Posts Tagged ‘siRNA’

Silencing is golden!

18 April, 2012

An excellent journal club article by Mark Whitehead:

Pavan Kumar, Sagar Subhash Pandit, Ian T. Baldwin. Tobacco Rattle Virus Vector: A Rapid and Transient Means of Silencing Manduca sexta Genes by Plant Mediated RNA Interference. PLoS ONE, 2012; 7 (2): e31347 DOI: 10.1371/journal.pone.0031347

Specific Insect Gene Silencing achieved by ingestion of plant produced dsRNA, via a transient viral vector platform.

RNAi- mediated gene silencing is an endogenous mechanism and has been utilised in reverse genetics in a number of organisms and it has the potential to be used as a tool for pest control.

The diagram below gives a good summary on the standard RNAi process. Briefly,  dsRNA produced in the nucleus is transported to the cytoplasm; alternatively, exogenous dsRNA can be taken up by cells with the help of a cell surface protein. In the cytoplasm, dsRNA is cleaved by RNaseIII type enzymes (dicers) to produce approximately 22 bp fragments, called small interfering RNAs (siRNAs). One strand of the siRNA (guide strand) is incorporated into the RNA-induced silencing complex (RISC) with the perfectly complementary site in a target mRNA to form a guide strand-target mRNA duplex. The target mRNA is then sliced by the Argonaute protein of RISC.

(With permission from http://www.RNAiweb.com.)

Plants have RNA-dependant RNA polymerases (RdRPs) that accentuate the process as they extend the bound guide strand to create more dsRNA that can then re-enter the RNAi cycle. dsRNA delivered to insects by various routes has been seen to induce RNAi, however insects lack RdRPs and therefore require a large constant supply of siRNAs for sustained gene silencing. Herbivorous insects feeding on stably transformed transgenic host plants have been seen to take up the produced dsRNA molecules into their gut cells, causing post transcriptional gene silencing. Generation of these stable transgenic plant lines is a time consuming task, while transient plant transformation offers a faster and more versatile approach, allowing for a number of dsRNA products to be created as a quicker screening method.

Larvae of the tobacco hornworm Manduca sexta contain genes that encode for nicotine-catabolising enzymes, rendering them resistant to the toxic nicotine alkaloid produced by their host plant Nicotiana attenuata. It was previously seen that some cytochrome P450 (CYP) genes were up-regulated in the larval gut in response to nicotine ingestion (CYP4M1 and CYP4M3 genes) and CYP6B46 was down-regulated when fed on nicotine suppressed plants.

In this paper the tobacco rattle virus (TRV) was used to transiently produce dsRNAs in Nicotiana attenuata – this approach was termed plant-virus based dsRNA producing system (VDPS) – in comparison to stably transformed plants – termed plant mediated RNAi (PMRi) for the silencing of these lepidopteran genes.

They initially checked to see if M. sexta could indeed take up the dsRNA and cause PMRi. It was observed that when the larvae were fed on a transgenic plant expressing dsRNA for the CYP6B46 gene, there was CYP6B46 smRNA found in the midgut and a reduction in the CYP6B46 transcript levels was observed, effectively causing silencing of the gene. It was very specific as the transcript levels of a similar gene (CYP6B45 – 80% similarity) was not affected. The VDPS was tested and compared to the PMRi for the same target and produced comparable silencing, that was also highly specific and did not cause any “off-target” effects.

Since the VDPS is a more rapid technique and was seen to be comparable to the PMRi, it was therefore used to screen the other gene targets – CYP4M1 and CYP4M3. Again the smRNAs for each were seen to be present in the midgut of the larvae when fed on the plants and the transcript levels were reduced with high specificity. The reduction of the CYP4M3 transcription levels also caused larval growth to decrease, indicating that this gene may a central role in nicotine tolerance.

The length of the dsRNA is known to have an effect on RNAi experiments and it would be ideal if the lengths were standardised. It is possible that the lepidopteran dicers that function in extremely alkaline environments of the midgut are specialized and possess different dicing properties than the plant dicers; consequently, insect-dicer diced smRNA might be more effective than the plant-dicer diced smRNA in gene silencing in insects.

Plant Dicers (DCLs) are involved in the biogenesis of smRNA by cleaving longer dsRNA. Four different types of DCLs are reported in higher plants. Their function has been found to overlap in plants, suggesting that one DCL can contribute to and/or compensate for the function of the others. Hence, more than one DCL might be involved in processing long dsRNA.

To address this they then silenced different combinations of the four N. attenuata’s Dicer genes in the transgenic PMRi lines producing CYP6B46 dsRNA. Long CYP6B46 transcript levels in the plants was found to be increased more than 50 fold when the DCL 1,3,4 or DCL 2,3,4 were co-silenced. These then lead to an enhanced silencing effect in the larvae midgut, indicating that there could be a preference for insect diced smRNAs or simply that the larger dsRNAs were more stable and the higher concentration enhanced the silencing effect. It also suggests that the plant and insect RNAi machinery respond differently to the dsRNA.

