Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections

Abstract

Background: Double-stranded RNA (dsRNA) is a potent initiator of gene silencing in a diverse group of organisms that includes plants, Caenorhabditis elegans, Drosophila and mammals. We have previously shown and patented that mechanical inoculation of in vitro-transcribed dsRNA derived from viral sequences specifically prevents virus infection in plants. The approach required the in vitro synthesis of large amounts of RNA involving high cost and considerable labour.

Bacterially Expressed Double-Stranded RNAs against Hot-Spot Sequences of Tobacco Mosaic Virus or Potato Virus Y Genome Have Different Ability to Protect Tobacco from Viral Infection

“…Recent studies have shown that bacterially produced dsRNA could interfere with virus infection [13,[30][31][32]. As a means of acquiring transgenic plants, using dsRNA transcripts provided by this strategy for RNA interference has apparent advantages.…”

“…Accumulation of hpRNAs in bacterial extracts were confirmed by treatment with 100 μg/mL RNase A under high salt conditions (0.3 mol/L NaCl, 0.03 mol/L sodium citrate) for 37°C, 1 h. Then, the dsRNAs were purified by gel-extraction, and reverse transcriptase PCR plus sequencing was done to make sure that the expressed dsRNAs have correct sequences. The correct strains were used to produce hpRNAs according to the method of Tenllado et al [13].…”

“…Later, Tenllado et al. [13] tested this method for the control of plant viruses and achieved positive results.…”

Strategic RNA Silencing for Plant Viral Resistance

“…The nontransgenic dsRNA approach, as confirmed in both a dicot plant (tobacco) and a monocot plant (maize), provided the possibility to protect a variety of crops from a diverse group of plant viruses (Gan et al 2010). However, in contrast to the heritable resistance mediated by transgene RNA silencing, the transient approach did not confer long-term protection (Tenllado et al 2003). Therefore, continuous spraying is required for the maintenance of protection.…”

“…Meanwhile, another study found that a bacterial strain lacking the dsRNA-specific endonuclease RNase III could be cultured to produce high levels of specific dsRNAs, and those dsRNAs could effectively trigger strong and gene-specific gene silencing when fed to Caenorhabditis elegans (Timmons et al 2001). Subsequently, this method was designed for bacterial systems to biosynthesize dsRNA in vivo, and crude extracts were inoculated into plants via spraying onto plant surfaces (Tenllado et al 2003). More studies on nontransgenic approaches were conducted, including producing dsRNA using different dsRNA prokaryotic expression systems ), resistant efficiency mediated by dsRNA derived from different viral function genes (Sun et al 2010b), and different regions of the same gene (Sun et al 2010a).…”