Isolation and characterization of a fatty acid- and retinoid-binding protein from the cereal cyst nematode Heterodera avenae

Accepted Manuscript Isolation and characterization of a fatty acid- and retinoid-binding protein from the cereal cyst nematode Heterodera avenae Xiuhu Le, Xuan Wang, Tinglong Guan, Yuliang Ju, Hongmei Li PII:

S0014-4894(16)30106-0

DOI:

10.1016/j.exppara.2016.05.009

Reference:

YEXPR 7253

To appear in:

Experimental Parasitology

Received Date: 28 January 2016 Revised Date:

22 April 2016

Accepted Date: 26 May 2016

Please cite this article as: Le, X., Wang, X., Guan, T., Ju, Y., Li, H., Isolation and characterization of a fatty acid- and retinoid-binding protein from the cereal cyst nematode Heterodera avenae, Experimental Parasitology (2016), doi: 10.1016/j.exppara.2016.05.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Full length article

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Isolation and characterization of a fatty acid- and retinoid-binding protein from

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the cereal cyst nematode Heterodera avenae

Xiuhu Le, Xuan Wang*, Tinglong Guan, Yuliang Ju and Hongmei Li

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Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Department of Plant

Pathology, Nanjing Agricultural University, Nanjing, China

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Correspondence authors: Department of Plant Pathology, Nanjing Agricultural University, Nanjing, 210095, China. E-mail addresses: [email protected] (X. Wang), [email protected] (H. Li) 1

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ABSTRACT A gene encoding fatty acid- and retinoid-binding protein was isolated from the cereal

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cyst nematode Heterodera avenae and the biochemical function of the protein that it encodes

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was analysed. The full-length cDNA of the Ha-far-1 gene is 827 bp long and includes a 22-

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nucleotide trans-spliced leader sequence (SL1) at its 5-end. The genomic clone of Ha-far-1

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consists of eight exons separated by seven introns, which range in size from 48 to 186 bp. The

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Ha-far-1 cDNA contains an open reading frame encoding a 191 amino acid protein, with a

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predicted secretory signal peptide. Sequence analysis showed that Ha-FAR-1 has highest

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similarity to the Gp-FAR-1 protein from the potato cyst nematode, Globodera pallida and that

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the protein was grouped with all homologues from other plant-parasitic nematodes in a

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phylogenetic analysis. Fluorescence-based ligand binding analysis confirmed that the

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recombinant Ha-FAR-1 protein was able to bind fatty acids and retinol. Spatial and temporal

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expression assays showed that the transcripts of Ha-far-1 accumulated mainly in the

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hypodermis and that the gene is most highly expressed in third-stage juveniles of H. avenae.

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Fluorescence immunolocalization showed that the Ha-FAR-1 protein was present on the

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surface of the infective second-stage juveniles of H. avenae. Nematodes treated with dsRNA

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corresponding to Ha-far-1 showed significantly reduced reproduction compared to nematodes

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exposed to dsRNA from a non-endogenous gene, suggesting that Ha-far-1 may be an effective

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target gene for control of H. avenae using an RNAi strategy.

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Keywords: Heterodera avenae, hypodermis, developmental expression, lipid-binding, RNA

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interference

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Introduction Plant-parasitic nematodes cause economic losses to crops throughout the world, with

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annual economic losses due to these pathogens thought to exceed $125 billion dollars (Nicol

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et al., 2011). The sedentary endoparasitic root-knot nematodes (RKNs, Meloidogyne spp.) and

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cyst nematodes (CNs, Globodera and Heterodera spp.) are the most economically important

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and wide spread genera. RKNs can have extremely broad host ranges, with some species able

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to infect almost all species of flowering plants (Bird et al., 2009). Four species of RKN, M.

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incognita, M. javanica, M. arenaria and M. hapla are generally considered to be the most

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widespread and damaging plant-parasitic nematodes. Cyst nematodes also contribute

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significantly to yield reduction in agriculture. For example, G. rostochiensis and G. pallida are

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estimated to cause annual losses of 9% of total potato production worldwide (Turner and

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Rowe, 2006). H. glycines is responsible for soybean losses worth $US1.5 billion each year in

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the USA (Jones et al., 2013). As a consequence of the economic importance of these species,

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much research is focused on them and thus substantial progress has been made over the past

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decades (Abad et al., 2008; Opperman et al., 2008; Cotton et al., 2014). In contrast, the

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importance and losses caused by the other nematode species probably has been

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underestimated.

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Heterodera avenae was first recognized as a parasite of cereals in Germany and has now

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been detected in more than 40 countries, including Australia, Canada, America and most

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European countries, as well as China, Japan, India and several countries within Western Asia

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and North Africa (Nicol and Rivoal, 2007). Yield losses caused by H. avenae can be in excess

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of 90% in some heavily infested fields (Nicol et al., 2011). Infection by H. avenae stimulates

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increased root production, resulting in the typical bushy-knotted root systems and stunted,

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pale yellowish green plants with a reduced number of tillers. These symptoms are easily be

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confused with nitrogen deficiency or poor soil condition.

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ACCEPTED MANUSCRIPT It is generally accepted that secretions produced by H. avenae, as is the case for other

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sedentary endoparasitic nematodes, are responsible for the successful parasitism of nematodes,

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and those expressed exclusively in the esophageal gland cells and secreted through the stylet

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may play key roles (Mitchum et al., 2013). Meanwhile, it has also been demonstrated that

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proteins present at the nematode surface are crucial for nematode survival (Blaxter et al., 1992;

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Jones et al., 2004). Gp-FAR-1, a retinol and fatty acid-binding protein (FAR) from the potato

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cyst nematode G. pallida, has been shown to accumulate on the nematode body surface and is

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thought to interfere with plant lipoxygenase (LOX)-mediated defense signaling (Prior et al.,

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2001). FAR proteins are also considered to play an important role in scavenging fatty acids

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and retinol (Garofalo et al., 2003), making them a key target for plant nematodes research.

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FARs have therefore been identified from several plant-parasitic nematodes (Cheng et al.,

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2013; Iberkleid et al., 2013; Zhang et al., 2015). More recently, it has been demonstrated that

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the FAR protein from root-knot nematode M. javanica may regulate expression of lipid-, cell

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wall-, stress- and phenylpropanoid-related genes during nematode infection of tomato

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(Iberkleid et al., 2015), possibly through interaction with the plant jasmonic acid (JA) based

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defence signaling pathway.

