title: 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.
authors.affiliation: The State Key Laboratory for Biology of Insect Pests and Plant Disease, Institute of Plant Protection, Chinese Academy of Agriculture Sciences, Beijing, 100193, P. R. China.
authors.forename: Fen
authors.initials: F
authors.lastname: Qiao
authors.affiliation: The State Key Laboratory for Biology of Insect Pests and Plant Disease, Institute of Plant Protection, Chinese Academy of Agriculture Sciences, Beijing, 100193, P. R. China.|College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, 430070, Hubei Province, P. R. China.
authors.forename: Lilian
authors.initials: L
authors.lastname: Luo
authors.affiliation: The State Key Laboratory for Biology of Insect Pests and Plant Disease, Institute of Plant Protection, Chinese Academy of Agriculture Sciences, Beijing, 100193, P. R. China.
authors.forename: Huan
authors.initials: H
authors.lastname: Peng
authors.affiliation: The State Key Laboratory for Biology of Insect Pests and Plant Disease, Institute of Plant Protection, Chinese Academy of Agriculture Sciences, Beijing, 100193, P. R. China.
authors.forename: Shujie
authors.initials: S
authors.lastname: Luo
authors.affiliation: The State Key Laboratory for Biology of Insect Pests and Plant Disease, Institute of Plant Protection, Chinese Academy of Agriculture Sciences, Beijing, 100193, P. R. China.
authors.forename: Wenkun
authors.initials: W
authors.lastname: Huang
authors.affiliation: The State Key Laboratory for Biology of Insect Pests and Plant Disease, Institute of Plant Protection, Chinese Academy of Agriculture Sciences, Beijing, 100193, P. R. China.
authors.forename: Jiangkuan
authors.initials: J
authors.lastname: Cui
authors.affiliation: The State Key Laboratory for Biology of Insect Pests and Plant Disease, Institute of Plant Protection, Chinese Academy of Agriculture Sciences, Beijing, 100193, P. R. China.
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.affiliation: The State Key Laboratory for Biology of Insect Pests and Plant Disease, Institute of Plant Protection, Chinese Academy of Agriculture Sciences, Beijing, 100193, P. R. China.
authors.forename: Lingan
authors.initials: L
authors.lastname: Kong
authors.affiliation: College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, 430070, Hubei Province, P. R. China.
authors.forename: Daohong
authors.initials: D
authors.lastname: Jiang
authors.affiliation: Nematology Laboratory, USDA-ARS, Building 011A, BARC-West, Beltsville, Maryland, 20705, United States of America.
authors.forename: David J
authors.initials: DJ
authors.lastname: Chitwood
authors.affiliation: The State Key Laboratory for Biology of Insect Pests and Plant Disease, Institute of Plant Protection, Chinese Academy of Agriculture Sciences, Beijing, 100193, P. R. China.
authors.forename: Deliang
authors.initials: D
authors.lastname: Peng
mesh_terms: D000818:Animals; D001665:Binding Sites; D018509:DNA, Helminth / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D050556:Fatty Acid-Binding Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017403:In Situ Hybridization; D010802:Phylogeny; D018517:Plant Roots / Q000469:parasitology; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D040521:Subcutaneous Tissue / Q000378:metabolism; D014908:Triticum / Q000469:parasitology; D014431:Tylenchoidea / Q000235:genetics* / Q000378:metabolism; D014801:Vitamin A / Q000737:chemistry / Q000378:metabolism
chemical_list: D018509:DNA, Helminth; D050556:Fatty Acid-Binding Proteins; D015801:Helminth Proteins; D011994:Recombinant Proteins; D014801:Vitamin A