Hidden Markov Models based search in combination with structural bioinformatics pipeline leads to the identification of DAF-12 distant orthologous in Meloidogyne incognita

Abstract

Root-knot nematode (RKN) Meloidogyne spp. is one of the most damaging parasites due to its wide range of hosts. Here, we report a C. elegans receptor DAF-12 ortholog gene in Meloidogyne incognita (DAF-12Minc), a promising molecular target to modify the RKN life cycle. Using a combination of Hidden Markov Models (HMM) based sequence search and phylogenetic analysis we identified three DAF-12Minc genes. Although the global sequence identity between previously reported DAF-12 genes and DAF-12Minc was acceptable, the correlation between binding site residues was low in the multiple sequence alignment (MSA). Since those residues are critical for DAF-12 interaction with its ligand, the dafachronic acids (DAs), and thus its biological role, we investigated whether even if the sequence conservation is low, the active site structure was conserved and thus able to bind DAs. For this purpose, we built accurate homology models of DAF-12Minc and used them to identify and characterize the ligand binding site (LBS) and its molecular interactions with DAs-like compounds. Finally, we cloned, expressed, and evaluated the biological role of DAF-12Minc in vitro and in vivo using a DAF-12 antagonist. These in vivo results suggest that our strategy was effective to find orthologous genes among species even when sequence similarity is low.