This document summarizes research on expressing protease inhibitors (PIs) from non-host plants in transgenic plants to develop resistance to the crop pest Helicoverpa armigera. The researchers screened various plant species to identify potent PIs against H. armigera gut proteinases. Non-host plant PIs from Datura alba and Capsicum annum showed the highest inhibition of gut proteinases in vitro and in vivo. Larvae fed diets containing these non-host PIs exhibited stunted growth compared to those fed host plant diets. The study demonstrates the potential of using non-host plant PIs to genetically engineer resistance to H. armigera in crops.

1. Expression of Non-host Plant Protease Inhibitors for
Developing Transgenic Plants Resistant to Helicoverpa armigera
Dec 2009
Vinod D Parde1,2
, Hari C Sharma1
and Manvendra S Kachole2
1
International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India.
2
Department of Biochemistry, Dr Babasaheb Ambedkar Marathwada University, Aurangabad 431 004, Maharashtra, India.
Introduction
Legume pod borer, Helicoverpa armigera
is one of the most important pests
of crops, such as cotton, chickpea,
pigeonpea, cereals and vegetable
and fruit crops (Figure 1). It causes
an estimated loss of over US$2 billion
annually in the semi-arid tropics, despite
US$500 million worth of pesticides
applied for controlling this pest (Sharma
2005). Natural defense of plants against
insects mediated by protease inhibitors
(PIs) can be used as one of the defense
mechanisms for insect control (Ryan
1990). The non-host plant PIs act against the proteinases of insect gut, and they can also protect the host
plants defense proteins from proteolysis, thus, giving the plant an edge over the insect pests. Therefore, we
screened a large number of diverse non-host plant species using in vivo and in vitro conditions to identify
potent inhibitors against H. armigera.
Figure 1. Pod borer, Helicoverpa armigera damage in
pigeonpea.
Materials and Methods
We used a simple, rapid and sensitive technique, called gel X-ray film contact print (GXCP) method for
estimating host and non-host serine protease inhibitor activity (Pichare and Kachole 1994). Host and
non-host plant PIs were impregnated into artificial diet and fed to H. armigera larvae. The resulting gut
proteinases were extracted in 0.2 M glycine-NaOH buffer, pH 10.0, and separated on 10% native-PAGE,
and contact printed by GXCP method. Inhibitory activity of PIs against trypsin and HaGPs was measured
by BApNAase and azo-caseinolytic assays, respectively. Studies on the effects of diet incorporated with
host and non-host plant PIs on growth and development were carried out on using third-instar larvae of
H. armigera.
Figure 2. (A) Detection of host and non-host plant
trypsin, chymotrypsin, and H. armigera gut proteinase
inhibitors by the dot-blot method. Spot 1, no inhibition
of gut proteinase activity; Spot 2, Inhibition of gut
proteinase activity by plant PIs. (B) Gut proteinase profile
of H. armigera fed on chickpea based artificial diet (PIs
removed). (C) Inhibition of H. armigera gut proteinase
by non-host plant PIs. Lane 1, Capsicum annum; lane
2, Datura alba ness; lane 3, Mucuna pruriens; lane 4,
Psophocarpus tetragonolobus.
Results and Discussion
Visualization of H. armigera gut proteinases
(HaGPs) profile on X-ray film. At least ten
proteinase activity bands were detected in
H. armigera gut extract, of which four were the
major proteinases (HaGPs 2, 5, 7 and 9), four
were relatively major proteinases (HaGPs 3, 4,
6 and 8), while the remaining two were minor
(HaGPs 1 and 10) [Figure 2(B)]. On the basis
of substrate specificity, inhibition by synthetic
inhibitors and their molecular weight, and position
of proteinase(s) in the gel, the HaGPs were
classified into two major serine proteinase families.
In vivo and in vitro inhibition of HaGPs by host
and non-host plant PIs. Inhibitors of H. armigera
gut proteinases from host and non-host plants
detected in spot test [Figure 2(A)] were further
analyzed for in vivo [Figure 3(A)] and in vitro
[Figure 3(B)] inhibition of HaGPs. Substrate assays
were used to determine the percentage inhibition
of total proteinase activity along with trypsin
isoforms activity (Figure 3), and the insensitive gut
proteinase profile electrophoretically [Figure 2(C)].
