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

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.


Introduction
Cereal cyst nematodes (CCNs, Heterodera spp.) are sedentary plant-parasitic nematodes that infect cereal food crops such as wheat (Triticum aestivum)), barley (Hordeum vulgare) and oat (Avena sativa) [1,2]. CCNs occur in most wheat-growing regions worldwide and cause serious yield losses globally, thereby being major pests affecting the world's food supply [3,4]. The CCNs are very complex taxonomically and include a group of 12 closely related species, thereby making the control of CCNs much difficult. Among these species, Heterodera avenae, H. filipjevi, and H. latipons are considered to be most important [4,5]. Unfortunately, wheat production in China is suffering major damage from the first two species, especially H. avenae, which has spread to Henan, Hebei, and 14 other provinces and cities in less than 30 years since the first report in Hubei Province in China; approximately 20 Mha, or 80% of the total wheat growing regions in China, are infested with H. avenae and incur yield losses of 20%-30% [2,[6][7][8]. Heterodera filipjevi is closely related to H. avenae in the H. avenae group complex and was first reported in Henan Province in China [9,10]. This species is also now considered to be an important pest of cereals worldwide, has induced average yield losses of 42% in Turkey and 48% in Iran, and is a great threat to wheat production in China [1,4,11,12].
Like other obligate sedentary endoparasitic nematodes, the life cycles of H. avenae and H. filipjevi largely occur within host roots, where ingenious feeding mechanisms have evolved whereby nematodes obtain nutrients from multinucleated syncytia [13,14]. Fatty acids and retinoids are essential compounds that play important roles in cell differentiation, tissue reparation, immune response and the supply of energy [15,16]. Nematodes appear to utilize hostsupplied fatty acids and retinoids to sustain their life activities and concurrently impair host lipid-based defenses by manipulating hormone balance [15,17,18]. Because fatty acids and retinoids are hydrophobic and often oxidation sensitive, specific carrier proteins are required for their transport and protection [19][20][21]. Various classes of lipid carrier proteins have been identified in nematodes, including the nematode polyprotein allergens/antigens (NPAs) and the fatty acid-and retinol-binding (FAR) proteins, which have no counterparts in their host [21][22][23][24][25]. The molecular weights of FAR proteins are about 20kDa, i.e., a little larger than NPAs, whose molecular weights are typically ca. 15kDa [22,26]. Functional analyses indicate that phytoparasitic nematodes secrete FAR proteins with high affinities for fatty acids and retinol and low equilibrium dissociation constants; overexpressing mj-far-1 in tomato roots lowers the expression of jasmonic acid-responsive genes and increases host susceptibility, thereby promoting the nematode development, reproduction and infection [15,17,27].
The first FAR protein identified and characterized in detail was Ov-FAR-1 (previously known as Ov20) from Onchocerca volvulus, which exists in two different molecular mass forms via extent of N-linked glycosylation [21,28]. Subsequently, several FAR proteins were discovered, including ones from other animal-parasitic, plant-parasitic and free-living nematodes [26,[29][30][31][32]. Unlike several other nematodes, in which only one FAR protein has been found, eight FAR proteins (Ce-FAR-1 to 8) occur in Caenorhabditis elegans [31], and two (Ac-FAR-1 and Ac-FAR-2) with 91 percent identity at the amino acid level occur in Ancylostoma caninum, which were supposed to possibly be the first reported far alleles in a nematode [26].
In the destructive CCNs H. avenae and H. filipjevi, study on FAR proteins is still in its infancy. The aim of this investigation was characterize the FAR genes from H. avenae and H. filipjevi. The objectives of our study included obtaining the full length cDNA of FAR genes from H. avenae and H. filipjevi by RT-PCR and RACE technology, detecting the mRNA with in situ hybridization and analyzing transcriptional levels using qRT-PCR, and measuring the binding activities with fluorescence-based assays.

