Chromosome-level genome assembly of the cereal cyst nematode Heterodera flipjevi

Abstract As an economically important plant parasitic nematode (PPN), Heterodera filipjevi causes great damage on wheat, and now it was widely recorded in many countries. While multiple genomes of PPNs have been published, high-quality genome assembly and annotation on H. filipjevi have yet to be performed. This study presents a chromosome-scale genome assembly and annotation for H. filipjevi, utilizing a combination of Illumina short-read, PacBio long-read, and Hi-C sequencing technologies. The genome consists of 9 pseudo-chromosomes that contain 134.19 Mb of sequence, with a scaffold N50 length of 11.88 Mb. In total, 10,036 genes were annotated, representing 75.20% of the total predicted protein-coding genes. Our study provides the first chromosome-scale genome for H. filipjevi, which is also the inaugural high-quality genome of cereal cyst nematodes (CCNs). It provides a valuable genomic resource for further biological research and pest management of cereal cyst nematodes disease

Hatching and development of maize cyst nematode Heterodera zeae infecting different plant hosts

The occurrence, distribution, and rapid molecular detection technology of Heterodera zeae Koshy et al. 1971, have been reported in China. We explored the biological characteristics of H. zeae sampled in Henan Province, China to understand its interaction with plants. Cysts and second-stage juveniles (J2s) were identified under an optical and scanning electron microscope, internal transcribed spacer (ITS) phylogenetic tree, and sequence characterized amplified region (SCAR)-PCR analyses. The optimum hatching temperatures of H. zeae were 30°C and 28°C, with cumulative hatching rates of 16.5 and 16.1%, respectively, at 30 days post-hatching (dph). The hatching rate of H. zeae eggs was improved by 20- and 50-time maize soil leachate and root juice, and 10-time root exudates. The hatching rate in 10-time root exudates was the highest (25.9%). The 10-time root exudates of maize and millet produced the highest hatching rate at 30 dph (25.9 and 22.9%, respectively), followed by wheat (19.9%), barley (18.3%), and rice (17.6%). Heterodera zeae developed faster in maize than in other crops. Fourth-stage juveniles (J4s) were detected in maize roots 8 days post-inoculation (dpi) at 28°C but not in other crops. Combined with hatching tests, the Huang–Huai–Hai summer maize region and the south and central-southwest mountainous maize areas are highly suitable for H. zeae in China. This is the first systematically study of the hatching and infection characteristics on different plant hosts of corn cyst nematode H. zeae in temperate regions. This study laid a theoretical foundation for the rapid spread and high environmental adaptability of corn cyst nematode

First report of sugar beet nematode, Heterodera schachtii Schmidt, 1871 (Nemata: Heteroderidae) in sugar beet growing areas of Sanliurfa, Turkey

WOS: 000386015700008The sugar beet nematode, Heterodera schachtii, is the major pest of sugar beet and causes serious yield losses of about 10-70%. Heterodera schachtii occurs in more than 50 countries and regions, however, there has be limited investigation of H. schachtii in Turkey. Therefore, a survey of H. schachtii in the sugar beet producing regions of Sanliurfa was conducted in 2014 and 2015 growing seasons. Using morphological and molecular methods, 12 samples collected from three districts, Bozova, Karakopru and Siverek, in Sanliurfa Province, Turkey, were identified as H. schachtii. In pathogenicity test, the seedling emergence was delayed and reduced, the seedlings were stunted and necrotic, and the white females of H. schachtii were evident 25 days after inoculation. Phylogenetic analyses were also conducted. The 12 H. schachtii populations from Sanliurfa Province clustered together with populations from Europe and Morocco at the value of 99%. Sugar beet is the second largest crop in Turkey with the annual production of more 16 Mt. To our best knowledge, this is the first report of H. schachtii in Sanliurfa Province of Turkey.Special Fund for Agro-scientific Research in the Public Interest in China [201503114]; National Key Basic Research Program of China (973 Program)National Basic Research Program of China [2013CB127502]This study was financially supported by the Special Fund for Agro-scientific Research in the Public Interest in China (#201503114) and the National Key Basic Research Program of China (973 Program, #2013CB127502). The authors would like to thank the Republic of Turkey Ministry of Food, Agriculture and Livestock and the International Wheat and Maize Improvement Center (CIMMYT, Turkey) for the help of sampling, Prof. Richard W. Smiley, Oregon State University, for his advice, and the help of Ms. Tian Zhongling from Zhejiang University and Ms. Li Shuhui from Henan Agricultural University

