Hidden Markov Models based search in combination with structural bioinformatics pipeline leads to the identification of DAF-12 distant orthologous inMeloidogyne incognita
Authors: Schuster Claudio David , Zabala Vanessa Judith Santillán , San Juan Rafael Betanzos , Sosa Ezequiel , Rodriguez Cristian , Kronberg Maria Florencia , Munarriz Eliana Rosa , Burton Gerardo , Castro Olga Alejandra , Modenutti Carlos Pablo 
Published: July 2023

Root-knot nematode (RKN)Meloidogynespp. is one of the most damaging parasites due to its wide range of hosts. Here, we report aC. elegansreceptor DAF-12 ortholog gene inMeloidogyne 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-12Mincgenes. Although the global sequence identity between previously reported DAF-12 genes and DAF-12Mincwas 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-12Mincand 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-12Mincin vitroandin vivousing a DAF-12 antagonist. Thesein vivoresults suggest that our strategy was effective to find orthologous genes among species even when sequence similarity is low.

Author summary

Root-knot nematodes are parasitic to plants and responsible for causing a significant loss of millions of dollars every year in crops worldwide, which makes it necessary to develop effective strategies to combat them. One popular approach is to identify genes that can serve as molecular targets. Typically, such molecular targets are discovered through basic research on model organisms. However, since they can be quite different from the target organism, conventional tools may not always be efficient in extrapolating results.

Dafachronic acids (DAs) are a crucial class of steroid hormones that regulate the development and physiology of nematodes. They are synthesized from cholesterol and are controlled by a nuclear hormone receptor known as DAF-12. This receptor acts as a master regulator of gene expression, playing a vital role in nematode biology, including development, reproduction, metabolism, stress response, and longevity. Therefore, DAF-12 is a promising molecular target for controlling parasitic nematodes.

Although DAF-12 was initially discovered in the model organismCaenorhabditis elegansand subsequently found in some parasitic nematodes, previous attempts to identify molecular targets in theMeloidogynegenus failed to detect DAF-12 orthologs. To address this gap, we employed a combination of sequence and structure analysis to identify potential candidates for DAF-12, a known and validated molecular target, which had not yet been found inMeloidogyne incognita. Our bioinformatics predictions were experimentally validated, which may serve as a starting point for future campaigns aimed at developing parasite control strategies based on this relevant molecular target.