Nematode Populations
The cysts of Heterodera avenae and H. filipjevi were isolated from wheat roots of Daxing district, Beijing, and Xuchang County of Henan province, China in 2010. Nematode species of H. avenae and H. filipjevi were clearly distinguished by species-specific PCR fragments with 242 and 170bp of ITS sequences, respectively [33]. Cereal cyst nematodes H. avenae and H filipjevi were maintained on a compatible wheat Triticum aestivum cv. Wenmai 19 at 16°C in a greenhouse for the first week and then 22°C for the remainder of the 6 weeks growth period, mature cysts were collected, and Cysts were incubated at 4°C for at least 8 weeks, and then transferred to 16°C for hatching, preparasitic second-stage juveniles (pre-J2) were hatched as previously described [34]. The freshly hatched pre-J2 were collected on 25μm aperture sieves, suspended in DEPC-treated water, counted under a microscope, and used for inoculation and DNA or RNA extraction.

DNA and RNA Extraction
Genomic DNA of H. avenae was extracted from mature cysts as described previously [35]. Total RNA of H. avenae and H. filipjevi were isolated from about 100,000 pre-J2s with TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions and then treated with RQ1 RNase-Free DNase (Promega, USA) to remove the genomic DNA. The concentration and quality of RNA was determined with a NanoDrop-1000 (Thermo Fisher Scientific Inc., Waltham, USA) and 1% agarose gel. cDNA was synthesized from 1μg total RNA with Oligo(dT) 18 primers by using SuperScript™ III First-Strand Synthesis kit (Invitrogen, USA) according to the manufacturer's instructions.

Sequence Analysis, Alignment and Phylogenetics
The sequence homology searches to non-redundant protein database (nr) and non-redundant nucleotide database (nt) was performed via the BLASTx and BLASTn programs at NCBI   Table 1) were used to obtain full-length cDNA exclusive of the putative signal peptides, then the amplification products were ligated to pGEM-T easy vector for sequence confirmation. The correct sequences were subcloned into pHAT2-His-tagged expression vector (kindly supplied by professor Jun-Feng Liu). Recombinant pHAT2 plasmids were introduced into E. coli BL21 (DE3) cells (Novagen, Germany). Recombinant proteins induced with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) were purified with Ni Sepharose High Performance (GE Healthcare, Sweden), and imidazole was removed with dialysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the purified recombinant proteins.

In Situ Hybridization
Gene-specific primers (iHa-far-1F, iHa-far-1R and iHa-far-2F, iHa-far-2R) ( Table 1) were designed to locate the mRNA of Ha-far-1 and Ha-far-2 by performing in situ hybridization as described previously [36]. PCR DIG Probe Synthesis Kit (Roche, Switzerland) was used to synthesize digoxigenin (DIG)-labeled sense and antisense cDNA, and DIG High Primer and Detection Starter Kit I (Roche, Switzerland) was used for hybridization following the manufacturer's instructions.

Expression Pattern of Ha-far-1 mRNA at Different Development Stages of H. avenae
Quantitative real-time reverse transcription-PCR (qRT-PCR) was performed to analyze the expression pattern of Ha-far-1 (Primers: qHa-far-1F and qHa-far-1R) and Ha-far-2 (Primers: qHa-far-2F and qHa-far-2R) ( Table 1) among different developmental stages. The β-actin gene (Primers: ActinF and ActinR) and glyceraldehyde 3 phosphate dehydrogenase gene (Primers: GAPDH-qS1 and GAPDH-qAS1) [37] (Table 1) were used as a reference gene. One or both of the specific primer pairs crossed two exons. Total RNA was extracted from pre-parasitic J2s, J3s, J4s and mature females of H. avenae, and parasitic J2s were from wheat root infested by H. avenae at 1dpi (day past inoculation). After removal of contaminating genomic DNA by RQ1 RNase-Free DNase (Promega, USA), 1μg total RNA was reverse transcripted into cDNA. The qRT-PCR was carried out with triplicate technical replicas by SYBR qPCR SuperMix-UDG w/ ROX (Invitrogen Corporation, Carlsbad, CA, USA) on an ABI 7500 Fast RT-PCR System (Applied Biosystems Inc., USA). The data were analyzed by the ΔΔCt method and standardized to the β-actin gene expression levels.