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

This is the <i>Heterodera flipjevi</i> genome of chromosome level, longest transcripts, predicted gene models and proteins.

Our study provides the first chromosome-scale genome for H. filipjevi, which is also the inaugural high-quality genome of cereal cyst nematodes (CCNs). It provides a valuable genomic resource for further biological research and pest management of cereal cyst nematodes disease.</p

Phylogenetic tree of the polysaccharide lyase family 3 generated using maximum likelihood of PhyML.

<p>The numbers next to the branches indicate the bootstrap value. GenBank accession numbers of pectate lyase proteins correspond to <i>Meloidogyne incognita</i> (MI-PEL-1, MI-PEL-2 and MI-PEL-3), <i>M</i>. <i>hapha</i> (Mh-ENG-1 and Mh-ENG-2), <i>M</i>. <i>javanica</i> (MJ-PEL-1), <i>Globodera rostochiensis</i> (GR-PEL-1and GR-PEL-2), <i>G</i>. <i>pallida</i> (Gp-ENG-1 and Gp-ENG-2), <i>Heterodera glycines</i> (HG-PEL-1, HG-PEL-2, HG-PEL-3, HG-PEL-4, HG-PEL-5, HG-PEL-6 and Hg-ENG-7), <i>H</i>. <i>schachtii</i> (HS-PEL-1 and HS-PEL-2), <i>H</i>. <i>avenae</i> (HA-PEL-1 and HA-PEL-2), <i>Aphelenchus avenae</i> (AA-PEL-1 and AA-PEL-2), <i>Bursaphelenchus xylophilus</i> (BX-PEL-and BX-PEL-2), and <i>B</i>. <i>mucronatus</i> (BM-PEL-1 and BM-PEL-2).</p

Alignment of the putative protein sequences of the pectate lyases that belong to the polysaccharide lyase family 3.

<p>HG-PEL-1 [AAK08974], HG-PEL-2 [AAM74954] and HG-PEL-5 [ADW77534] correspond to <i>Heterodera glycines</i>; HS-PEL-1 [ABN14273] and HS-PEL-2 [ABN14272] correspond to <i>H</i>. <i>schachtii</i>; GR-PEL-1 [AAF80747] and GR-PEL-2 [AAM21970] correspond to <i>Golobedera rostochiensis</i>; GP-PEL-2 [ACU64826] corresponds to <i>G</i>. <i>pallida</i>; BX-PEL-1 [BAE48369] and BX-PEL-2 [BAE48370] correspond to <i>Bursaphelenchus xylophilus</i>; BM-PEL-1 [BAE48373] and BM-PEL-2 [BAE48375] correspond to <i>B</i>. <i>mucronatus</i>; AA-PEL-1 [BAI44999] and AA-PEL-2 [BAI44997] correspond to <i>Aphelenchus avenae</i>; MI-PEL-1 [AAQ09004] and MI-PEL-2 [AAQ97032] correspond to <i>Meloidogyne incognita</i>. Identical residues are highlighted in black. Black bars (I to IV) indicate the conserved regions characteristic of the PL3 pectate lyases. The asterisk (*) indicates conserved cysteine residues. The positions of the intron <i>hg-pel-3</i>, <i>hg-pel-4</i> and <i>hg-pel-7</i>are indicated by Hg, that of <i>hg-pel-6</i> by Hg6, that of <i>Bx-pel-1</i>, <i>Bx-pel-2</i>, <i>Bm-pel-1</i> and <i>Bm-pel-2</i> by B, that of <i>Gr-pel-1</i> by Gr1, that of <i>Mi-pel-1</i> by Mi1and that of <i>Mi-pel-2</i> by Mi2. Diamonds, black triangles and white triangles represent phase 0, 1 and 2 introns, respectively.</p