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Here we report the cloning of the first FAR gene from H. avenae, the spatial and

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temporal transcription patterns of this gene, the fatty acid binding characteristics of

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recombinant FAR protein, as well as the effects of gene silencing on reproduction of

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nematodes. These data suggest that FAR gene may represent an excellent target for control of

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H. avenae.

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2.

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2.1.

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Materials and methods Nematode culture The cereal cyst nematode, H. avenae, was maintained on compatible wheat (Triticum

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ACCEPTED MANUSCRIPT aestivum cv. Aikang58) in a greenhouse as described by Ferris et al. (1989). Cysts were

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extracted by floating the air-dried soil and washing the debris onto a moistened filter paper in

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a Buchner funnel (Krusberg et al., 1994). Preparasitic second-stage juveniles (pre-J2s) were

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collected by hatching eggs pre-treated at 5 oC for 8 weeks on 25-µm-pore sieves in sterile

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water in plastic bowls at 15 oC. Freshly hatched pre-J2s were pipetted into the soil near the

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roots of seedlings and infected roots were harvested 10, 20 or 30 days post-inoculation (dpi).

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Different life stages of H. avenae were collected by blending the roots and sieving followed

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by picking the various life stages by hand from the resulting homogenate (De Boer et al.,

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1999). Adult females were directly hand-picked from root surfaces under a dissecting

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microscope at 60 dpi.

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2.2.

Nucleic acid extraction and cDNA synthesis

Genomic DNA was isolated from pre-J2 of H. avenae as described by Ray et al. (1994).

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Total RNA was isolated from different stages of H. avenae with Trizol (Invitrogen, USA)

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according to the manufacturer’s instructions after grinding in liquid nitrogen with a mortar

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and pestle. The mRNA was purified with the Oligotex mRNA Mini Kit (QIAGEN, Germany)

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and first-strand cDNA was generated by reverse transcription using the PrimeScript™ II 1st

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Strand cDNA Synthesis Kit (TaKaRa, Japan) following the manufacturer’s instructions.

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2.3.

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Isolation of cDNA and genomic DNA clone

5’ RACE primers (Far-R1 and Far-R2) and 3’ RACE primers (Far-F1 and Far-F2) were

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designed from an expressed sequence tag generated from a cDNA library of H. avenae

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(unpublished data). 5’ and 3’ RACE were performed using cDNAs from H. avenae with a

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SMART RACE cDNA Amplification kit (Clontech, Japan). Both of the PCR reaction

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products were cloned into the pMD19-T vector (TaKaRa, Japan) and then transformed into

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ACCEPTED MANUSCRIPT Escherichia coli JM109 competent cells. The positive clones were sent to Sunbiotech (Beijing,

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China) for sequencing. The complete sequence of the Ha-far-1 gene was generated from the

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overlapping sequences of both amplification products using BioEdit Version 7.0.1 (T.A. Hall,

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North Carolina State University, USA). The specific primers of FarFL-F and FarFL-R were

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designed and used to amplify the full-length cDNA and genomic clone of Ha-far-1. All

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primers used in this study are listed in Table 1.

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2.4.

Sequence analysis

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Sequence similarity searches were conducted using BLASTN and BLASTX

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(http://blast.ncbi.nlm.nih.gov/Blast.cgi). Signal peptide prediction was made using SignalP

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4.1 Server (http://www.cbs.dtu.dk/services/SignalP/) (Petersen et al., 2011). The protein

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molecular weight, theoretical isoelectric point, hydrophobicity and glycosylation sites were

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predicted through ExPASy available at the Bioinformatics Resource Portal

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(http://www.expasy.ch/tools/). Sequence alignments were conducted using CLUSTALW1.82

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(Thompson et al., 1994). The phylogenetic analysis of FAR proteins from different nematode

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species was conducted by the Neighbor-joining method using PAUP*4.0b10 (Swofford, 2002).

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Support for the inferred clusters was evaluated with 1000 bootstrap replicates.

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2.5.

Developmental expression analysis

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The transcription of Ha-far-1 in different developmental stages of H. avenae (eggs,

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pre-parasitic J2, parasitic J2, J3, J4 and adult females) was analyzed using the specific primers

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FarRT-F and FarRT-F by real-time quantitative reverse transcription PCR (RT-qPCR). Two

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housekeeping genes actin (GenBank no. JQ074059) and elongation factor-1α (EF-1α; In this

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study) were used as the references. The RT-qPCRs were performed in the Applied Biosystems

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7500 System (Applied Biosystems, USA) using SYBR Premix ExTaq (TaKaRa, Japan). Three

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biological replicates were conducted and each RT-qPCR reaction was run in triplicate. Ct

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values of Ha-far-1 were normalized to those of actin and EF-1α genes. Relative transcript

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levels were calculated by the 2–∆∆Ct method (Pfaffl, 2001).

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2.6.

In situ hybridization

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A fragment of the Ha-far-1 gene used as probe template was amplified from the cDNA

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template of H. avenae pre-J2s using the specific primers FarIS-F and FarIS-R. The sense and

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antisense cDNA probes were digoxigenin (DIG)-labeled with PCR DIG Probe Synthesis Kit

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(Roche, SWI) by asymmetric PCR (Huang et al., 2003). In situ hybridization was performed

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as described by De Boer et al. (1998) with minor modifications. Briefly, mixed stages of H.

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avenae were fixed in 4% paraformaldehyde at 5◦C for 18 h, followed by an additional

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incubation at room temperature for 4 h. Then in situ hybridization was performed with the

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digoxigenin (DIG)-labeled sense and antisense cDNA probes at 55 ◦C overnight.

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Hybridization signals within the nematodes were detected with alkaline

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phosphatase-conjugated anti-digoxigenin antibody and substrate, and specimens were

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observed with an Olympus BX51 microscope, and images were captured with a DP72

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Digital Camera (Olympus, Japan).

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Western blot and Immunolocalization The preparation of homogenate from H. avenae J2s and western blot were performed as

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described previously (De Boer et al., 1996). For immunodetection, the PVDF membranes

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(Millipore, USA) were incubated with 1 : 200 rabbit anti-rGp-FAR-1 serum (Prior et al.,

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2001), followed by a 1 : 2000 diluted HRP-conjugated monoclonal goat anti-(rabbit IgG) Ig

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(Tiagen Biotech, China). The membrane was developed with the SuperSignal West Pico

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HRP substrate kit (Thermo Scientific, USA) and photographed on a Tanon 5200 multi

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Chemiluminescent Imaging System (Tanon Science and Technology, CN).

