Research Groups
Plant Biology: Insect Resistance
Research Interests and Description
Group Leader: Raj Kamal Bhatnagar, PhDGroup Members
Research Interests
Bacillus thuringiensis, Insectidal proteins, mode of action, mosquitometagenomics, RNAi in insects.
Description of Research
Current research is focused on understanding the molecular basis of Bt. toxin receptor interaction specificity and metagenomics of malaria transmission vector Anopheles stephensi.Toxin receptor binding is a two step process involving reversible and irreversible association of interacting ligands. To elucidate molecular details of this interaction we have employed heterologously expressed and purified Cry1C and receptor aminopeptidase N (SIAPN). Insecticidal protein Cry1C consists of three distinct domains, D-1, D-2 and D-3. Using individually expressed and purified domains we established that D-2 and D-3 are involved in the receptor recognition and binding and D-2 has higher binding affinity than D-3. The receptor recognizing epitopes of Cry1C were further identified by panning immobilized receptor with phage library. Our results revealed that the loop 2 and loop 3 of D-2 and Loop-a of D-3 are involved in binding to SIAPN. Using Cry1C protein as the immobilized ligand in the phage display system we identified a putative SIAPN binding epitope (128HLHFHLP134) and demonstrated its involvement in recognition and toxicity. To identify specific residues of Cry1C involved in toxin activity we created a battery of substitution and deletion mutations in Loop 2 and 3 of D-2 and Loop a, of D-3 and analyzed their effects on biological activity, cytotoxicity and binding efficiency. Deletion of Loop 2 or Loop 3 or Loop a, and alanines substitution of residues 533VIVL536 resulted in nearly complete loss of toxicity, while substitution of residues 373QQ37, 379PP380, 436QR437, 38SG439, 537TG538 and 541ST542 resulted in upto 25 times lower toxicity of Cry1C. Loop 2 and Loop 3 mutants, affected in toxicity, bound to SIAPN with affinities upto 4-fold lower than native toxin. In contrast, all loop a, mutants bound to SIAPN with affinities similar to native toxin. However only 62%, 60%, 45% and 42% of the TG-AA, ST-AA, loop a DEL and VIVL-AAAA mutants were able to associate irreversibly with SIAPN. Our results with receptor interacting epitope implicate residues H128, H130, H132 and P134 of SIAPN in the binding and toxicity of Cry1C to insect larvae.
Metagenomics and immune components of malaria parasite vector Anopheles sp.
To elucidate and identify mosquito associated bacterial flora, we conducted a screen for midgut bacteria from lab reared and wild An. stephensi mosquitoes using culture dependent and culture independent approach.
A total of 22 and 32 distinct phylotypes were identified from lab reared adult A. stephensi. Distinctinctive bacterial population was observed in the female and male mosquitoes. In lab reared male A. stephensi isolates, 3 major groups were: CFB, α-, and γ- proteobacteria, whereas in lab reared female β- proteobacteria was also identified. In culturable subset of lab reared adult A. stephensi, most abundant and diverse members were of γ- proteobacteria particularly, Pseudomonas mendocina and S. marcescens, as a dominant group. Interestingly, Agrobacterium and Klebsiella sp. were present exclusively in male mosquitoes whereas Commamonas sp. was found only in female mosquitoes. The bacterial diversity of larvae was distinct from corresponding adult mosquitoes. Bacterial sp. unique in larvae were tentatively identified as, Calothrix sp., Brevibacterium sp., Dysqonomonas sp., Streptococcus sp., Lactobacillus sp., Azoarcus sp., Leptothrix sp., Exiguobacterium sp., Hydroxenophaga sp., Aeromonas sp., Thorsellia sp., and Deinococcus spp. The functional significance of such diversity is being investigated.
Recent Publications
Rajagopal, R., Arora, N., Sivakumar, S., Rao, N.G.V., Nimbalkar, S.A., Bhatnagar, R.K. 2009. Resistance of Helicoverpa armigera to Cry1Ac toxin from Bacillus thuringiensis is due to improper processing of the protoxin. Biochem J. 419, 309-316
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. 9, 96
Singh, G., Popli, S., Hari, Y., Malhotra, P., Mukherjee, S., Bhatnagar, R.K. 2009. Suppression of RNA silencing by Flock house virus B2 protein is mediated through its interaction with the PAZ domain of Dicer. FASEB J. 23, 1845-1857
Agrawaj, N., Adak, T., Chauhan, V.S., Bhatnagar, R.K. 2008. Int Propeptide-mediated specific inhibition of a recombinant serine protease from Indian Malaria vector Anopheles culicifacies. Int. J. Pep. Res. Therap. 14, 21-28
Singh, C.K., Ojha, A., Bhatnagar, R.K., Kachru, D.N. 2008. Detection and characterization of recombinant DNA expressing vip3A type insecticidal gene in GMO’s- standard single, multiplex and construct specific PCR assays. Anal. Bioanal. Chem. 390, 377-387