Results
Three Full-Length FAR Genes from H. avenae and H. filipjevi    (Fig 2). The tree indicated that Ha-FAR-1, Ha-FAR-2 and Hf-FAR-1were present in one group constructed of 13 FAR proteins from plant and animal-parasitic nematodes. Ha-FAR-1 and Hf-FAR-1 adjoined each other in the plant-parasitic nematode branch, but Ha-FAR-2 seemed to be self-contained within the plantparasitic nematode and the animal-parasitic nematode clusters (Fig 2). SignalP and PSORT II Prediction program analyses showed that Ha-FAR-1, Ha-FAR-2 and Hf-FAR-1 possessed 21, 23 and 21 aa cleavable hydrophobic secretary signal peptides at the N terminus, respectively, suggesting that these three proteins are secreted like other FARs (Fig 3). Protein secondary structures predictions performed at PBIL showed that the three proteins were alpha-helix-rich and no beta-sheet was detected, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich (Fig 3). Furthermore, sequence analysis has showed that Ha-FAR-1, Ha-FAR-2 and Hf-FAR-1 all contained a conserved domain of Gp-FAR-1, spanning from amino acids 31-182 (E-value 4.04e-58), 49-190 (E-value 1.14e-24), and 31-182 (E-value 1.73e-57), respectively, indicating that they belong to the fatty acid-and retinoid-binding (FAR) family of proteins (Fig 3). Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites but no N-glycosylation site (Fig 3). In contrast, Ha-FAR-2 was predicted to have N-glycosylation sites, but no casein kinase II phosphorylation site (Fig 3).

Ligand Binding
The His fusion recombinant proteins of Ha-FAR-1, Ha-FAR-2 and Hf-FAR-1 were expressed in E. coli BL21 (DE3) cells. SDS-PAGE analysis showed the proteins existed in the supernatant, thus indicating that they were soluble proteins (Fig 4: lane 1, 3, 5). After purification, only single bands for Ha-FAR-1, Hf-FAR-1 and Ha-FAR-2 were observed and were approximately 17 kDa,17 kDa and 30 kDa, respectively (Fig 4: lane 2, 4, 6), indicating that the purification was effective. All three purified proteins had binding activities to DAUDA with blue shifts in their peak emission, but degrees were quite diverse. For absence and presence of Ha-FAR-1, the peak fluorescence emission shifted from 553 nm to 489 nm, and for the presence of Hf-FAR-1, from 553 nm to 491 nm, but for the presence of Ha-FAR-2 only to 528 nm, thus indicating that Ha-FAR-1and Hf-FAR-1 had similar polar binding sites but that the polar binding site of Ha-FAR-2 was much weaker (Fig 5A). Fluorescence emission intensity was increased after addition of the purified proteins into solutions of retinol, indicating that retinol had been removed from the solvent buffer by protein polar binding sites (Fig 6A). Similarly, the much The binding affinities of FAR proteins to DAUDA and retinol were further measured by vitro titration analysis. The in vitro equilibrium dissociation constants (Kds) for interaction with DAUDA by Ha-FAR-1, Hf-FAR-1 and Ha-FAR-2 were 1.63x10 -6 , 1.84x10 -6 and 6.91x10 -6 , respectively (Fig 5B-5D). The values for interaction with retinol by Ha-FAR-2, Hf-FAR-1 and Ha-FAR-1 were 3.15x10 -6 , 1.97x10 -6 , and 2.04x10 -6 , respectively (6B-D) which were all within micromolar ranges as other reported FARs [15]; however, binding ability of Ha-FAR-2 to DAUDA and retinol was significantly poorer than Ha-FAR-1and Hf-FAR-1. Competition experiments showed that fluorescence intensity of DAUDA or retinol produced a pronounced drop after adding oleic acid, and for DAUDA, red shifts in their peak emission from 499nm to 530nm were detected (Fig 7), indicating that DAUDA and retinol have the same or interactive binding sites and can be competitively displaced by oleic acid.

Expression and Localization of Ha-far-1 mRNA
The mRNA of Ha-far-1 and Ha-far-2 were detected by in situ hybridization in pre-parasitic juveniles of H. avenae. The result showed that the DIG-labeled antisense probes of Ha-far-1and Ha-far-2 both hybridized in the hypodermis (Fig 8A and 8C). No signal in control groups was detected by hybridization with the DIG-labeled sense probes (Fig 8B and 8D). The qRT-PCR results showed that Ha-far-1and Ha-far-2 were expressed in all developmental stages, while the expression level of Ha-far-1 was significantly greater than that of Ha-far-2 in all developmental stages (Fig 9). The highest expression level of Ha-far-1 was observed in J4, whereas for Ha-far-2 the highest expression level was observed in J2 (Fig 9).

