://doi.org/10.2478/s11658-011-0041-9

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Key words

  • BSMV
  • Cereals
  • Functional analysis
  • Phospholipase D
  • Phytoene desaturase
  • Silencing
  • Wheat
  • Vector stability
  • VIGS