Stephen Jenkins and Dr. Byron Adams, Biology
Plant-parasitic nematodes cause an estimated $125 billion worth of crop losses globally each year. Fifteen percent of nematode species are plant-parasitic nematodes, and the most significant species parasitize roots. While the parasites don’t always kill the plants, they disrupt water transport and divert nutrients to themselves, causing considerable impact on crop yields. The most significant contributors are root-knot nematodes and cyst nematodes.
Historically plant-parasitic nematodes have been managed with chemical nematicides, which have adverse environmental effects. DBCP causes male sterility and methyl bromide is an ozone depleter. Crop rotation is also used to manage pests, but some nematodes, like M. incognita can parasitize up to 3,000 plant species. A newer technique for controlling pests is the use of transgenic crops. Plants can be engineered to express Bt toxins, which are very effective, but already a list is emerging of pests that have evolved resistance to Bt crops (Fuller 2008). A more robust strategy is needed to manage plant-parasitic pests.
RNAi has been proposed as a method for conferring pests resistance to plants. RNAi is post-transcriptional gene-silencing triggered by dsRNA. It evolved in eukaryotes as a defense against viral agents, but is also involved in developmental timing and heterochromatin modification (Steeves et al. 2006) RNAi is widely conserved across the eukaryotic phylogeny, with some estimates indicating that the common ancestor of eukaryotes had a functional RNAi pathway over a billion years ago (Roger and Hug, 2006). RNAi was first demonstrated by Andrew Fire and Craig Mello in the nematode C. elegans, for which they received the Nobel Prize for Medicine and Physiology in 2006.
RNAi is triggered when double stranded RNA (dsRNA) is introduced into cells. The dsRNA is cleaved by the endonuclease Dicer into short-interfering RNAs (siRNAs), which guide the RNA-induced silencing complex (RISC) to complementary mRNAs. RISC degrades target mRNA transcripts. If the organism has RNA dependent RNA Polymerase (RdRP), then the RNAi effect can be amplified, and more siRNAs are synthesized and exported to neighboring cells. This results in the systemic spread of the knockout effect. A special case of systemic RNAi, called environmental RNAi, occurs when an organism ingests or absorbs exogenous dsRNA from its environment (Whangbo, 2008). This allows for a simple dsRNA delivery system, which would be perfect for pest control.
Significant research has already been done with engineering transgenic plants to express dsRNA as a method of pest control. Scientists can target multiple parasitism genes in the pest, making it a very robust strategy. The first reported use of in planta RNAi was in the root-knot nematode. Yadav et al (2006) engineered tobacco plants that “provided effective resistance against the parasite.” In the same year Huang et al. (2006) targeted four root-knot nematode genes with transgenic Arabidopsis, conferring resistance to the plant. Research has also been done with the soybean cyst nematode, Heterodera glycines. Steeves et al. (2006) targeted the major sperm protein gene in the SCN with transgenic soybeans, and noted a significant reduction in reproductive potential. Work has also been conducted with the sugar beet cyst nematode, with encouraging preliminary results (Sindhu, 2009).
Any time selective pressure is imposed on a population, there is a chance that organisms will evolve. We wanted to know the likelihood that the nematode pests will evolve resistance to environmental RNAi. Nematodes will most likely not evolve resistance through point mutations in the target genes because RNAi can knock out multiple genes at once. If the nematode has a point mutation in the RNAi pathway, however, then the RNAi machinery will no longer function, and the pest will be resistant to the effects of in planta RNAi. With enough selective pressure, this new mutation could spread rapidly to the rest of the population.
We want to know the fitness costs associated with acquiring resistance to RNAi. By examining the fitness of the mutants and comparing it to the wildtype, we can get an idea of how mutations in the RNAi pathway genes affect the overall fitness of nematodes.
Nematodes were reared at ten degrees Celsius on agar plates with OP50 E.coli as a food source. When there were a large number of gravid females, eggs were extracted by soaking eggs in sodium hydroxide/bleach solution. This degrades the adults, but because the eggs have a protective shell, they are not damaged. Following extraction, eggs were placed in a buffer solution over night. When the eggs hatched they were arrested in the L1 stage until given food. This synchronizes their life cycles. After two days I transferred the maturing adults to new plates. Two days later, I transferred each nematode to a new plate and counted the offspring left behind. Two days later I repeated the process and continued until the nematodes ceased laying eggs. I counted up the total offspring per nematode and calculated the average for each strain.
The general trend seems to show that the mutants have reduced fitness when compared to the wildtype nematode. This means that the resistant alleles will probably be rare in the population, at the rate of a spontaneous mutation. Under normal circumstances the resistant phenotype frequencies will stay low. However, when high selection pressure is imposed by transgenic crops, we would predict that those frequencies will rise. If all the wildtype pests are selected against, the resistant mutants would be the only organisms enjoying reproductive success, and the trait could spread rapidly to the whole population.
We need to do additional bio assays and examine things like gender ratios, longevity and tolerance to environmental extremes. We also need to find homologous genes for the RNAi pathway in plant-parasitic nematodes to see whether they would behave similarly. While RNAi is a creative solution to pest control, it is not invulnerable to the possibility of resistance, and my data show that transgenic crops may not be worthy of investment.
References
- Fuller, Victoria, et al. (2008) “Nematode Resistance.” New Phytologist. 180:27-44.
- Huang, Guozhong. (2006) “Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene.” Proceedings of the National Academy of Sciences of the United States of America. 103:14302-14306.
- Sindhu, Anoop S., et al. (2009) “Effective and specific in planta RNAi in cyst nematodes: expression interference of four parasitism genes reduces parasitic success.” Journal of Experimental Botany. 60:315-324.
- Steeves, Ryan M. et al. (2006) “Transgenic soybeans expressing siRNAs specific to major sperm protein gene suppress Heterodera glycines reproduction. Funct. Plant Biol. 33:991-999.
- Whangbo, Jennifer and Craig P. Hunter (2008) “Environmental RNAi.” Cell.
- Yadav, B.C. et al. (2006) Host-generated double stranded RNA induces RNAi in plant-parasitic nematodes and protects the host from infection. Mol. Biochem. Parasitol. 148, 219-222.