Download Fighting enemies under the ground

Fighting enemies under the ground
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!
Figure 1. A corn field infected by plant-parasitic nematodes.
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.
mouth
ovary
oocyte
intestine
tail
ovary
pharynx
intestine
embryos
spermatheca
embryos
vulva
oocytes
spermatheca
Figure 2. The free-living soil nematode Caenorhabditis elegans. Headto-tail length is about 1 mm.
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.
RNA-mediated interference
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 [1]. 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 RNAmediated 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 [2]. 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 [3]. Our group, at IITKanpur, 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 [4]. 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 knot-like structures formed
due to infection by the parasite. Inset shows a single knot at a higher magnification.
Scale bar for the inset: 200 mm.
References
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 (In press; available at
www.sciencedirect.com)