Characterization of Three Novel Fatty Acid- and Retinoid-Binding Protein Genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the Cereal Cyst Nematodes Heterodera avenae and H. filipjevi

Abstract

Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 (KU877266), Ha-far-2 (KU877267), Hf-far-1 (KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in all stages. The highest expression level of Ha-far-1 was observed in fourth-stage juvenile (J4), whereas the highest expression level of Ha-far-2 occurred in second-stage juvenile (J2). In conclusion, we have identified two novel far genes from H. avenae and one from H. filipjevi and have provided further indication that nematode far genes are present in a variety of nematode species, where the FAR proteins share similar basic structure, expression pattern and biochemical activities.

Characterization and silencing of the fatty acid‐ and retinol‐binding Pp‐far‐1 gene in Pratylenchus penetrans

“…Pp-FAR-1 encodes an 185 amino acid peptide, possessing an N-terminal secreted signal peptide and no transmembrane domain. Among plantparasitic nematodes, FAR-1 homologues have been identified for both sedentary nematodes, such as cyst nematodes [G. pallida (Prior et al, 2001), H. avenae (Le et al, 2016;Qiao et al, 2016), H. filipjei (Qiao et al, 2016)] and root-…”

“…Statistical significant differences were calculated by ANOVA, using Tukey's test, with different letters indicating significant differences (P < 0.05 (Iberkleid et al, 2013) and Meloidogyne hispanica (Duarte et al, 2014)], and for migratory species such as R. similis (Zhang et al, 2015), A. besseyi (Cheng et al, 2013) and B. xylophilus (Espada et al, 2016). More recently, two FAR genes (Ha-far-1 and Ha-far-2) have been identified in the cyst nematode H. avenae (Qiao et al, 2016). When compared with other nematode FARs, Pp-FAR-1 showed higher sequence identity to other Pratylenchidae species, followed by cyst (Heteroderidae) and root-knot (Meloidogynidae) nematodes.…”

“…In plant-parasitic nematodes, a single FAR gene has been reported for both sedentary [e.g. Globodera pallida (Prior et al, 2001), Meloidogyne javanica (Iberkleid et al, 2013), Heterodera avenae (Le et al, 2016) and Heterodera filipjevi (Qiao et al, 2016)] and migratory [e.g. Aphelenchoides besseyi (Cheng et al, 2013), Radopholus similis (Zhang et al, 2015)] species.…”

“…More recently, comparative transcriptome and genomic data suggested that plantparasitic species (e.g. Bursaphelenchus xylophilus and H. avenae) have additional members of this FAR family (Dillman et al, 2015;Espada et al, 2016;Qiao et al, 2016). In entomopathogenic nematode species of the genus Steinernema, a wide expansion of the number of genes within the FAR family has been reported, ranging between 38 and 54 gene members of this family (Dillman et al, 2015).…”

Putative Inhibitors of SARS-CoV-2 Main Protease from A Library of Marine Natural Products: A Virtual Screening and Molecular Modeling Study

“…The protonation states were calculated, assuming a neutral pH. The PM3 Hamiltonian, as implemented in the MOPAC package (MOPAC2016 v. 18.151, Stewart Computational Chemistry, Colorado Springs, CO, USA) [38][39][40], was then used to further optimize the 3D-structures before the alignment for the docking calculations.…”