Research Groups

Plant Biology: Insect Resistance

Research Interests and Description

Research Interests

Bacillus thuringiensis, Insectidal proteins, mode of action, mosquitometagenomics, RNAi in insects.

Description of Research

Insecticidal proteins of Bacillus thuringiensis: application and mode of action
The Group works on insect pests of agronomic relevance and mosquito-vector transmitters of human pathogens. For agriculturally important pests, we have developed a biopesticidal formulation and constructed Bt insecticidal protein encoding plant transformation vectors. Following Bt-protein coding, genes have been assembled, evaluated for insecticidal activity and transferred to several ICGEB Member States for application in their relevant crops, Cry1Ac, Cry2Ab, Cry1Ia5, Vip, Cry1Ac and Cry6B. Transgenic cotton and rice plants bearing relevant genes are in advance stages of evaluation for protection against insect pests. We have also analyzed the mechanism of action of Cry1Ac and Vegetative Insecticidal Protein (VIP). The receptors to these proteins in susceptible larvae have been identified by RNAi directed gene silencing and in vitro analysis. The molecular basis of specificity of their interaction has been examined by phage display strategy and site directed mutagenesis of receptor and toxins. In laboratory-simulated conditions, we have examined the basis of development of resistance in Helicoverpa armigera against Cry1Ac protein. Our results revealed that the Cry1Ac resistant population acquired the ability to degrade insecticidal protein by upregulating a specific protease. This protease processed the Cry1Ac protein imprecisely leading to the generation of inactive toxin.
In addition, the Group is exploring identification of other insecticidal proteins. We have examined the insecticidal activity of pro-regions of protease and phenol oxidase of Helicoverpa armigera. At least two pro-region peptides have been identified that specifically kill neonate larvae of Helicoverpa armigera. Another hydrolytic enzyme, chitinase, was found to follow a cyclic pattern of appearance viz. growth of larvae. By subtractive micro RNA library screening, we have identified a specific miroRNA that regulates expression of chitinase in developing larvae. We are further evaluating its role in insect larvae molting and development.
Metagenomics and immune components of malaria parasite vector Anopheles sp.
With regard to vectors transmitting diseases of human health, we have been working on the metagenome of mosquito and the elucidation of molecular events in a malaria transmitting vector that facilitates normal development of the parasite. To elucidate and identify mosquito associated bacterial flora, we conducted a screen for midgut bacteria from lab-reared and wild An. stephensi mosquitoes using a culture dependent and culture independent approach. A total of 22 and 32 distinct phylotypes were identified from lab reared adult A. stephensi. A distinctinctive bacterial population was observed in female and male mosquitoes.
Genome wide screening of RNAi factors in Sf21 cells
We have completed, and annotated in silico, the sequencing of the genome of Sf 21 cells and identified several RNAi factors. An RNAi sensor cell line has been developed by integrating gfp-expressing gene in the genome of Sf21 cells. This cell line expresses gfp as a FACS linked, scorable marker. In addition, we have developed a second RNAi sensor cell line that has shRNA-GFP integrated in the genome together with gfp. This cell line does not express gfp but in the presence of suppressor proteins in trans the RNAi is functionalized and GFP expression is restored. These cell lines are being employed to identify virus encoded RNAi suppressor proteins.

Recent Publications

Sharma, A., Kumar, S., Bhatnagar, R.K. 2011. Bacillus thuringiensis protein Cry6B (BGSC ID 4D8) is toxic to larvae of Hypera postica. Curr Microbiol 62, 597-605 PubMed link

Rani, A., Sharma, A., Adak, T., Bhatnagar, R.K. 2010. Pseudoxanthomonas icgebensis sp. nov., Isolated from the Midgut of Anopheles stephensi Field Collected Larvae. J Microbiology 48, 601-606 PubMed link

Singh, G., Korde, R., Malhotra, P., Mukherjee S., Bhatnagar, R.K. 2010. Systematic deletion and site- directed mutagenesis of FHVB2 establish the role of C-terminal amino acid residues in RNAi  suppression. 2010. Biochem Biophys Res Commun 398, 290-295 PubMed link

Singh, G., Sachdev, B., Seth, R., Sharma, K.L., Bhatnagar, Raj K. 2010. Mode of action of vegetative  insecticidal protein of Bacillus thuringiensis: interaction with ribosomal S2 protein triggers larvicidal  activity in Spodoptera. Appl Environ Microbiol 76, 7202-9 PubMed link

Sree, K., Sowjanya, S. Sachdev, B., Padmaja, V., Bhatnagar, R.K. 2010. Electron spin resonance spectroscopic studies of free radical generation and tissue specific catalase gene expression in Spodoptera litura (Fab.) larvae treated with the mycotoxin, destruxin. Pesticide Biochem Physiol 97, 168-176

Rani, A., Sharma, A., Rajagopal, R., Adak, T., Bhatnagar, R.K. 2009. Bacterial diversity analysis of larvae  and adult midgut flora using culture-dependent and culture-independent methods in lab-reared and field collected Anopheles stephensi an Asian malarial vector. BMC Microbiol 19, 96 PubMed link