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Expression of non-host plant protease inhibitors for developing transgenic plants resistant to Helicoverpa armigera
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Expression of non-host plant protease inhibitors for developing transgenic plants resistant to Helicoverpa armigera

  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 A B
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