Cloning and characterization of genes encoding two detoxifying enzymes, glutathione s-transferase and carboxylesterase, from burrowing nematode (Radopholus similis)

Abstract

Radopholus similis (Cobb) Thorne is a migratory endoparasitic nematode infesting several tropical and sub-tropical plant species. Computational screening and conserved domain annotation of assembled EST sequences from the burrowing nematode (R. similis) revealed seven contigs similar to glutathione S-transferases (GSTs) and four contigs for carboxylesterases / cholinesterases (CESs). One of the contigs corresponding to each gene were cloned from R. similis cDNA (KM670018, KP027005) and characterized by phylogeny and structural motif comparison. Glutathione S-transferase is a critical antioxidant and detoxification enzyme and carboxylesterase is responsible for controlling the nerve impulse, detoxification and various developmental functions. Detoxifying ability of these proteins makes them as major targets of pesticides used for plant parasitic nematode (PPN) management. Methodology: In the present work, both molecular biology and bioinformatics approaches have been used to study two potential target genes of R. similis. The study presents 3D-structural models for Rs-GST and Rs-CES proteins using conventional molecular modeling techniques and structural motifs have been characterized with motif elucidation and sequence analysis methods. Subsequently, consense based phylogenetic analysis approach was followed to define the evolutionary relationships for each target proteins. Results: We report for the first time the presence and amplification of two novel target genes (GSTs and CESs) from R. similis. The structural motif characterization of the two genes with corresponding nematode genes indicated the functional diversity of the conserved motifs present in Rs-GST and Rs-CES. The search for protein signature motifs through InterProScan analysis confirmed the presence of thioredoxin-like fold (IPR012336), glutathione S-transferase, N-terminal (IPR004045), glutathione S-transferase, C-terminal (IPR010987) and glutathione S-transferase domain (PF00043) for Rs-GST and carboxylesterase, type B (IPR002018), alpha/beta hydrolase fold (IPR029058) and carboxylesterase domain (PF00135) for Rs-CES. The 3D protein models of each protein were developed through homology modeling and the active-site residues were predicted. Phylogenetic analysis revealed the evolutionary relationships of each target proteins. Conclusions: Identifying and cloning of genes involved in nematode survival and determining their functions are vital to elucidate the parasitism and host invasion processes of PPNs. The comparative structural analysis and motif characterization of studied targets will probably offer a novel approach for controlling plant nematodes.