Plant-parasitic nematodes – enemies under the ground
While traveling by train or road, you may have noticed agricultural fields with patches of stunted crops, like the one in figure 1. Ever wondered what was wrong with these plants? Most probably, the stunted plants you observed were suffering infection by one or more of a variety of tiny round worms called nematodes. Nematodes appeared about 700 million years ago and they are now the most numerous of all organisms, occupying all types of habitats. Several species of these round worms parasitize a wide variety of organisms including many important crop plants, cattle and human. To get an idea of their success as parasites, consider this: nearly half the human population suffers from nematode infection at some point during their life time, and world-wide agriculture looses over US$100 billion to these worms!
Despite the enormous impact nematodes have on our lives, we do not yet have an effective and safe method to control them. We have not made much progress towards understanding their biology either. One of the major hurdles is their parasitic style of living. Since they need a host to feed and reproduce, attempts to grow them in the laboratory have not been successful so far. This has severely hampered investigations that rely on traditional biochemical and genetic tools – the two most common types of tools biologists employ to identify genes responsible for any biological process.
Nematodes: An introduction
What are nematodes? As I mentioned earlier, these are round worms, but very different, biologically, from the earthworm that you may be familiar with. Unlike the earthworm, which has a segmented body, nematodes do not have segments. Simply put, their body, which is usually transparent, consists of two tubes – intestine and the gonad – within a third tube, the outer cuticle or skin. Apart from these, they also possess nervous, excretory and chemosensory systems. Their size ranges from less than a millimeter to about a meter and contain a defined number of cells.
Not all nematodes are parasites. For example, the widely used experimental model Caenorhabditis elegans is a free-living soil nematode (figure 2). Several features of this remarkably small (head-to-tail length – 1 mm) worm make it an ideal model organism for various experimental investigations. The adult body of every C. elegans worm contains exactly 959 cells. These cells arise exactly in the same way in every one of the embryos following an invariant pattern of cell division. It takes about 2.5 days for a newly laid embryo to develop into an adult that lives for about 17 days. Primarily feeding on soil bacteria, each adult worm produces approximately 300 offsprings. Its genetic information content is 30 times smaller than the human. Since reviewing the immense body of literature available on the biology of C. elegans is beyond the scope of this article, I shall limit myself here to just one major recent development that is central to our work on parasitic nematodes.
Before we continue the story on nematodes, let us take a short detour to molecular biology to familiarize ourselves with the central dogma of biology: genetic information – the information required for the formation and functioning of organisms – is encoded in molecules called DNA. This genetic code is ternary: DNA is a polymer consisting of four different monomers called shortly as A, T, G and C. The order, or sequence, of these four monomers in a given segment of DNA represents a unique genetic information. For example, the genetic message contained in the sequence GAATTC is different from the one in AAGCTT. The genetic information is first transcribed from DNA into another type of molecules called mRNAs, which are then used for the synthesis of a third variety of biomolecules called proteins. Most biological functions are carried out by proteins. A segment of DNA that contains a single genetic message is called as a gene. Most often, a gene represents the information to produce a single protein. In the cell, DNA exists in a double-stranded form, where the A in one strand interacts with the T in the other strand and G with C. This A-T and G-C pairing allows us to predict the sequence of one strand from the other strand. The same principle is used by our cells to copy the genetic information as well as to transcribe genes to produce mRNAs, which are single-stranded.
Now, back to worms: Fire and co-workers, working at the department of embryology at the Carnegie Institution of Washington in Baltimore, observed that the injection into C. elegans of double-stranded RNA (dsRNA) corresponding to any one of its genes specifically depleted the mRNA molecules transcribed from that gene . In the absence of mRNA, the corresponding protein is not produced and, therefore, this ultimately results in the loss of function of the targeted gene. This phenomenon is termed as RNA-mediated interference or, shortly, RNAi. The cells of organisms such as nematodes, plants and human do not produce dsRNA. However, many viruses produce dsRNA. Thus, the RNAi machinery seems to have evolved as a defense mechanism against viruses. Scientists have exploited RNAi as a powerful tool to disrupt the functions of specific genes to uncover their functions. Since its original discovery in C. elegans, RNAi has been observed in many other organisms as well . Now that the complete DNA sequence (genome) is available for many organisms, RNAi can be applied in all these organisms for revealing the biological functional information contained in their genomes. In fact, this has already revolutionized the field of functional genomics – an area of biology focused on the characterization of gene function in large scale.
RNA-mediated interference: an effective new weapon against parasitic nematodes
An amazing aspect of RNAi in C. elegans is that it can be triggered when the worm feeds on bacteria that produce the dsRNA of the worm's gene . Our group, at IIT-Kanpur, reasoned that a similar delivery of dsRNA of parasite's genes through host organisms is likely to induce RNAi in the parasites, and if we target genes that are essential for the survival of the parasite, then it may protect the host from infection. In addition, this approach could be a powerful tool to the functional genomics of parasites. We engineered tobacco plants – a good plant model organism amenable for genetic transformation – to produce dsRNA of two essential genes of the parasitic nematode Meloidogyne incognita, which infects a wide range of agriculturally important plants. As we predicted, the transgenic tobacco plants very effectively resisted M. incognita infection (figure 4). A closer observation of the nematodes in these plants revealed that their development was severely impaired. Further, these nematodes were specifically deficient in the mRNA of targeted genes, indicating that the dsRNA produced in plants did indeed trigger RNAi response in the nematode . Ongoing efforts in our laboratory aim to apply this technology to characterize the functions of plant-parasitic nematodes as well as to produce nematode-resistant transgenic crop plants.
Figure 3. Host plant-generated dsRNA triggers RNAi in parasitic nematodes. Roots of control (A) and transgenic (B) plants 45 days after inoculation with 2500 M. incognita juveniles. Scale Bar: 1cm. Arrows point to know-like structures formed due to infection by the parasite. Inset shows a single knot at higher magnification. Scale bar for the inset: 200 mm.
Ukrainian translation of this article by Martha
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1. Fire, A., et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998. 391(6669): p. 806-11.
2. Hannon, G.J., RNAi: A guide to gene silencing. 2003, Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
3. Timmons, L., D.L. Court, and A. Fire, Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene, 2001. 263(1-2): p. 103-12.
4. Yadav, B.C., K. Veluthambi, and K. Subramaniam, Host-generated double stranded RNAinduces RNAi in plant-parasitic nematodes and protects the host from infection. Molecular and Biochemical Parasitology, 2006, 148, p. 219-222.
Department of Biological Sciences and Bio Engineering