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ACCEPTED MANUSCRIPT Freshly hatched H.avenae J2s were fixed for 18 h at 4°C in phosphate-buffered saline

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(PBS) containing 2% paraformaldehyde followed by an additional incubation for 4 h at room

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temperature. Immunolocalizations were performed as described by Semblat et al. (2001).

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Anti-rGp-FAR-1 serum was used at a 1:50 dilution in PBS (containing 0.1% horse serum

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and phenylmethylsulphonyl fluoride), followed by a 1 : 200 diluted Dylight 488 monoclonal

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goat anti-(rabbit IgG) Ig (Abbkine, USA) in PBSTB buffer (PBS containing 0.1% Tween-20

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and 0.1% bovine serum albumin). Control samples were incubated in pre-immune serum or

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diluted Dylight 488 alone. Signals were observed and photographed using an

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epifluorescence microscopy equipped differential interference contrast optics (Olympus

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BX51, Japan).

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2.8.

Recombinant protein expression and ligand binding assay

A fragment derived from full-length cDNA of Ha-far-1 gene lacking the predicted signal sequence was amplified using the primers FarEc-F and FarEc-R. The amplified product was

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cloned into pMD19-T and confirmed by sequencing, and then subcloned into BamHI/ HindIII

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sites of pET-28a(+) expression plasmid vector (Novagen, USA). The construct pET28a

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(+)-Ha-far-1 was transformed into E. coli BL21 (DE3) for protein induction. Expression of

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the recombinant protein was induced by adding 1 mM isopropyl-β-D-1-

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thiogalactopyranoside (IPTG) at 37°C for 6 h and purified by affinity chromatography using

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Ni Sepharose High Performance (GE Healthcare, Sweden) according to the manufacturer’s

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instructions and confirmed by SDS-PAGE.

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The fatty acid binding activity of rHa-FAR-1 was measured using the fluorescent

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analogs 11-(5-dimethylaminonaphthalene-1-sulfonyl amino) undecanoic acid (DAUDA)

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(Sigma, USA), cis-parinaric acid (Molecular Probes) (Cayman, USA), retinoic acid (Sigma,

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USA) and oleic acid (Sigma, USA) as previously described (Prior et al., 2001). DAUDA,

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ACCEPTED MANUSCRIPT cis-parinaric acid and oleic acid were prepared at 10 mM stock solution in ethanol and used at

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1:1000 dilutions in PBS except for cis-parinaric acid, which was diluted at 1: 2000 in PBS.

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Retinol was freshly prepared at 10 mM in ethanol and used at a 1:1000 dilution in ethanol and

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added directly to the protein solutions. Fluorescence emission spectra were recorded at 25°C

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with a SpectraMax M5 (Molecular Devices, USA) and the excitation wavelengths used for

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DAUDA, retinol and cis-parinaric acid were 345, 350 and 319 nm, respectively.

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2.9.

Gene silencing in vitro and inoculation

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A 420 bp fragment was amplified from the full-length cDNA of Ha-far-1 using the

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specific primers FarT7-F and FarT7-R which incorporated a T7 promoter sequence. The

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fragment amplified from the pBin-GFP vector with the primers GFPT7-F/R was used as a

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negative control. The dsRNAs were synthesized from the PCR products using T7

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RiboMAXTM Express RNAi System Kit (Progema, USA) according to the manufacturer’s

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instructions. The RNAi soaking method was performed according to Urwin et al. (2002).

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Freshly hatched J2s (approximately 10,000) of H. avenae were soaked in soaking buffer (50

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mM octopamine in M9 buffer containing dsRNA at 2 µg/µl) for 4 h at room temperature on a

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rotator. After soaking, nematodes were washed three times with nuclease-free water to remove

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any external exogenous dsRNA. The treated J2 were further incubated in nuclease-free water

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for 24 h at room temperature and then assayed by using RT-qPCR and infection of wheat roots

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(800 J2 per plant). Treated nematodes were inoculated into pouches near the root of

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two-week-old wheat seedlings of Aikang58. The number of females on the infected plant root

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surface as well in the soil was analyzed 60 days after inoculation. The experiment was

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repeated three times and the data were analysed using a t-test (P < 0.05).

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3.

Results

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3.1.

Cloning of the full-length Ha-far-1 gene Products generated by 3’ and 5’ RACE procedures were combined to generate full-length

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cDNAs of the far-1 gene from H. avenae. The cDNA designated as Ha-far-1 contains 827

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nucleotides with an 573-bp open reading frame (ORF). The 5’ untranslated regions (UTR) of

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the cDNA was 63 bp long and included the spliced leader SL1 sequence, while the 3’ UTRs

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contained a polyadenylation signal (attaaa) followed by a polyA tail. Ha-far-1 genomic clone

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was obtained by PCR amplification from H. avenae genomic DNA, resulting in a 1391 bp

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gene (GenBank accession number KR864864), which contained 7 introns ranging in size

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from 48 to 186 bp. The sequences of all exon-intron junctions conformed to the GT/AG rule

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except that the splice donor site of exon 2 was GC instead of GT (Blumenthal and Steward,

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1997) (Fig. 1).

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3.2

Sequence and phylogeny analyses of Ha-far-1

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The ORF present in the Ha-far-1 cDNA encoded a deduced protein of 191 amino acids

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with a theoretical molecular mass of 21 kDa and pI of 5.62, as well as a predicted signal

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peptide with a cleavage site between Ala21 and Ala22. A motif search revealed that

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Ha-FAR-1 had conserved casein kinase II phosphorylation sites at residues 50, 52, 81, 107

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and 138, but did not have predicted glycosylation sites (Fig. 1). A protein similarity search of

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Ha-FAR-1 in GenBank using BLASTP showed high similarity with Gp-FAR-1 from G.

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pallida (78% identity and 85% similarity, E-value 2e-92). Ha-FAR-1 also showed

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27-72% identity and 44-83% similarity to other homologues from plant-parasitic, animal,

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human and free-living nematodes.

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The sequences of Ha-FAR-1 and homologues from other nematodes downloaded from

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NCBI were used to conduct a phylogenic analysis. The tree was generated with amino acid

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ACCEPTED MANUSCRIPT sequences without any signal peptides identified by SignalP. The result shows that all FARs

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from plant-parasitic nematodes formed a well-supported clade, in which Ha-FAR-1 and

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Gp-FAR-1 formed a tight cluster that was separated from sequences from Aphelenchoides,

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Radopholus and Meloidogyne. The phylogenetic relationship of FARs between animal and

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human nematodes was complicated; the distribution of FAR proteins from animal parasitic

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nematodes was decentralized in the tree with some of the FARs from animal parasites

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grouped with those from human nematodes (Fig. 2).

