Chapter: Biotechnology Applying the Genetic Revolution: RNA-Based Technologies

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RNAi in Plants and Fungi

RNAi was actually first observed in plants, where it was named posttranscriptional gene silencing (PTGS). The phenomenon was first noted when some early transgenic experiments in plants gave strange results.

RNAi IN PLANTS AND FUNGI

RNAi was actually first observed in plants, where it was named posttranscriptional gene silencing (PTGS). The phenomenon was first noted when some early transgenic experiments in plants gave strange results. When an extra copy of a gene was inserted to increase production of a particular protein, both the inserted gene (i.e., the transgene) and the resident gene copy were silenced. The result was a plant that made less of the target protein rather than more.

 

For example, in 1990, researchers inserted a gene to make petunia flowers a darker purple. Instead, the plant made white flowers. Both the transgene and the endogenous gene were suppressed, leaving the flower without any pigment. A similar phenomenon was seen in Neurospora, where it was called quelling. After the discovery of RNAi in Caenorhabditis elegans, it was recognized that RNAi, PTGS, and quelling all operate via the same mechanism. Initially, none of these processes affected the level of transcription. There was plenty of mRNA produced from the transgene. After some time, the mRNA for the transgene was found in two fragments, suggesting an endonuclease cleaved it in two. Later, the target mRNA was found in smaller and smaller fragments, suggesting that exonucleases were digesting the large mRNA segments. Finally, the genes were converted into heterochromatin. 


How does an extra copy of a gene induce a system that is triggered by dsRNA? One theory is that overproduction of certain mRNAs triggers RdRP to make dsRNA from the excess. This dsRNA activates Dicer to create siRNAs that quench mRNA, both from the transgene and from any closely related endogenous gene. Alternatively, when certain transgenes are expressed, some regions of the mRNA may fold back on themselves to form hairpins. These double-stranded segments may also activate Dicer. Genetic analysis of the model plant, Arabidopsis, has shown that the RdRP encoded by the SDE1 gene is necessary for transgene silencing but is not needed for antiviral RNAi. (In the latter case, the virus RNA polymerase would make dsRNA and the plant RdRP enzyme would therefore not be necessary.) This favors the first model for transgene-triggered silencing.


The most interesting trait of PTGS is the ability of silencing to propagate from one part of the plant to the next. Plants can be grafted, that is, a leaf or stem can be attached to a different plant. If the graft has a transgene silenced by PTGS, the scion (piece of grafted plant) will then silence the corresponding endogenous gene. The effect of RNAi travels through the vascular system of the plant, and affects regions without the transgene. RNAi in C. elegans also has the ability to spread, not only from tissue to tissue, but also from parent to progeny. It does not appear that mammalian systems have the ability to spread the RNAi signal.

The ability to spread may not rely solely on siRNA. In plants, the potyviruses produce an inhibitor of RNAi called helper component proteinase (Hc-Pro). This protein blocks the accumulation of siRNA. Despite this, the RNAi signal still spreads to other parts of the plant and triggers methylation of DNA, thus turning it into heterochromatin. Other viral genes that inhibit different steps of the RNAi process will, it is hoped, illuminate the mechanism of spread.

 

Other terms used to describe variants of RNAi are transcriptional gene silencing, co-suppression, and virus-induced silencing. Virus-induced silencing occurs when the viral genome has a double-stranded RNA intermediate, which triggers Dicer and RISC. Co-suppression is an early name for PTGS. Transcriptional gene silencing refers to the silencing of gene expression by converting the gene into heterochromatin. A comprehensive term, GENE impedance (GENEi), has been proposed to encompass all these phenomena but is rarely used.


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