Non-host plant, Datura alba ness PIs showed
significant inhibition of gut proteinase activity in in
vivo as well as in vitro [Figure 2(C); Figure 3; Table
1], followed by PIs from Capsicum annum, Mucuna
pruriens, and Psophocarpus tetragonolobus.
Among the host plants, Cicer arietinum and
Cajanus cajan PIs exhibited low inhibitory activity
against HaGPs.
Effect of host and non-host plant PIs on
H. armigera growth and development.
The H. armigera larvae reared on a diet with
non-host plant PIs (eg, D. alba ness) showed
stunted growth of larvae (Figure 4). Larval growth
was significantly reduced by non-host plant PIs
compared to the larvae fed on host plant diet
(Table 2). However, starvation and added stress
on gut proteinase expression system resulted in
synthesis of new and/or higher amounts of proteinases [Figure 2(C)], indicating that non-host plant PIs have
potent anti-metabolic activity towards H. armigera.
Figure 3. (A) In vivo and (B) in vitro inhibition of H. armigera gut proteinase activity by host and non-host
plant PIs.
Table 1. Electrophoretic detection of in vivo and in vitro inhibition of HaGPs by host and
non-host plant PIs.
Host and non-host
plant PIs
In vitro inhibition of HaGP In vivo inhibition of HaGP
1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
Cajanus cajan - - - - - - - + - + - - - - - - - - - +
Capsicum annum + + + + - + + + + + + + + + - + + + - +
Cicer arietinum - - - - - - - + - - - - - - - - - - - -
Datura alba ness + + + + + + + + + + + - + + - + + + + +
Mucuna pruriens + + + + - + + + + + + + + + - - + + + +
Psophocarpus
tetragonolobus
+ + + + - + + + + + + + + + - - - + + +
‘+’ Inhibition of HaGP, ‘-’ No inhibition of HaGP.
Table 2. Helicoverpa armigera fed on host and non-host plant PIs for inhibition of gut
proteinase activity.
Host and non-host
plant PIs
Initial weight
(mg)
Final weight
(mg)
Weight gain
(mg)
Growth rate
(%)
Cicer arietinum* 22.24 426.8 404.6 1902.5
Cajanus cajan 51.27 369.2 317.9 723.9
Capsicum annum 26.6 56.7 29.7 102.7
Cicer arietinum 25.5 377.4 351.9 1436.5
Datura alba ness 25.18 39.1 13.7 53.8
Mucuna pruriens 26.26 70.6 44.3 181
Psophocarpus
tetragonolobus
23.32 94 71.4 334.3
SE ± 1.025 5.4 5.34 30.3
*PIs removed from the sample.
These studies demonstrated the efficacy of non-host plant PIs
against H. armigera larvae in feeding assays, which corresponded
to their effectiveness as inhibitors of gut proteinases, as estimated
by in vitro inhibition assays. Non-host PIs from D. alba ness,
C. annum, M. pruriens and P. tetragonolobus inhibited more than
80% of the total proteolytic (azo-caseinolytic) activity of
H. armigera larvae in vivo. Therefore, non-host plant PIs can
be used as potential candidates for genetic transformation as a
protective mechanism to impart resistance to H. armigera.
Acknowledgments
We thank the staff of entomology for help with the insect culture, and
Dr Vivek Thakur for his cooperation in preparing this poster.
References
Pichare MM and Kachole MS. 1994. Detection of electrophoretically
separated proteinase inhibitors using X-ray film. Journal of biochemical
and biophysical methods. 28:215–224.
Figure 4. Development of H. armigera
fed on diets with and without plant PIs.
Larvae fed on control diet-containing
chickpea (PIs removed) showed
normal growth (upper row), while the
larvae fed on diet-containing Datura
alba ness showed retarded growth
(lower row).
Ryan CA. 1990. Proteinase inhibitors in plants: genes for improving defenses against insects and pathogens. Annual
Review of Phytopathology. 28:425-449.
Sharma HC (ed.). 2005. Heliothis/Helicoverpa Management: Emerging Trends and Strategies for Future Research.
New Delhi, India: Oxford and IBH Publishing. 469 pp.
For more information, contact: HC Sharma, Principal Scientist (Entomology), e-mail: h.sharma@cgiar.org
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