Raman Bansal Ohio State University Adopting high

Raman Bansal
Ohio State University
Adopting high-throughput and non-destructive RNAi technology in soybean aphid
Goal: Through IGTRCN’s peer-to-peer training program, my overall goal is to learn and
adapt a novel RNAi technology for use in the soybean aphid, an important insect pest
where gene silencing has been unsuccessful. This RNAi technology involves a unique
method of delivering small interfering RNAs (siRNAs), coupled with nanoparticles, through
aerosolization with a nebulizer. Specifically, siRNA-nanoparticles complexes are delivered into
the insect in the form of an aerosol spray that penetrates the spiracles and travel through the
tracheal respiratory system to silence targeted gene expression. This technology has been
developed recently and was successfully demonstrated in the honey bee (Li-Byarlay et al.,
2013) and, if successful, can vastly improve functional genetic research in pest species. Overall,
my proposed research will lead to the adoption and implementation of innovative RNAi
technology in a non-model system and discovery of new genes for developing RNAi-based
aphid control technologies.
Scientific merit: RNAi not only serves as a tool for functional gene analysis but also holds
promise for management of aphids which are arguably the most insidious pests on crops
worldwide (Gordon and Waterhouse, 2007; Emden and Harrington, 2007). Nevertheless, aphid
RNAi is marked by a considerable degree of inconsistency and poor silencing efficiency
(Sapountzis et al., 2014). Moreover in the soybean aphid, RNAi success has remained elusive
mainly due to the lack of an efficient delivery method. Microinjections for dsRNA (or siRNA)
delivery have proved too stressful for soybean aphid to survive due to its small (1.5 mm
maximum length, 0.6 mm maximum width) and soft body. Furthermore, due to it extreme
soybean specificity, standardized artificial diet for soybean aphid is not available, thus delivering
dsRNA (or siRNA) through the ingestion is not feasible. Recent development of the abovementioned RNAi technology presents an ideal opportunity to test a unique way of dsRNA (or
siRNA) delivery in soybean aphid. Successful execution of this non-destructive delivery method
in soybean aphid is not only expected to result in significant RNAi silencing but also allow for a
rapid evaluation of a large number of genes. In the long term, this technology carries potential to
revolutionize the basic as well as applied research on soybean aphid which is a serious pest in
soybean growing regions of US and Canada.
Significance to the applicant and host laboratory: The durability of aphid resistant soybean
varieties is questionable since this insect has evolved virulent biotypes which overcome natural
resistance (Bansal et al., 2013a). As a postdoctoral researcher, the goal of my research is to
understand the genetic basis of soybean aphid biotype evolution in the general framework of
aphid-plant interactions. My research has led to the identification of several candidate genes
which are involved in biotype evolution as well as in mediating the aphid-plant interaction.
Additionally, I have characterized genes for other aphid traits [e.g. chitin synthase (Bansal et al.,
2012), trehalase (Bansal et al., 2013b)] which are promising for its field management. However,
the lack of a successful RNAi methodology in soybean aphid has proved to be a big impediment
for functional analysis of candidate genes and in turn, the understanding of molecular
mechanisms in this insect. I believe that the learning and successful adoption of this novel RNAi
approach through the proposed training will lead to greater advancement of available genetic
technologies in soybean aphid which will be highly beneficial to meet my research goals and
towards improving soybean production.
Raman Bansal
Ohio State University
The host laboratory, the Hansen laboratory, is located at University of Illinois at UrbanaChampaign; the institute where the abovementioned RNAi technology was developed, first in
the honeybee in Gene Robinson's lab (Li-Byarlay et al., 2013), and recently for the pea aphid by
the Allison Hansen lab (Hansen lab in prep). Dr. Hansen has agreed to provide the requisite
training to adapt this technique to soybean aphid. Dr. Hansen is working to elucidate the
complex molecular mechanisms underlying pea aphid-symbiont co-evolution and its impact on
aphid biology. Our concerted research efforts will allow comparative genomic analysis in two
divergent aphid species, which will help to develop broader understanding of gene function in
aphids and their tri-trophic interactions with symbionts and host plants. Because we share
research interests in the functional genomics of divergent aphid species, I believe the proposed
training will serve as an excellent opportunity to explore future research collaborations between
us.