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3.3.

Localization and expression of Ha-far-1

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The tissue localization of the Ha-far-1 transcripts was analysed by in situ mRNA

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hybridization. The results showed that the signal from the Ha-far-1 mRNA was dispersed in

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different areas of the nematode hypodermis (Fig. 3). This is consistent with the surface

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localization reported for the Gp-FAR-1 protein of G. pallida (Prior et al., 2001). The

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expression pattern of Ha-far-1 was quantified in different life stages of H. avenae using

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RT-qPCR. The mRNA transcript of Ha-far-1 was present in all developmental stages. The

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highest level of transcript accumulation was detected in third-stage juveniles (J3), in which

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expression was approximately fourteen fold higher than in eggs. Expression levels of

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Ha-far-1 in pre-parasitic second-stage juveniles (Pre-J2), parasitic second-stage juveniles

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(Par-J2), fourth-stage juveniles (J4) and females showed increases of 2.5, 7.9, 5.0 and 2.2 fold

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compared to those in eggs, respectively (Fig. 4).

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3.4. Immunoassay of Ha-FAR-1

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Ha-FAR-1 was detected in nematode homogenate of H.avenae by Western-blot analysis

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using a polyclonal antiserum raised against a recombinant FAR protein from G. pallida

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(rGp-FAR-1). The polyclonal antiserum reacted specifically with a protein whose molecular

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ACCEPTED MANUSCRIPT mass is approximately 20 kDa, which is similar to the calculated molecular mass of the

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predicted mature protein of Ha-FAR-1. No signal was observed on western blot treated with a

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preimmune serum (Fig. 5a). Immunolocalization assays of Ha-FAR-1 on freshly hatched

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pre-parasitic J2s showed that the rGp-FAR-1 antiserum mainly labelled the surface of the

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anterior end of the nematodes (Fig. 5a). No binding was detected with either the pre-immune

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serum or the secondary antibody alone (data not shown).

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3.5.

Ligand binding assay

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The Ha-FAR-1 recombinant protein (rHa-FAR-1) was analysed after heterologous

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expression in E. coli BL21 (DE3) and purification by Ni2+-chelating Sepharose column. A

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single band of approximately 21 kDa was visible after SDS PAGE, which is consistent with

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the theoretical molecular mass of Ha-far-1 coupled to a His-tag (Fig. 6a). The rHa-FAR-1 was

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found to bind the fluorophore-tagged fatty acid DAUDA to produce a significant blue shift in

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its peak emission from 556 nm to 492 nm, which revealed that rHa-FAR-1 has a highly apolar

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binding site (Macgregor and Weber, 1986). The addition of oleic acid to rHa-FAR-1 and

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DAUDA complexes led to a substantial drop in the fluorescence intensity, indicative of

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competitive displacement of DAUDA by oleic acid (Fig. 6b). The rHa-far-1 protein also

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bound the intrinsically fluorescent retinol and cis-parinaric acid, resulting in substantial

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increases in fluorescence emission intensity. The addition of oleic acid could displace retinol

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and cis-parinaric from the binding site (Fig. 6c, 6d). Ha-FAR-1 is therefore a functional lipid

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and retinol binding protein.

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3.6.

Effect of Ha-far-1 silencing on the pathogenicity of H. avenae

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The Ha-far-1 transcripts expressed in juveniles after soaking in dsRNA were detected by

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RT-qPCR (Fig. 7a). The relative expression of Ha-far-1 mRNA in nematodes treated with

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ACCEPTED MANUSCRIPT Ha-far-1 dsRNA showed a significant reduction of 57.8% (P < 0.05) compared with the blank

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control that was soaked in water. No significant difference was seen in the expression of

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Ha-far-1 in juveniles treated with gfp dsRNA and/or water. The reproduction of H. avenae on

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seedlings of T. aestivum was investigated 60 days after being inoculated with J2s treated with

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the dsRNAs or water. The number of cysts produced by the nematodes treated with Ha-far-1

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dsRNA significantly decreased by 62.1% compared with the control (P < 0.05), while the

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number of cysts produced by the nematodes treated with gfp dsRNA did not show a

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significant difference compared with the control (Fig. 7b).

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4.

Discussion

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Nematodes cannot synthesize all of the lipids required for their metabolic and

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developmental demands and parasites have to obtain these molecules from their hosts. Lipid

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binding proteins, which allow hydrophobic lipids to be transported in the aqueous

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environment of the cell, play important roles in this process. The FAR proteins of nematodes,

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are structurally distinct ~20 kDa lipid binding proteins, which have no counterparts in plants

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or animals. Almost all parasitic nematodes studied to date possess only one type of FAR

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protein (although different alleles may be present) (Basavaraju et al., 2003). However, the

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genome of one human parasitic nematode, the blood-feeding intestinal parasite Necator

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americanus, contains at least seven more paralogues of the FAR (Tang et al., 2014), give it a

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FAR complement similar to that of free-living nematode C.elegans (Garofalo et al., 2003).

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The phylogenetic analysis in this study shows that the FAR proteins of N. americanus fall into

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several distinct groups, and all of them are located far from those of plant-parasitic nematodes.

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It is not clear what causes this difference in members of FAR family between this parasitic

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nematode and others, whether they had followed different evolutionary paths or there are

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more FAR proteins that have not yet been identified in other nematodes. Recently, the 3D

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ACCEPTED MANUSCRIPT structure of a FAR proein from N. americanus has been determined by NMR (nuclear

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magnetic resonance) spectroscopy and X-ray crystallography (Rey-Burusco et al., 2009),

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combined with the Ce-FAR-7 whose structure has previously been solved (Jordanova et al.,

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2015), providing us with the possibility of predicting the structure of Ha-FAR-1. The 3D

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structure of Ha-FAR-1 predicted by homology modeling using Na-FAR-1 showed the typical

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folding of FAR proteins, which is composed of thirteen alpha helices of various lengths. The

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first three helices in N-terminal are co-planar with the C-terminal helices α12 and α13, the

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other five helices α5, α7, α8, α9 and α10 are co-planar, together with α6 and α7 sealed by α4

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and formed a wedge-shaped internal cavity. Compared to Na-FAR-1, the two additional

10

helices of Ha-FAR-1 create more complex internal ligand binding cavity that might be give

11

rise to flexibility of ligand-binding.

