Structural characterization and localization analysis of the root-knot nematode Meloidogyne javanica fatty acid and retinol binding protein (Mj-FAR-1)

Abstract

Plant-parasitic nematodes are extremely destructive pathogens with a cosmopolitan distribution and a host range that affects most crops. They are characterized by distinct parasitic lifestyles, e.g., as sedentary or migratory endo- or ectoparasites, resulting in high losses in yield and revenue. Possessing limited lipid metabolism, they produce one or two structurally unique classes of small α-helix-rich fatty acid and retinol binding (FAR) proteins that have no counterpart in other organisms. We investigated the sequence and structural characteristics of the FAR protein of the root-knot nematode Meloidogyne javanica (Mj-FAR-1) in comparison to other studied FAR proteins. Protein sequence analyses enabled phylogenetic clustering according to trophic groups and lifestyles. Bioinformatics analysis of the FAR protein sequences revealed ten likely core amino acids representing the trophic-group clustering. Clear modifications of four of these amino acids from less reactive (nonpolar, with aliphatic R group) to more reactive (positively or negatively charged R groups, or uncharged polar R groups) might distinguish free-living from parasitic nematode species. Structural predictions of the mature Mj-FAR-1 protein and its ligand-binding pockets, suggest that adaptation toward parasitism is associated with increased reactivity of the second pocket residues, as well as those on the protein surface. Subcellular localization of Mj-FAR-1 with or without its signal peptide was determined by Agrobacterium infiltration of N-terminal mCherry-tagged protein into Nicotiana benthamiana leaves. Intact Mj-FAR-1 with its signal peptide was predominantly localized along the plasma membrane surrounding plant cells while removing the signal peptide resulted in additional localization within the cell nucleus. The nuclear localization agreed with in-silico analysis of the Mj-FAR-1 sequence and sheds new light on its function in manipulating the plant response. Our study provides the first basic structural information and subcellular localization of the plant-parasitic Mj-FAR-1 protein.