Discussion
FAR proteins are unique lipid carrier proteins in nematodes and are essential for the infection process and for completing the nematode life cycle [29,32]. Retinoids appear to be one class of molecules of importance to nematodes; for example. host nodules induced by the river blindness nematode Onchocerca volvulus contained eight times more retinol than the surrounding skin [38].With respect to plant-parasitic nematodes, much less was known about FAR proteins and lipid synthesis in plant nematodes than in animal parasites. But overexpression of mj-far-1 in tomato roots regulated plant cell wall-, hormone-and fatty acid-related genes and suppressed the expression of jasmonic acid-responsive genes, thereby increasing host susceptibility and promoting nematode development, reproduction and infection [16,17]. Whether plant-or animal-parasitic, nematodes may acquire fatty acids and retinoids to meet their developmental and metabolic needs and to disturb host physiology. Consequently, the investigation of FAR proteins is important for understanding host response and nematode pathogenesis.
In this study, we identified two novel functional FAR proteins (Ha-FAR-1 and Ha-FAR-2) from H. avenae and one FAR protein (Hf-FAR-1) from H. filipjevi. The primary and secondary structures of these proteins shared much in common with FAR proteins secreted by other nematodes (Fig 3) which are alpha-helix-rich and contain a conserved FAR domain indicating that they belong to the fatty acid-and retinoid-binding (FAR) family of proteins [31,32]; Ha-FAR-2 had a long peptide at the C terminus which was random-coil rich (Fig 3). Both Ha-FAR-1 and Hf-FAR-1 apparently have casein kinase II (CKII) phosphorylation sites but no N-glycosylation site, which is conserved in many FAR proteins [26,27]. In the eight C. elegans FAR proteins (Ce-   Three Novel Fatty Acid and Retinol Binding Protein from Heterodera avenae, H. filipjevi FAR-1 to -8), the CKII phosphorylation site is conserved within the family and the ligand binding activity of Ce-FAR-7 increases after phosphorylation by this kinase [39]. Interestingly, the Ha-FAR-2 sequence predicts it has N-glycosylation sites but no CKII phosphorylation site (Fig 3).
The FAR proteins from O. volvulus occur in two isoforms with different molecular masses of 20 and 22 kDa, which results from different levels of N-linked glycosylation rather than sequence variation [40]. A study of FAR proteins from filarial nematodes showed that FAR proteins are differentially regulated by post-translational modification but that biochemical activities are strongly conserved [41]. It is possible that Ha-FAR-1 and Ha-FAR-2 are also modified post-translationally differently. Our ligand binding assays showed that Ha-FAR-1, Ha-FAR-2 and Hf-FAR-1 exhibited significant binding activities to fatty acids and retinol (Figs 5 and 6), thereby indicating that these three proteins might be involved in sequestering lipids from the nematode host. Similarly, FAR proteins from Aphelenchoides besseyi (Ab-FAR-1), Radopholus similis (Rs-FAR-1) and Ancylostoma caninum (Ac-FAR-1) have high affinity for fatty acids and retinol [15,25,26]. Interestingly, compared to Ha-FAR-1 and Hf-FAR-1, the binding of Ha-FAR-2 is much weaker (Figs 5 and 6), perhaps due to the extended C-terminal fragment or different post-translational modification or perhaps do to its preference for structurally different ligands. Competition assays in this study showed oleic acid competitively displaced not only DAUDA but also retinol (Fig 7), thereby indicating that the two distinct binding pockets might be interactive. Similarly, previous reports indicated that Gp-FAR-1, Ab-FAR-1 and Ce-FAR-7 have distinct binding pockets for fatty acids and retinoids [15,27,31]. The result of in situ hybridization showed that the mRNAs encoding Ha-FAR-1 and Ha-FAR- Three Novel Fatty Acid and Retinol Binding Protein from Heterodera avenae, H. filipjevi 2 were present in the hypodermis (Fig 8) and had hydrophobic secretory signal peptides. These results are consistent with investigations of other nematode FAR proteins. For example, the Gp-FAR-1 protein was detected on the surface of freshly hatched preparasitic J2 of G. pallida by immunolocalization studies and its mRNA was present in the hypodermis [27]. Similar results with hypodermal localization of far mRNA were obtained with A. besseyi FAR-1 [15,42]. The hypodermis is very active metabolically and plays an important roles in absorption of compounds from the external environment and in storage of metabolic reserves [43]. The excretory/secretory (ES) products of several mammal-parasitic nematodes are known to contains FAR proteins [44]; for example, the occurrence of Ac-FAR-1 in A. caninum \ES products and somatic extracts [26] indicates that secreted FAR protein exerts a role in host tissue. In H. avenae, far genes are transcribed and translated in the hypodermis and then released through the cuticle, as often occurs in many nematodes. In M. hispanica, the mRNA of FAR-1 was localized in the subventral esophageal glands and possible secretion into host tissue through the stylet [45] as are other phytoparasitic nematode secretory proteins.