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The first described FAR family member was Ov-FAR-1 from the filarial nematode

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Onchocerca volvulus, which causes human river blindness (Kennedy et al., 1997).

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Immunolocalization and in situ hybridization studies showed that Ov-FAR-1 is transcribed

15

and expressed in the body wall of adult females and present in all life-cycle stages of the

16

parasite (Tree et al., 1995). Similar results have also been reported from the plant parasitic

17

nematode G. pallida (Prior et al., 2001). The cuticle surface-localization suggests that these

18

proteins might be secreted by the hypodermis onto the parasite surface where they interact

19

with the external environment. Furthermore, the mRNA of Ab-far-1 gene encoding a FAR

20

protein from rice white tip nematode, A. besseyi, was also present in the ovaries of females

21

and the testes of males (Cheng et al., 2013), suggesting that the FAR protein in this species

22

may be involved in parasite reproduction. This localization is also consistent with the protein

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transporting lipids required for development of the reproductive tissues.

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More recently, a gene from the migratory endoparasitic nematode Bursaphelenchus

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xylophilus, similar in sequence to a putative fatty acid and retinoid-binding protein, was

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ACCEPTED MANUSCRIPT localized in the pharyngeal gland cells (Espada et al., 2016). This localization implies that the

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B. xylophilus FAR protein may play a role in parasitism. In this study, strong Ha-far-1 mRNA

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expression observed in the hypodermis of H. avenae, which is consistent with findings for

4

Gp-far-1 in G. pallida. Immunoassays using the antiserum against rGp-FAR-1 also revealed

5

that Ha-FAR-1 is present on the parasite surface, as has been shown for the G. pallida protein.

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Occasionally some Ha-far-1 signal in situ hybridization was seen in the area of the rectum.

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Although ascribing this expression to a specific tissue is difficult, this may also represent

8

expression in the hypodermis as this tissue extends to the posterior end of the nematode body.

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No expression was detectable in the pharyngeal gland cells.

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FAR proteins identified from a wide range of nematodes, including parasites of humans,

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animals, and plants, have been found to have ligand-binding properties (Kennedy et al., 1997;

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Prior et al., 2001; Basavaraju et al., 2003; Cheng et al., 2013; Iberkleid et al., 2013; Zhang et

13

al., 2015). This was also the case in our study; fluorescence-based ligand binding analysis

14

confirmed that the recombinant Ha-FAR-1 binds the environment-sensitive fluorescent

15

ligands DAUDA, cis-parinaric acid and retinol, causing a dramatic blue shift in the

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wave-length of peak fluorescence emission. These data show that rHa-FAR-1 is a functional

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lipid-binding protein.

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After being treated with dsRNA homologous to Ha-far-1 and inoculated onto wheat

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seedlings, H. avenae had significantly lower reproduction than those treated with gfp dsRNA

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or water. All the results demonstrated that Ha-far-1 may play an important role during

21

parasitism of H. avenae, which was also well supported by the expression dynamics of

22

Ha-far-1 gene as Ha-far-1 transcripts levels in parasitic stage were higher than those in

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non-parasitic stages. Nematode lipid binding proteins may function in transport of fatty acids

24

and retinols, which are used in the metabolic and developmental processes of embryogenesis,

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glycoprotein synthesis, growth and cellular differentiation (McDermott et al., 1999; Kennedy,

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ACCEPTED MANUSCRIPT 2000), and these proteins may also inhibit the plant defense reaction by impeding the

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metabolism of lipids by lipoxygenase, an early stage of the JA synthesis pathway in plants

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(Prior et al., 2001; Iberkleid et al., 2013). Therefore, the reduction of reproduction observed

4

here may either be due to the disturbance of intrinsic metabolism or the weakening of the

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nematode’s ability to suppress the host immune system, or both. Given that FAR proteins

6

seem to be confined to nematodes and play an important role in nematode biology, the

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inhibition of far expression by Gene silencing appear to be an effective and specific approach

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to the control of H. avenae, as well as other plant-parasitic nematodes.

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5.

Conclusion

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In conclusion, Ha-FAR-1 is a functional lipid and retinol binding protein and highly

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expressed during the cyst nematode parasitism, which may play an important role in

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nematode biology and appear to be an effective target to control the parasite by gene

14

silencing.

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Acknowledgements

This work was supported by the Natural Science Foundation of China (Grant No. 21471751)

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and the Special Fund for Agro-scientific Research in the Public Interest (Grant No.

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201503114). The authors thank Prof. John Jones for providing the Gp-FAR-1 antiserum and

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proof reading a draft of this manuscript.

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Disclosure The authors have declared that no competing interests exist.

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References

2

Abad, P., Gouzy, J., Aury, J.M., Castagnone-Sereno, P., Danchin, E.G., Deleury, E., Perfus-Barbeoch, L., Anthouard, V., Artiguenave, F., Blok, V.C., Caillaud,

4

M.C., Coutinho, P.M., Dasilva, C., De Luca, F., Deau, F., Esquibet, M., Flutre,

5

T., Goldstone, J.V., Hamamouch, N., Hewezi, T., Jaillon, O., Jubin, C., Leonetti,

6

P., Magliano, M., Maier, T.R., Markov, G.V., McVeigh, P., Pesole, G., Poulain,

7

J., Robinson-Rechavi, M., Sallet, E., Ségurens, B., Steinbach, D., Tytgat, T., Ugarte,

8

E., van Ghelder, C., Veronico, P., Baum, T.J., Blaxter, M., Bleve-Zacheo, T., Davis,

9

E.L., Ewbank, J.J., Favery, B., Grenier, E., Henrissat, B., Jones, J.T., Laudet, V., Maule,

10

A.G., Quesneville, H., Rosso, M.N., Schiex, T., Smant, G., Weissenbach, J., Wincker, P.,

11

2008. Genome sequence of the metazoan plant-parasitic nematode Meloidogyne

12

incognita. Nature Biotechnol. 26, 909–915.

SC

M AN U

13

RI PT

3

Basavaraju, S., Zhan, B., Kennedy, M.W., Liu, Y., Hawdon, J., Hotez, P.J., 2003. Ac-FAR-1, a 20 kDa fatty acid- and retinol-binding protein secreted by adult Ancylostoma caninum

15

hookworms: gene transcription pattern, ligand binding properties and structural

16

characterisation. Mol. Biochem. Parasitol. 126, 63–71.

