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RNAi at work: Targeted pest control

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Ex 2B 89

Advances in RNA interference: dsRNA Treatment in Trees

and Grapevines for Insect Pest Suppression
Author(s): Wayne B. Hunter, Eitan Glick, Nitzan Paldi and Blake R. Bextine
Source: Southwestern Entomologist, 37(1):85-87. 2012.
Published By: Society of Southwestern Entomologists
DOI: http://dx.doi.org/10.3958/059.037.0110
URL: http://www.bioone.org/doi/full/10.3958/059.037.0110

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VOL. 37, NO. 1

SOUTHWESTERN ENTOMOLOGIST
SCIENTIFIC NOTE

MAR. 2012

Advances in RNA interference: dsRNA Treatment in Trees and Grapevines for

Insect Pest Suppression
Wayne B. Hunter1, Eitan Glick2, Nitzan Paldi2, and Blake R. Bextine3
RNA interference (RNAi) is a breakthrough technology that has significantly
impacted contemporary approaches to control the damage caused by insect pests.
The method permits functional genomics studies in many organisms that are difficult
to study, moving science beyond model systems and laboratory studies. Reviews
on applications of RNAi promise to improve human health and agricultural
production (Castanotto and Rossi 2009, Siomi and Siomi 2009, Bells 2010,
Huvenne and Smagghe 2010). To move RNAi into real-world applications such as
Citrus sp. and viticulture, we evaluated the movement and persistence of dsRNA in
citrus trees and grapevines (Hunter et al. 2010ab). Most well-known RNAi studies
continue to rely on injecting the dsRNA molecules directly into the organism; this
approach is not suitable for use in the field. If host-delivered RNAi-based
management approaches are to be implemented, plants must successfully uptake
the dsRNA and retain it long enough for the target insects to ingest it through
feeding (Hunter et al. 2010c, Huvenne and Smagghe 2010).
We propose the development of a new class of environmentally-friendly,
Highly Specific Pest Control (HiSPeC) substances. HiSPeCs are highly specific,
with no adverse effects on non-target species. Equally important, these substances
are environmentally friendly and safe to handle. This report entails current efforts of
HiSPeCs that use plant-systemic delivery of an RNAi agent. To facilitate a realworld implementation of a host-delivered RNAi strategy, robust intake of the dsRNA
and system-wide spread of the silencing action must be achieved. In some
invertebrates, there exists a systemic RNAi pathway (Jose et al. 2009); other
invertebrates depend on receptor-mediated endocytosis for cell internalization of the
dsRNA (Saleh et al. 2006, Jose and Hunter 2007). The dsRNA can have very low
LD-50, in the pictograms; results showed specificity to silence arginine kinase, AKtranscripts in psyllids and leafhoppers with the corresponding dsRNA experiments
in lab tests. RNAi strategies will demand mass-production of dsRNA, efficient
delivery methods, and methods to validate its environmental stability (Hunter et al.
2010c). The longevity of the dsRNA in Citrus trees showed suitability to develop an
area-wide pest suppression approach. This study reports the robust nature and
persistence of the RNAi pathway metabolites in whole-plant systems targeting
insects. Persistence of dsRNA in psyllids and leafhoppers was detectable for 5-8
days post ingestion from plants, while detection in treated Citrus was at least 57 days
post treatment (Fig. 1). Small interfering RNA (siRNA) was detectable into the third
month.
________________________
1

USDA, ARS, 2001 S. Rock Rd, Ft Pierce, FL 34945. Correspondent: Wayne.hunter@ars.usda.gov

Beeologics LLC., 11800 SW 77th Ave. Miami, FL 33156.
3
University of Texas at Tyler, 3900 University Blvd., Tyler, TX 75799.
2

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Fig. 1. Detection of dsRNA molecules in Citrus trees by Real time RT-PCR. Meltcurve analyses are shown of replicated samples using dsRNA-specific primers on
total RNA isolated at 35 and 57 days post-treatment, dsRNA (81C). Melt Curve
temperatures listed at top of curves are mean values of three repeated samples,
from two replicates. Control samples for citrus dehydrin occurred at (84.8C).
RNAi effects from species specific dsRNAs, made to arginine kinase, under
laboratory conditions, reduced survival of two psyllids species: Asian citrus psyllid,
Diaphorina citri Kuwayama, and potato psyllid, Bactericera cockerelli (Sulc)
(Hemiptera: Psyllidae), and the glassy-winged sharpshooter, Homalodisca
vitripennis (Germar) (Hemiptera: Cicadellidae). The RNAi-increased mortality,
worked in feeding assays of dsRNAs in sucrose solutions, cut plant tissue
absorption, and rooted plantlets. Treatments of Citrus trees and grapevines
demonstrated uptake into whole plant systems by root drench and injections.
Introduced dsRNA was detected in Citrus trees, 6-year-old Mexican limes,
approximately 2.5 m tall, 7 weeks post treatment, after a single treatment (2 g
dsRNA in 15 liters of water). Previous work supporting the efficacy of RNAi
technology also showed silencing endogenous insect genes both by injection and
feeding, to a wide range of insects; honey bees, Apis mellifera L.; beetles;
whiteflies; mosquitoes; flies; and others (Price and Gatehouse 2008, Tomoyasu et
al. 2008, Maori et al. 2009, Whyard et al. 2009, Hunter et al. 2010c). The evidence
supports development of RNAi approaches for area-wide, host-delivered, population
suppression strategies of insect pests. RNAi-based pest suppression, HiSPeC,
may be suitable to reduce insects and transmitted disease pathogens (i.e.,
Huanglongbing in citrus, Pierces disease of grapevine, and zebra chip of potato,
Solanum tuberosum L.). In conclusion, this is the first report on the delivery and
stability of dsRNA treatments in field conditions, i.e., Citrus trees and grapevine,

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over a 3-month period. This discovery, while not yet fully understood, is a
breakthrough that expands current understanding of the use and applications of
RNAi management of insect pests and plant diseases.
Acknowledgment
The authors thank Maria Gonzalez, Biological Science Technician, USDA,
ARS, U.S. Horticultural Research Lab, Fort Pierce, FL 34945.
References Cited
Bells, X. 2010. Beyond Drosophila: RNAi in vivo and functional genomics in
insects. Annu. Rev. Entomol. 55: 111-128.
Castanotto, D., and J. J. Rossi. 2009. The promises and pitfalls of RNAinterference-based therapeutics. Nature 457: 426-433.
Hunter, W. B., B. R. Bextine, R. G. Shatters, Jr., and D. G. Hall. 2010a. Emerging
psyllid genomes increasing RNAinterference targets for insect management.
DSP1. 93rd Annual Meeting Florida Entomol. Soc., 25-28 July.
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Hunter, W. B., E. Glick, B. R. Bextine, and N. Paldi. 2010b. RNAinterference to
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Williams, I. Sela, E. Maori, J. Pettis, D. Cox-Foster, and N. Paldi. 2010c.
Large-Scale Field Application of RNAi Technology Reducing Israeli Acute
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Huvenne, H., and G. Smagghe. 2010. Mechanisms of dsRNA uptake in insects
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Jose, A. M., and C. P. Hunter. 2007. Transport of sequence-specific RNA
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Jose, A. M., J. J. Smith, and C. P. Hunter. 2009. Export of RNA silencing from C.
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Maori E., N. Paldi, S. Shafir, H. Kalev, E. Tsur, E. Glick, and I. Sela. 2009. IAPV, a
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Andino. 2006. The endocytic pathway mediates cell entry of dsRNA to induce
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act as species-specific insecticides Ins. Biochem. Mol. Biol. 39: 824-832.

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