In conclusion PMRi can be a specific and robust system of gene silencing in M. sexta. PMRi would be the method of choice for crop protection in countries which allow the growth of transgenic crops. While retaining all the virtues of PMRi, VDPS promises to be a rapid and high throughput alternative, suitable for ecological research.

This article has been a short review of the journal article stated below. For more in depth information on this research, follow the link and download the freely available journal article.

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Silence(d) is Golden (mosaic)…

12 October, 2011

Geminivirus particle: characteristic doubled icosahedron containing a single ssDNA (courtesy Russell Kightley)

About that title…I read in my Nature News on the iPad about the use of siRNA in transgenic beans to silence expression of the Bean golden mosaic begomovirus, and I irresistibly thought of this…B-)

To serious matters – said article reported the following:

“Brazilian scientists roll out a transgenic pinto bean (Phaseolus vulgaris) engineered to fend off one of the crop’s most devastating enemies: the golden mosaic virus. Approved on 15 September by the Brazilian National Technical Commission on Biosafety (CTNBio), the transgenic bean uses RNA interference to shut down replication of the virus [reported originally in Mol Plant Microbe Interac in 2007].”

This paper reported the following:

“…we explored the concept of using an RNA interference construct to silence the sequence region of the AC1 viral gene and generate highly resistant transgenic common bean plants. Eighteen transgenic common bean lines were obtained with an intron-hairpin construction to induce post-transcriptional gene silencing against the AC1 gene. One line (named 5.1) presented high resistance (approximately 93% of the plants were free of symptoms) upon inoculation at high pressure (more than 300 viruliferous whiteflies per plant during the whole plant life cycle) and at a very early stage of plant development. “

OK, some background: Bean golden mosaic virus (BGMV) is a begomovirus, a representative of the largest genus of the Geminiviridae, and one of the more devastating viral plant pathogens on the planet.  It is a single-stranded circular DNA virus with a very distinct particle morphology, which replicates its genome by a rolling circle mechanism shared by all geminiviruses, nanoviruses, circoviruses, microviruses and pretty much any other ssDNA virus, as well as some plasmids.

RNA silencing – once known as post-transcriptional gene silencing, before the field was usurped by non-plant virologists – is a natural mechanism used by plants in particular as an adaptive immune response to plant viruses, as well as to control gene expression.  It is a complicated process, involving the formation of double-stranded RNAs from complementary sequences, transcribed from DNA or RNA genomes, which are then chopped up into shorter 21-25 base-length sequences.  These small interfering (si) RNAs are dissociated, and are free to bind to complementary sequences in the plant cell cytoplasm – and target them for degradation by a particular set of enzymes.  This happens frequently in transgenic plants, where the desired over-expression of a particular gene may be frustrated by the plant promptly silencing it.  It is also part of an arms race between plant viruses and plants, with nearly all plant viruses demonstrating some ability to interfere with siRNA silencing.

Geminiviruses are no exception: a number of papers have explored silencing suppression by geminiviruses, with a review by Dave Bisaro prominent among them.  Who is also famous for singing “Born to be Wild” in a Spanish karaoke bar in 1994 with a number of other geminivirologists, who called themselves “Subgroup IV” – but I digress.

It is interesting, then, that one can make transgenic plants expressing siRNA specific for a geminivirus gene – and get silencing of viral expression, and effective immunity to the virus: this would seem to have potential for a deathmatch, with the plant trying to silence virus-coded RNA, while the virus tries to suppress RNA silencing by the plant…as well as the fact that it is a DNA virus, and silencing is mediated at the level of cytoplasmic RNA.

But it obviously works – and probably because the siRNA is being expressed constitutively, meaning the virus infecting the first cell(s) gets shut down before it has a chance to get expression going.  The choice of gene – the “AC1” or Rep – is also important, as expression of mRNA from this is at a very low level, and it is crucial for virus genome replication.  This means that shutting it down stops any DNA replication from occurring.

So Viva! Brasil, Viva! as we South African are fond of saying.  Southern hemisphere rules geminivirus resistance, OK…because we have more than a passing interest in the same phenomenon…B-)

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dsRNA: new-new therapy, or…?

1 April, 2010

We plant virologists – or almost-former plant virologists, in my case – have a slightly cynical attitude towards the all-new, all-encompassing craze among cell biologists and mainstream virologists that is siRNA, and all its purported applications.

This is because the phenomenon of RNA silencing was first discovered in the context of unexpected resistance in transgenic plants to the homologous virus, mediated by transgenes that either produced no measurable amount of protein, or mRNAs that were not translatable.  The phenomenon was known as “post-transcriptional gene silencing” back then, among plant molecular biologists and virologists – until, that is, it was taken up by the mammalian and insect cell biology folk, whereupon its origins were quickly forgotten, and the Nobels went to…well, let us just say, not to whom some folk thought they ought.