TE D

14

Bird, D., Opperman, C.H., Williamson, V.M., 2009. Plant infection by root-knot nematode. In:

18

Berg, R.H., Taylor, C.G. (Eds.), Cell Biology of Plant Nematode Parasitism, Springer,

19

Heidelberg, Germany, pp. 1–13.

21 22

AC C

20

EP

17

Blaxter, M.L., Page, A.P., Rudin, W., Maizels, R.M., 1992. Nematode surface coats: actively evading immunity. Parasitol. Today 8, 243–247. Blumenthal, T., Steward, K., 1997. RNA processing and gene structure. In: Riddle, D.L.,

23

Blumenthal, T., Meyer, B.J., Priess, J.R. (Eds.), C. elegans II. Cold Spring Harbor

24

Laboratory Press, Cold Spring, pp. 117–146.

25

Cheng, X., Xiang, Y., Xie, H., Xu, C., Xie, T., Zhang, C., Li, Y., 2013. Molecular

17

ACCEPTED MANUSCRIPT 1

characterization and functions of fatty acid and retinoid binding protein gene (Ab-far-1)

2

in Aphelenchoides besseyi. PLoS One 8, e66011.

3

Cotton, J.A., Lilley, C.J., Jones, L.M., Kikuchi, T., Reid, A.J., Thorpe, P., Tsai, I.J., Beasley, H., Blok, V., Cock, P.J., van den Akker, S.E., Holroyd, N., Hunt, M., Mantelin,

5

S., Naghra, H., Pain, A., Palomares-Rius, J.E., Zarowiecki, M., Berriman, M., Jones,

6

J.T., Urwin, P.E., 2014. The genome and life-stage specific transcriptomes of Globodera

7

pallida elucidate key aspects of plant parasitism by a cyst nematode. Genome Biol, 2014,

8

15: R43.

SC

9

RI PT

4

De Boer, J.M., Smant, G., Goverse, A., Davis, E.L., Overmars, H.A., Pomp, H., van-Gent, P.M., Zilverentant, J.F., Stokker-mans, J.P., Hussey, R.S., Gommers, F.J., Bakker, J.,

11

Schots, A., 1996. Secretory granule proteins from the subventral esoph-ageal glands of

12

the potato cyst nematode identified by monoclonal antibodies to a protein fraction from

13

second-stage juveniles. Mol.Plant Microbe Interact. 9, 39–46.

15

De Boer, J.M., Yan, Y., Smant, G., Davis, E.L., Baum, T.J., 1998. In situ hybridization to

TE D

14

M AN U

10

messenger RNA in Heterodera glycines. J. Nematol. 30, 309–312. De Boer, J.M., Yan, Y., Wang, X., Smant, G., Hussey, R.S., Davis, E.L., Baum, T.J., 1999.

17

Developmental expression of secretory β-1,4-endoglucanases in the subventral

18

esophageal glands of Heterodera glycines. Mol. Plant-Microbe Interact. 12, 663–669. Espada, M., Silva, A.C., van den Akker, S.E., Cock, P.J.A., Mota, M., Jones, J.T., 2016.

AC C

19

EP

16

20

Identification and characterization of parasitism genes from the pinewood nematode

21

Bursaphelenchus xylophilus reveals a multilayered detoxification strategy. Mol. Plant

22

Pathol. 17, 286–295.

23 24 25

Ferris, V., Faghihi, J., Ireholm, A., Ferris, J., 1989. Two-dimensional protein patterns of cereal cyst nematodes. Phytopathology 79, 927–933. Garofalo, A., Kennedy, M.W., Bradley, J.E., 2003. The FAR proteins of parasitic nematodes:

18

ACCEPTED MANUSCRIPT 1

their possible involvement in the pathogenesis of infection and the use of Caenorhabditis

2

elegans as a model system to evaluate their function. Med. Microbiol. Immunol. 192,

3

47–52. Huang, G., Gao, B., Maier, T., Allen, R., Davis, E.L., Baum, T.J., Hussey, R.S., 2003. A profile

5

of putative parasitism genes expressed in the esophageal gland cells of the root-knot

6

nematode Meloidogyne incognita. Mol. Plant-Microbe Interact. 16, 376–381.

7

RI PT

4

Iberkleid, I., Sela, N., Miyara, S.B., 2015. Meloidogyne javanica fatty acid- and

retinol-binding protein (Mj-FAR-1) regulates expression of lipid-, cell wall-, stress- and

9

phenylpropanoid-related genes during nematode infection of tomato. BMC Genomics 16,

11

272.

M AN U

10

SC

8

Iberkleid, I., Vieira, P., de Almeida Engler, J., Firester, K., Spiegel, Y., Horowitz, S.B., 2013.

12

Fatty acid-and retinol-binding protein, Mj-FAR-1 induces tomato host susceptibility to

13

root-knot nematodes. PLoS One 8, e64586.

15 16

Jones, J.T., Reavy, B., Smant, G., Prior, A.E., 2004. Glutathione peroxidases of the potato cyst

TE D

14

nematode Globodera rostochiensis. Gene 324, 47–54. Jones, J.T., Haegeman, A., Danchin, E.G., Gaur, H.S., Helder, J., Jones, M.G., Kikuchi, T., Manzanilla-López, R., Palomares-Rius. J.E., Wesemael, W.M., Perry, R.N., 2013.

18

Top 10 plant-parasitic nematodes in molecular plant pathology. Mol. Plant Pathol. 14,

19

946–961.

AC C

20

EP

17

Jordanova, R., Groves, M., Kostova, E., Woltersdorf, C., Liebau, E., Tucker, P.A., 2009.

21

Fatty acid- and retinoid-binding proteins have distinct binding pockets for the two types

22

of cargo. J. Biol. Chem. 284, 35818-35826.

23 24 25

Kennedy, M.W., 2000. The polyprotein lipid binding proteins of nematodes. Biochim. Biophys. Acta 1476, 149–164. Kennedy, M.W., Garside, L.H., Goodrick, L.E., McDermott, L., Brass, A., Price, N.C., Kelly,

19

ACCEPTED MANUSCRIPT 1

S.M., Cooper, A., Bradley, J.E., 1997. The Ov20 protein of the parasitic nematode

2

Onchocerca volvulus: a structurally novel class of small helix-rich retinol-binding

3

proteins. J. Biol. Chem. 272, 29442–29448.

7 8 9 10

RI PT

6

counting of nematode cysts. J. Nematol. 26, 599.