Activity plan and timetable: All chemical reagents including siRNA and nanoparticles will
either be synthesized or ordered as described in Li-Byarlay et al., (2013). Initially, we plan to
screen two candidate genes encoding for enzymes (aspartate transaminase and branch chain
amino-acid transaminase) involved in the shared insect-microbe amino-acid metabolism
(Hansen and Moran, 2011). Due to the silencing of these genes, we expect to observe a
significant reduction in gene expression of aspartate transaminase and branch chain amino-acid
transaminase, which are involved in essential amino acid biosynthesis. We predict that this
reduction in essential aphid nutrients will reduce aphid body weight and ultimately fitness. One
day following the aerosol sprays on age-synchronized soybean aphids (~6 day old), we will
determine which insect tissues and cells siRNA-nanoparticle complexes are penetrated into
using FISH (fluorescent in situ hybridization) (similar to Li-Byarlay et al., (2013)). Data on aphid
fecundity and survival will be recorded daily whereas body weight will be measured at 5 and 10
days post treatment. To evaluate whether or not aphid gene expression of target genes is
reduced, aphid samples will be collected at 2, 4, 8, and 12 day intervals post-treatment for qRTPCR analysis. Overall, the proposed work will take approximately one month and will be
conducted between mid-January and mid-February 2015.
Justification and relation to goals of the IGTRCN: Through the proposed training program, I
intend to learn the execution and implementation of a novel RNAi technology in an insect
system where RNAi is not successful so far. The stated goal of my training is strongly consistent
with the IGTRCN objective of promoting the development and adaptation of genetic
technologies in insect systems where that technology is not well established. As IGTRCN aims
to create opportunities for sharing of genetic technologies among researchers, successful
completion of my training in the Hansen laboratory will be an important step towards the spread
and adoption of an innovative RNAi technology across molecular entomologists.
Data dissemination plan: Immediately after the completion of training program, I plan to
submit a comprehensive post-visit report to IGTRCN. This report will cover a project
introduction, objectives, methodology (including the detailed protocol), results and conclusions
of the conducted research. Additionally, I plan to present my research findings at the Arthropod
Genomics Symposium (June 2015) and at the annual meeting of Entomological Society of
America (November 2015). Lastly, I intend to publish to my findings in a scientific journal
together in collaboration with the Hansen laboratory.
Raman Bansal
Ohio State University
Literature cited:
Bansal, R., Mian, M. R., Mittapalli, O., & Michel, A. P. (2012). Characterization of a chitin
synthase encoding gene and effect of diflubenzuron in soybean aphid, Aphis glycines.
International journal of biological sciences, 8(10), 1323.
Bansal, R., Jun, T. H., Mian, M. A. R., & Michel, A. P. (2013a). Developing host-plant resistance
for hemipteran soybean pests: lessons from soybean aphid and stink bugs. Soybean-Pest
Resistance. InTech, Rijeka, Croatia, 19-46.
Bansal, R., Mian, M. A., Mittapalli, O., & Michel, A. P. (2013b). Molecular characterization and
expression analysis of soluble trehalase gene in Aphis glycines, a migratory pest of
soybean. Bulletin of entomological research, 103(03), 286-295.
Emden, H. F. V., & Harrington, R. (Eds.). (2007). Aphids as crop pests. CABI.
Gordon, K. H., & Waterhouse, P. M. (2007). RNAi for insect-proof plants. Nature biotechnology,
25(11), 1231-1232.
Hansen, A. K., & Moran, N. A. (2011). Aphid genome expression reveals host–symbiont
cooperation in the production of amino acids. Proceedings of the National Academy of
Sciences, 108(7), 2849-2854.
Li-Byarlay, H., Li, Y., Stroud, H., Feng, S., Newman, T. C., Kaneda, M., ... & Robinson, G. E.
(2013). RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative
splicing in the honey bee. Proceedings of the National Academy of Sciences, 110(31),
12750-12755.
Sapountzis, P., Duport, G., Balmand, S., Gaget, K., Jaubert-Possamai, S., Febvay, G., ... &
Calevro, F. (2014). New insight into the RNA interference response against cathepsin-L
gene in the pea aphid, Acyrthosiphon pisum: Molting or gut phenotypes specifically induced
by injection or feeding treatments. Insect biochemistry and molecular biology, 51, 20-32.

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