1. Designing Small Aptamers to Impair Rep Activity in the Cabbage Leaf Curl Virus
Aparna Krishnamurthy1, Vincent Green1, Monica Metro1, Christian Colon1,
Nathan Lauer1, Maria Reyes2, Ben Bobay1, Linda Hanley-Bowdoin2 and José T. Ascencio-Ibáñez1
1Department of Molecular and Structural Biochemistry and 2Department of Plant and Microbial Biology
North Carolina State University, Raleigh NC 27695
Abstract
Aptamers are small chains of amino acids
that have been used to reduce replication
of geminiviruses. The overall goal of this
project is to determine how effectively new
and modified peptide aptamers can repress
the replication mechanisms of the Rep
protein in the Cabbage leaf curl virus
(CaLCuV) infection in Arabidopsis. To
achieve this goal, we designed primers with
the targeted peptide sequences, then
amplified and cloned on a geminivirus
vector instead of transforming plants, to
verify if this method of testing allows for a
speedier testing. Then, using a low-
pressure gene gun, we inoculated the
vector containing the peptide aptamers into
a plant host. Finally, we will analyze the
effect of the presence of peptide aptamers
in the development of the geminivirus
infection.
Vector used: 007
Testing the system
These represent the inoculation of Arabidopsis plants by aptamers
previously found to reduce the rate of infection of the Cabbage leaf curl
virus. Compared to C008, A1825 and A1843 showed the best results.
Though A1841 showed higher infection rates, there was no significant
difference in its plant heights. Other aptamers will be tested to determine
their strength of reduction in the infection rate.
Future Impact
In the United States alone, agriculture accounts for
approximately 80% of the nation’s consumptive water use.
The potential impact of synthetic peptide aptamers may
provide a model of a more sustainable type of crop protection
that increases crop yields and reduces the strain placed on
the environment by mitigation of chemical use on soils and
the overuse of agrochemicals.
Future research includes performing ligations and
transformations on the rest of our aptamers. We will also test
the ability of these aptamers’ to resist the rate of infection by
bombardment via gold particles into arabidopsis plants.
Future research also includes the comparison of the
effectiveness of peptide aptamers without thioredoxin
scaffolding versus peptide aptamers that have not been
modified. These methods can be assessed though
comparison of stem height, symptoms, and viral load. Run
test again with higher numbers of replicate plant samples in
order to see if significant impact is observable.
All new aptamers will be
cloned onto the 007 polylinker
to be delivered at the same
time as the virus.
First we inoculated arabidopsis plants with a control that
includes a silencing molecule for Mg Chelatase that will show
as bleaching (to verify we actually infected plants). Then we
tested three aptamers, A1825, A1844 and A1843 and follow
the infection. Results of the average heights of the infected
plants are shown here. The height of the floral stems reflect
the health of the plant. Therefore, longer plants indicate the
virus was not as effective.
0
5
10
15
20
25
30
35
40
C008 A1825 A1841 A1843
Heightcm
C008
* There were no statistically significant
differences between the average heights of
these plants at the 95% confidence level.
Future Research
Citations
• Reyes MI1, Nash TE, Dallas MM, Ascencio-Ibáñez JT, Hanley-Bowdoin L.
“Peptide Aptamers That Bind to Geminivirus Replication Proteins Confer a
Resistance Phenotype to Tomato Yellow Leaf Curl Virus and Tomato Mottle
Virus Infection in Tomato.” J. Virol. 2013 Sep; 87(17):9691-706.
• Lopez-Ochoa L, Ramirez-Prado J, Hanley-Bowdoin L. “Peptide aptamers that
bind to a geminivirus replication protein interfere with viral replication in plant
cells.” J. Virol. 2006; 80:5841–5853.
• Plant transformation bombardment diagram.
http://www.nepadbiosafety.net/subjects/biotechnology/plant-transformation-
bombardment
Acknowledgements
We would like to thank the NC State Undergraduate Research
Department for funding this research project. We also would like
to thank Dr. Niki Robertson for the 007 vector.
A1825
A1841 A1843