But I digress:  suffice it to say that siRNA has now been amply demonstrated to be not only the eukaryotic cell’s (and especially those of plants) adaptive nucleic acid-based immune response to virus infection, but also a widely used means of regulation of gene expression (see here).  Needless to say, its potential uses for gene and disease therapy are also multiplying daily – which is when people forget the roots of the science.   At first sight, the New Scientist issue of 23rd March – which has an excellent article on the use of siRNA-based strategies to combat insect pests – goes some way to redressing that, given that the science has found its way back to plants.  It is especially interesting that delivering siRNA-eliciting constructs in insects can be achieved by simply feeding them dsRNA of the appropriate sequence, rather than by use of chemically-altered or encapsulated material.

 However, and here’s where one can see that no-one except plant virologists reads the plant virology literature (the converse being untrue, naturally), a problem crops up when the article goes on to discuss whether or not dsRNA is safe.  In a side box in the article, this is said:

Is it safe?

Using gene silencing, or RNA interference (RNAi) to target specific pests while leaving other species unharmed sounds like an enormous step forward. But can we be sure that the key ingredient – double-stranded RNA (dsRNA) – won’t have unexpected side effects in people?

Although most RNA in cells is single-stranded, all plants and animals also produce dsRNA to regulate the activity of their own genes. “There are lots of dsRNAs in the plant and animal products that we eat every day,” says Michael Czech, a molecular biologist at the University of Massachusetts Medical School in Worcester, who is exploring ways to use RNAi to treat type II diabetes. “Those RNA molecules are rapidly chopped up by the enzymes in our gut and are non-toxic.”

There is also a second line of defence in the form of enzymes in our blood that break down dsRNA. “You could inject dsRNA into primates at moderate doses and nothing would happen, [my emphasis]” says Daniel Anderson of the David H. Koch Institute for Integrative Cancer Research at the Massachusetts Institute of Technology, who is designing drugs based on RNAi.

Ummmm…sorry, that simply isn’t true: as long ago as the early 1970s, plant and fungal virologists had hit on the idea of using antibodies specific for dsRNA to detect the molecules in extracts of hosts infected with dsRNA (and even ssRNA) viruses.  Here’s two examples of papers from then:

Detection of mycoviruses using antiserum specific for dsRNA.
Moffitt EM, Lister RM.  Virology. 1973 Mar;52(1):301-4.

Immunochemical detection of double-stranded ribonucleic acid in leaves of sugar cane infected with Fiji disease virus.
Francki RI, Jackson AO.  Virology. 1972 Apr;48(1):275-7.

And of course, using antibodies to dsRNA implies that one can raise them in the first place…which, as I recall (my career started shortly afterwards, and I read these papers), was as a consequence of raising antisera to dsRNA-containing phytoreovirus and cryptovirus virions.  It was later found that sera to synthetic ds oligoribonucleic acids also detect dsRNAs – all of which means that injection of dsRNAs is likely to elicit antibodies against them.  With who knows what corollaries…because not too many plant virologists were too worried about long-term effects in the mainly bunnies that they injected.

Of course, the route to dsRNA pesticide effects is via the insect gut – and the same New Scientist article has this to say about dosing humans thus:

“There are lots of dsRNAs in the plant and animal products that we eat every day,” says Michael Czech, a molecular biologist at the University of Massachusetts Medical School in Worcester, who is exploring ways to use RNAi to treat type II diabetes. “Those RNA molecules are rapidly chopped up by the enzymes in our gut and are non-toxic.”

Ye-es…they would say that, wouldn’t they?  And no-one knew you could make Abs to dsRNA before someone noticed phytoreovirus antiserum bound dsRNA – so it would be a very interesting exercise to assay sera from individuals or animals previously exposed to multiple rounds of dsRNA rotavirus infection, and see whether these contained dsRNA-specific Abs, wouldn’t it?  The NS article negates some of its own reassurances by saying:

So there is good reason to think sprays containing dsRNAs lethal to insects, or plants modified to produce them, will pass all the safety tests. However, if the RNA was altered in a way that allows it to get into human cells, perhaps as a result of changes intended to make it persist longer in the environment, it might cause problems. “If you modify the dsRNA – by encapsulating it or changing the RNA molecule – then you are imposing a new chemistry that could have toxic effects on humans,” Czech cautions.

Yeah – like encapsidating it like a virus does….

And it is all probably a bit of a sideshow, in that we are in fact exposed to dsRNAs in our diet all the time: especially if one eats organic, the fresh fruits and uncooked vegetables will be rife with dsRNA-containing fungal viruses; even material containing ss+RNA viruses contains significant amounts of replicative form dsRNA  (including insect material, BTW) – so a little more used as pesticide probably wouldn’t hurt.

We hope.

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