Macgregor, R.B., Weber, G., 1986. Estimation of the polarity of the protein interior by optical spectroscopy. Nature 319, 70–73.

McDermott, L., Cooper, A., Kennedy, M.W., 1999. Novel classes of fatty acid and retinol

SC

5

Krusberg, L.R., Sardanelli, S., Meyer, S.L.F., Crowley, P., 1994. A method for recovery and

binding protein from nematodes. Mol. Cell. Biochem. 192, 69–75.

Mitchum, M.G., Hussey, R.S., Baum, T.J., Wang, X., Elling, A.A., Wubben, M., Davis, E.L.,

M AN U

4

11

2013. Nematode effector proteins: an emerging paradigm of parasitism. New Phytol. 199,

12

879–894.

13

Nicol, J.M., Rivoal, R., 2007. Integrated management and biocontrol of vegetable and grain crops nematodes. In: Ciancio, A., Mukerji, K.G., Eds. Global Knowledge and Its

15

Application for the Integrated Control and Management of Nematodes on Wheat.

16

Springer, The Netherlands, pp 243–287.

Nicol, J.M., Turner, S.J., Coyne, D.L., den Nijs, L., Hockland, S. and Maafi, Z.T. (2011)

EP

17

TE D

14

Current nematode threats to world agriculture. In: Genomics and Molecular Genetics of

19

Plant–Nematode Interactions (Jones, J.T., Gheysen,G. and Fenoll, C., Eds), Heidelberg,

20

Springer. pp. 21–44.

21

AC C

18

Opperman, C.H., Bird, D.M., Williamson, V.M., Rokhsar, D.S., Burke, M., Cohn, J., Cromer,

22

J., Diener, S., Gajan, J., Graham, S., Houfek, T.D., Liu, Q., Mitros, T., Schaff, J., Schaffer,

23

R., Scholl, E., Sosinski, B.R., Thomas, V.P., Windham, E., 2008. Sequence and genetic

24

map of Meloidogyne hapla: A compact nematode genome for plant parasitism. Proc. Natl.

25

Acad. Sci. U.S.A. 105, 14802–14807.

20

ACCEPTED MANUSCRIPT 1

Petersen, T.N., Brunak, S., von Heijne, G., Nielsen, H., 2011. SignalP 4.0: discriminating

2

signal peptides from transmembrane regions. Nature Methods 8, 785–786.

3

Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time

5

RT-PCR. Nucleic Acids Res. 29, e45. Prior, A., Jones, J.T., Blok, V.C., Beauchamp, J., McDermott, L., Cooper, A., Kennedy, M.W.,

RI PT

4

2001. A surface-associated retinol-and fatty acid-binding protein (Gp-FAR-1) from the

7

potato cyst nematode Globodera pallida: lipid binding activities, structural analysis and

8

expression pattern. Biochem. J. 356, 387–394.

SC

6

Ray, C., Abbott, A.G., Hussey, R.S., 1994. Trans-splicing of a Meloidogyne incognita mRNA

10

encoding a putative esophageal gland protein. Mol. Biochem. Parasitol. 68, 93–101.

11

Rey-Burusco, M.F., Ibanez-Shimabukuro, M., Gabrielsen, M., Franchini, G.R., Roe A.J.,

12

Griffiths, K., Zhan, B., Cooper, A., Kennedy, M.W., Corsico, B., Smith, B.O., 2015.

13

Diversity in the structures and ligand-binding sites of nematode fatty acid and

14

retinol-binding proteins revealed by Na-FAR-1 from Necator americanus. Biochem. J.

15

471, 403-414.

TE D

16

M AN U

9

Semblat, J.P., Rosso, M.N., Hussey, R.S., Abad, P., Castagnone-Sereno, P., 200. Molecular cloning of a cDNA encoding an amphid secreted putative avirulence protein from the

18

root-knot nematode Meloidogyne incognita. Mol.Plant Microbe Interact. 14, 72–79.

19

Swofford, D., 2002. PAUP* Version 4.0. Phylogenetic Analysis Using Parsimony (and Other

AC C

20

EP

17

Methods). Sinauer Associates, Inc., Sunderland.

21

Tang, Y.T., Gao, X., Rosa, B.A., Abubucker, S., Hallsworth-Pepin, K., Martin, J., Tyagi, R.,

22

Heizer, E., Zhang, X., Bhonagiri-Palsikar, V., Minx, P., Warren, W.C., Wang, Q., Zhan,

23

B., Hotez, P.J., Sternberg, P.W., Dougall, A., Gaze, S.T., Mulvenna, J., Sotillo, J.,

24

Ranganathan, S., Rabelo, E.M., Wilson, R.K., Felgner, P.L., Bethony, J., Hawdon, J.M.,

25

Gasser, R.B., Loukas, A., Mitreva, M., 2014. Genome of the human hookworm Necator

21

ACCEPTED MANUSCRIPT 1

americanus. Nat. Genet. 46, 261-271.Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994.

2

CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment

3

through sequence weighting, position-specific gap penalties and weight matrix choice.

4

Nucleic Acids Res. 22, 4673–4680. Tree, T.I.M., Gillespie, A.J., Shepley, K.J., Blaxter, M.L., Tuan, R.S., Bradley, J.E., 1995.

RI PT

5

Characterisation of an immunodominant glycoprotein antigen of Onchocerca volvulus

7

with homologues in other filarial nematodes and Caenorhabditis elegans. Mol. Biochem.

8

Parasitol. 69, 185–195.

SC

6

Turner, S.J., Rowe, J.A., 2006. Cyst nematodes. In: Perry, R.N., Moens, M., Eds. Plant

10

Nematology. Wallingford, Oxfordshire, UK, CAB International, pp. 90–122.

11

M AN U

9

Urwin, P.E., Lilley, C.J., Atkinson, H.J., 2002. Ingestion of double-stranded RNA by

12

preparasitic juvenile cyst nematodes leads to RNA interference. Mol. Plant-Microbe

13

Interact.15, 747–752.

Zhang, C., Xie, H., Cheng, X., Wang, D., Li, Y., Xu, C.L., Huang, X., 2015. Molecular

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identification and functional characterization of the fatty acid- and retinoid-binding

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protein gene Rs-far-1 in the burrowing nematode Radopholus similis (Tylenchida:

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Pratylenchidae). PLoS One 10, e0118414.

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Legends of figures

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Fig. 1. Complete nucleotide and deduced amino acid sequences of the Ha-far-1 cDNA. The

4

spliced leader sequence SL1, start codon, stop codon (TAA) and the putative polyadenylation

5

signal sequence (attaaa) are underlined. The intron splice sites are shown with vertical arrows.

