Science Pic of the Day: Virus-Induced Gene Silencing


What's up with these plants?!?

Plants have evolved sophisticated mechanisms that help to keep pathogens at bay. One major group of pathogens comprise viruses - extremely small particles that infect host cells, use the host's machinery to replicate and spread throughout the plant. Viral replication comes at a cost to the plant, which in part, has led to the emergence of defense mechanisms that aim to suppress or eliminate viral replication or spread.

There are many viruses that can infect and propagate on plants. Some of these viruses have RNA genomes rather then DNA-encoded genomes (which is what we have). When these RNA viruses enter plant cells, they multiply by getting the plant cell to produce the required proteins while also replicating the viral genomes. This allows the movement of the virus and its rapid accumulation. How does the plant combat RNA viruses?

One mechanism, deployed against RNA-viruses, is Virus-Induced Gene Silencing (VIGS).

The Image: Plants that feature low levels of Phytoene Desaturase (PDS) gene expression. Plants on the fringes of this image are empty vector controls (wildtype virus) and look normal. Source: My own collection.

One universal feature that immune systems have in common is the recognition of molecules that are NOT host-derived. Microbes and viruses carry molecules that are not present in a (non-infected) host, meaning that the very presence of "non-self" or foreign molecules, signals danger or infection. The hyper-accumulation of viral RNA in the host cell is such a signal.

When viruses hijack the host cell machinery, it comes at an enormous cost to the plant as it has to make loads of proteins and RNA molecules it really does not need. To counter RNA viruses, plants have therefore adopted an RNA based and sequence-specific process to suppress the accumulation of viral RNAs. It does this by chopping up the viral RNA and using the degraded small RNA molecules to target more of the same viral RNAs. Thus, the defense is induced by the virus and is targeted against the viral genome sequence. A key and very useful feature of this defense mechanism is that it acts in a sequence-specific way. It is this sequence specificity that has made VIGS such a useful and malleable trick that researchers can use to their benefit.

The key premise to VIGS technology can be presented as a question: What would happen if a piece of host mRNA ended up in the viral RNA genome?

The answer is: it would target it too!! If plants target viruses in a sequence-specific way, they can be tricked into "thinking" that their own genes (or mRNAs to be precise) are viral RNAs. If that were to be the case (which it is), one can "silence" any plant gene using VIGS and ask questions about the role of the protein each given transcript encodes.

This has led researchers to adopt a functional tool where they:

(1) Clone a gene fragment into the genome of an RNA virus

(2) Inoculate plants with this virus (in this case a relative of tobacco called "Nicotiana benthamiana")

(3) Wait for the viral RNAs to accumulate (including the piece of plant-derived RNA) and the silencing machinery to reduce viral RNA accumulation (this takes between 2-3 weeks)

(4) Assess whether the levels of mRNAs for the endogenous gene in question (corresponding to the fragment cloned into the viral genome), is reduced (silenced).

The image shown is that of the PDS gene (A phytoene desaturase) which is a key enzyme in pigment biosynthesis. when it is not present, chlorophyll will bleach and leaves will turn white. It is a control that we often use to see whether our silencing experiment is working.

As you can see, the introduction of this small piece of PDS sequence into the viral genome has led to the silencing of its own PDS gene, giving a phenotype that is consistent with its functional role. VIGS is a powerful tool to study gene function in plants.

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