Novel Pectate Lyase Genes of <i>Heterodera glycines</i> Play Key Roles in the Early Stage of Parasitism

<div><p>Pectate lyases are known to play a key role in pectin degradation by catalyzing the random cleavage of internal polymer linkages (endo-pectinases). In this paper, four novel cDNAs, designated <i>Hg-pel-3</i>, <i>Hg-pel-4</i>, <i>Hg-pel-6</i> and <i>Hg-pel-7</i>, that encode pectate lyases were cloned and characterized from the soybean cyst nematode, <i>Heterodera glycines</i>. The predicted protein sequences of HG-PEL-3, HG-PEL-4 and HG-PEL-6 differed significantly in both their amino acid sequences and their genomic structures from other pectate lyases of <i>H</i>. <i>glycines</i> (HG-PEL-1, HG-PEL-2 and HG-PEL-7). A phylogenetic study revealed that the pectate lyase proteins of <i>H</i>. <i>glycines</i> are clustered into distinct clades and have distinct numbers and positioning of introns, which suggests that the pectate lyase genes of <i>H</i>. <i>glycines</i> may have evolved from at least two ancestral genes. A Southern blot analysis revealed that multiple <i>Hg-pel-6</i>-like genes were present in the <i>H</i>. <i>glycines</i> genome. <i>In situ</i> hybridization showed that four novel pectate lyases (<i>Hg-pel-3</i>, <i>Hg-pel-4</i>, <i>Hg-pel-6</i> and <i>Hg-pel-7)</i> were actively transcribed in the subventral esophageal gland cells. A semi-quantitative RT-PCR assay supported the finding that the expression of these genes was strong in the egg, pre-parasitic second-stage juvenile (J2) and early parasitic J2 stages and that it declined in further developmental stages of the nematode. This expression pattern suggests that these proteins play a role in the migratory phase of the nematode life cycle. Knocking down <i>Hg-pel-6</i> using <i>in vitro</i> RNA interference resulted in a 46.9% reduction of the number of nematodes that invaded the plants and a 61.5% suppression of the development of <i>H</i>. <i>glycines</i> females within roots compared to the GFP-dsRNA control. Plant host-derived RNAi induced the silencing of the <i>Hg-pel-6</i>gene, which significantly reduced the nematode infection levels at 7 Days post inoculation (dpi). Similarly, this procedure reduced the number of female adults at 40 dpi, which suggests the important roles of this gene in the early stages of parasitism. Our combined data suggest that two types of pectate lyases are present in the <i>H</i>. <i>glycines</i> genome and may have different roles during infection.</p></div

Developmental expression of <i>hg-pel-3</i>, <i>hg-pel-4</i>, <i>hg-pel-6</i> and <i>hg-pel-7</i> in different stages of <i>Heterodera glycines</i>.

<p>Pre-J2, pre-parasitic second stage juveniles; J2, parasitic second stage juveniles; J3, parasitic third stage juveniles; J4, parasitic forth stage juveniles; Female, adult females: eggs; N, negative control (genomic DNA of soybean roots). Maker, DL2000 DNA ladder. Actin, the <i>H</i>. <i>glycines</i> β-actin genem was used as positive control for each cDNA templates.</p

Localization by <i>in situ</i> hybridization of <i>hg-pel-3</i>, <i>hg-pel-4</i>, <i>hg-pel-6</i> and <i>hg-pel-7</i> in pre-parasitic second stages of <i>Heterodera glycines</i>.

<p>Left panel: antisense <i>Hg-pel-3</i>, <i>Hg-pel-4</i>, <i>Hg-pel-6</i> and <i>Hg-pel-7</i> probes; right panel: corresponding sense probes. S, stylet; M, median bulb; SVG, subventral esophageal cell. Scale bar: 20 μm.</p