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PCR primer sites for RACE amplification are boxed. The predicted secretion signal sequence

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is shaded, the consensus casein kinase II phosphorylation sites (CK2) are double underlined.

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Fig. 2. Phylogenetic analysis based on the amino acid sequences of fatty acid and retinoid

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binding proteins from nematodes. The Neighbor-joining phylogenetic tree rooted against the

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FAR protein sequences of the free-living nematode C. elegans was generated using

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PAUP*4.0b10 program and the values supported in 1000 bootstrap replications are shown at

13

each node. Ha-Far-1 is underlined.

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Fig. 3. Localization of Ha-far-1 mRNA in H. avenae using in situ hybridization. (a), (b) and

16

(c): Signal was present in the hypodermis of nematodes hybridized with the

17

digoxigenin-labelled antisense cDNA probe. (d) Signal was also detected in the region of the

18

rectum of nematodes. (e) Hybridization with the digoxigenin-labelled sense cDNA probe. S:

19

stylet; M: metacorpus; H: hypodermis. Scale bars = 10 µm.

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Fig. 4. Expression of the Ha-far-1 through the H. avenae life cycle. The transcripts of

22

Ha-far-1 were detected in eggs, preparasitic second-stage juveniles (Pre-J2), parasitic

23

second-stage juveniles (Par-J2), third-stage juveniles (J3), fourth-stage juveniles and females.

24

The relative amounts of Ha-far-1 in each stage of H. avenae were normalized with the

25

geometric mean of reference genes actin and EF-1α and are presented as the ratio relative to

23

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the amount in eggs. Each bar represents the means ± S.D. of triplicate experiments;

2

statistically significant differences using a Student’s t-test (P < 0.05) are indicated with

3

different letters.

4

Fig. 5. Protein blotting and immunolabeling of Ha-FAR-1 with an antiserum raised against

6

rGp-FAR-1. (a) Western blot detection of homogenates from H. avenae J2s. A single band of

7

molecular mass approx. 20 kDa was detected. Lane M: Protein marker; Lane 1: Sample

8

probed with the rGp-FAR-1 antiserum; 2: Sample probed with the preimmune serum; (b) and

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(c) Immunofluorescence localization of Ha-FAR-1 within H. avenae J2. Target protein is

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present at the surface of the J2. (b) Bright-feld image. (c) Fluorescence image. S: stylet.

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Fig. 6. Recombinant protein expression and ligand binding of Ha-FAR-1. (a) SDS-PAGE of

13

rHa-FAR-1. Lane M: Protein marker; Lane 1: E. coli BL21 (DE3) transformed with the empty

14

expression vector pET28a (+) and treated with IPTG; Lane 2: E. coli BL21 (DE3)

15

transformed with the pET28a (+)-Ha-far-1 and treated with IPTG; Lanes 3 and 4: Purified

16

rHa-FAR-1 protein. (b) The fluorescence emission of DAUDA was 556 nm in buffer alone,

17

but moved to 492 nm upon addition of rHa-FAR-1, the addition of oleic acid to rHa-FAR-1

18

and DAUDA complexes lead to a substantial drop in the fluorescence intensity. (c) The

19

emission peak of cis-parinaric acid occurred at 470 nm alone, but moved to 464 nm upon

20

addition of rHa-FAR-1 and dropped in the fluorescence intensity after further addition of oleic

21

acid. (d) The emission peak of retinol appeared at 412 nm alone, but moved to 416 nm upon

22

addition of rHa-FAR-1 and dropped in the fluorescence intensity after further addition of oleic

23

acid.

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Fig. 7. Gene silencing of Ha-far-1 and infection assay. (a) Ha-far-1 transcript abundance in J2 24

ACCEPTED MANUSCRIPT treated with Ha-far-1 dsRNA, gfp dsRNA and deionized water, respectively. The relative

2

amounts of Ha-far-1 in J2 soaked in different kinds of dsRNA were determined with respect

3

to the actin and EF-1α internal standards and are presented as the ratio relative to the amount

4

of those in deionized water. (b) The number of cysts produced on roots of wheat after

5

inoculation with J2 treated with Ha-far-1 dsRNA, gfp dsRNA and deionized water,

6

respectively. Each bar represents the means ± S.D. of triplicate experiments; Asterisk indicate

7

statistically significant differences using a Student’s t-test (P < 0.05).

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ACCEPTED MANUSCRIPT Table 1 Primers used in this study

FarT7-R GFPT7-F GFPT7-R

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Actin-F Actin-R EF1α-F EF1α-R

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Sequences（5'- 3'） GACCGAGGAGGACAAACAGG CTTTGCCAATGCTTGGAACT AGGAATTTGTCACCGGGATG CAGTTCGTTGGCCGTCTTGC CAACGGAAGTGACCGACTTT AGCTCGACCGCTTTGTTGTA CAATTCCATTTTCAACAAAC AAGAAATGTTGGTGAAAATGC TGACATCAACTCCATCCCGC CCTCCAGATTCGGCTTCTCT CGGGATCCATGGCCACTTTGCCACCCATT CCCAAGCTTGGCGGCTGGTGCGGCACCC GATCACTAATACGACTCACTATAGGGGGAAAATTGACGCTTTG GAG GATCACTAATACGACTCACTATAGGGGCACAAATTCGATGTGTT GG GATCACTAATACGACTCACTATAGGGCACAAGTTCAGCGTGTC CG GATCACTAATACGACTCACTATAGGGTGGGTGCTCAGGTAGTG GTT CTCCTCGCTGGAGAAGAGTT GTAAGTGGACTCGTGGATGC GTTTAAGGGATGGAGCGTTGAG CGGCTTGTCTGTTGGTCTTT

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Primer name Far-F1 Far-R1 Far-F2 Far-R2 FarRT-F FarRT-R FarFL-F FarFL-R FarIS-F FarIS-R FarEc-F FarEc-R

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BamHI ⁄ HindIII sites are underlined, T7 promoter sequences are italicized.

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1. First fatty acid- and retinoid-binding protein gene isolated from Heterodera avenae. 2. Expression of the far gene accumulated mainly in the hypodermis. 3. Far gene highly expressed in during the cyst nematode parasitism. 4. Recombinant protein of the far gene was able to bind fatty acids and retinol. 5. Silencing of the far gene results in a significant reduction of nematode parasitism.