Sadam Munawar¹, Muhammad Qasim², Muhammad Sarwar Khan¹, Muhammad Amjad Ali³, Faiz Ahmad Joyia^1*

¹Centre of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad-38040, Pakistan ²Department of Bioinformatics and Biotechnology, Government College University, Faisalabad-38040, Pakistan ³Department of Plant Pathology, University of Agriculture, Faisalabad-38040, Pakistan

Abstract

Bacillus thuringiensis Cry proteins are used for biological control of insect pests; however, insect pests are developing resistance against these proteins especially Cry1Ac. We hypothesized that the problem of resistance development can be overcome by mutating hot spot amino acid residues of Cry1Ac protein conferring enhanced toxicity against resistant insects. Cadherin protein region CD7-CD8 is known to act as primary insect midgut receptors involved in Cry1Ac binding. Amino acid mutations in this region may evolve resistance against Bt toxins. Here we have identified three key amino acid residues A1264, H1436 and L1461 of cadherin receptor protein from Helicoverpa armigera genome and in silico protein-protein interaction studies revealed their role in insect resistance against Cry1Ac. Three mutations viz A1264P, H1436L and L1461V showed significantly high ∆∆G values as 9.3, 6.0 and 5.9 respectively indicating destabilization of cadherin protein which reduced its binding with Cry1Ac resulting in resistance development. Further, molecular docking of these mutated amino acid residues revealed lack of interaction with amino acid residues of Cry1Ac viz Q509, Y513, W544, N547 and I585 essential for cadherin-Cry1Ac binding in susceptible insects. In second part of our study, we identified two hot spot amino acid residues of Cry1Ac viz S548, I586 whose mutation viz S548H or S548W and I586Y brought about strong interaction with midgut receptors of resistant insects having mutated cadherin. Based on these results, we sugest, reported Cry1Ac hot spot amino acids if mutated can help to overcome resistance mechanism. Here we laid a foundation for further experiments to modify the Cry1Ac hot spot residues which bind with the resistant receptor binding protein (cadherin) more strongly to perform efficient insecticidal activity against resistant strains of Helicoverpa armigera.

Keywords: Multiple sequence alignment, Cadherin, Aminopeptidase-N, Alkaline phosphatase

Sidra Hussan Saeed¹, Ghulam Mujtaba Shah Gillani¹, Uneb Gazder², Shahida Shaheen³, Alia Gul¹,

Md.  Arifuzzaman^4*, Afzal Haq Asif⁵, Alifa Nasrin⁶, Md. Asaduzzaman⁷, Qaisar Mahmood^{8, 9*}

¹Department of Botany, Hazara University, Mansehra, Pakistan

²Department of Civil Engineering, University of Bahrain, Isa Town 32038, Bahrain

³Department of Environmental Sciences, Kohsar University, Murree, Pakistan

⁴Department of Civil and Environmental Engineering, College of Engineering, King Faisal University, Al-Ahsa 31982, Saudi Arabia

⁵Pharmacy Practice Department, College of Clinical Pharmacy, King Faisal University, Al-Ahsa 31982, Saudi Arabia

⁶Combined Military Hospital, Dhaka, Bangladesh

⁷National Heart Foundation Hospital & Research Institute, Dhaka, Bangladesh

⁸Department of Environmental Sciences, COMSATS University, Abbottabad Camps 22060, Pakistan

⁹Department of Biology, College of Science, University of Bahrain, Sakhir 32038, Bahrain

Abstract

Phenolic and flavonoid content in plants are important abiotic stress biomarkers. The individual and combined impacts of toxiferous metals Arsenic (As), Cadmium (Cd) and (Copper) Cu were employed in recent studies to investigate their effect on Total flavonoids content (TFC) and total phenolic content (TPC) in various parts of Hydrocotyle umbellata L., to explore the role of plant in abating metal contamination. Folin-Ciocalteu and AlCl₃ methods were used to study TPC and TFC, respectively. Two-way analysis of variance (ANOVA) and a classification and regression tree (CART) model was employed for statistical analysis. Highest TPC was observed in decreasing order as leaf > stem > root for all the metals stress. Whereas, highest TFC was found in all plant parts when subjected to As toxicity, and the lowest TFC was found in stem of the plant under Cu toxicity. There was significant effect on TPC when subjected to Cu and As stress; in addition, significant effects of Cd and combined metal stress were also evident. Treatment concentration had non-significant effect on TPC under single metal but had significant effect in case of combined metal stress. Similarly, in case of TFC no significant effect was recorded under all the stress types. Metal type had significant effect on TPC and TFC. Whereas plant part had significant impact on TPC but non-significant values were observed on TFC. This study epitomized TPC and TFC in H. umbellata L. as effective and viable tool to pertain its role in phytoremediation against contamination of Cd, Cu and As.

Keywords: Metal toxicity, Phytoremediation, Secondary plant metabolites, Water pennywort