The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages examined, indicating that both are essential for the entire nematode life cycle. The two far genes have discrete transcriptional patterns, with Ha-far-1 particularly greater than Ha-far-2 in all developmental stages (Fig 9). Curiously, the highest expression level of Hafar-1 was observed in J4, whereas for postparasitic J2 exhibited the highest expression level of Ha-far-2. The results demonstrate that the two genes perhaps possess different biological functions, with Ha-far-1 playing a key role in J4 an important pre-reproduction Ha-far-2 playing a key role in the postparasitic J2 establishing and maintaining infection. Similarly, members of the far family of genes are differentially expressed in different developmental stages of C. elegans [31]. In animal-parasitic nematodes, the Hc-far-1 gene expression in Haemonchus contortus was higher in adults than in larvae [24]; in A. ceylanicum, Ac-far-1 mRNA expression was lowest in males [26]. In plant-parasitic nematodes, the highest level far transcription in A. besseyi occurred in females [15]. The Mj-far-1gene in M. javanica was highly expressed in the second-stage juveniles [17]. In conclusion, the expression pattern of FAR genes varies among species and developmental stages in accordance with their biological functions.
Our study is the first to reveal two distinct FAR proteins within the same plant-parasitic nematode species: Ha-FAR-1 and Ha-FAR-2 had 28 percent identity at the amino acid level. In contrast, only one FAR protein has been reported in other species of phytoparasitic nematodes, such as Mj-FAR-1 from M. javanica, Rs-FAR-1from R. similis, Gp-FAR-1 from G. pallida and Ab-FAR-1 from A. besseyi [15,25,27,42], and the H. filipjevi reported herein. Among other nematodes, C. elegans notably produces multiple isoforms of FAR proteins [31], and the animal parasite A. caninum produces two FAR orthologues (Ac-FAR-1 and Ac-FAR-2) suggested to be controlled by alleles. Sequence identity between Ac-FAR-1and Ac-FAR-2 is 91 percent at the amino acid level, and no significant difference was found in molecular mass [26,29]. In contrast, our discovered sequence identity (28%) and molecular mass differences between Ha-FAR-1 (17kDa) and Ha-FAR-2 (30kDa) are dramatic. Moreover, the two proteins were encoded by distinct genes; phylogenetic analysis indicated that Ha-FAR-1, Ha-FAR-2 and Hf-FAR-1were present in one group, Ha-FAR-2 was in a self-contained branch within the plantand animal-parasitic nematode clusters (Fig 2). These results suggest a paralogous rather than an allelic relationship, a possibility that requires confirmation by genomic localization and evolutionary analysis. The theoretical pI of Ha-FAR-1 and Ha-FAR-2 was 5.62 and 9.02, respectively, indicating that the two proteins may play a role in different cellular environments. H. avenae and H. filipjevi are closely related species with very minor molecular and morphological differences [5,33,46], and sequence similarities illustrated by sequence alignment and phylogenetic analysis showed that identity between Ha-FAR-1 and Hf-FAR-1 was significantly high (96%), and grouped together in the phylogenetic tree (Fig 2), thereby indicating that the function, origin and evolution of the two genes were similar. Remarkably, no Hf-FAR-2 was detected in H. filipjevi, neither by searching its transcriptomic database (unpublished data) nor by PCR with the primers designed based on the cDNA sequence of Ha-far-2. It is possible that, like other plant-parasitic nematodes, H. filipjevi contains only a single FAR protein.
In conclusion, we made a preliminary attempt to characterize and understand far genes in cereal cyst nematodes. Our results provide the first indication that plant-parasitic nematodes may possess two FAR proteins originating from different genes and that both gene duplication and post-transcriptional modifications may be used to generate diverse FAR proteins.