Genetic Characterisation of Vitellogenin Egg Proteins in Heterodera schachtii

The beet cyst nematode, Heterodera schachtii, is an economically important crop pest. As part of the cyst nematode life cycle, the juvenile nematode develops inside the egg, where it is surrounded by perivitelline fluid. This fluid contains essential nutrients for embryogenesis; one of the most abundant proteins found is vitellin, formed from a precursor protein vitellogenin. In Caenorhabditis elegans, vitellogenin has been shown to have a role in nematode development by transporting lipids, providing amino acids, influencing post-embryonic phenotypes, and providing some environmental protection. Vitellogenin has not yet been fully identified and characterised in many species of plant-parasitic nematodes (PPN); some encoding genes have been identified within the genomes of some species, but the protein has only been partially characterised in Heterodera glycines. The objective of this work was to investigate and characterise vitellogenin in the closely related species H. schachtii and to elucidate its function in nematode development. Initially using a bioinformatic approach, the presence of vitellogenin (vit) genes within species of cyst, root-knot (RKN) and root-lesion nematodes (RLN) was determined. The vit gene sequences from both Globodera ellingtonae and Pratylenchus scribneri were predicted, as these gene sequences had not previously been identified. The phylogenetic relationship between the PPN and Caenorhabditis spp. vit genes illustrated that the genes were clustered by their groups (cyst nematode, RKN, RLN or Caenorhabditis spp.) but were not clustered by species within these groups. It was shown that there was sequence conservation between the PPN species and both C. elegans and C. briggsae. All predicted genes contained the same protein domains as C. elegans: Vitellogenin_N, vitellogenin open beta-sheet and von Willebrand factor domain type D. Also, the gene ontology annotation of many of the PPN vit genes corresponded to the C. elegans vit genes of lipid transport and nutrient reserve. The gene expression of PPN vit genes was investigated by utilising expression data available in the public domain. It was shown that vit genes were most highly expressed at the adult female stage of the nematode’s lifecycle and this expression was significantly greater than in earlier stages, such as the second-stage juvenile (J2) and the egg. In contrast to previous research, it was also shown that vit genes were also expressed at low levels in J2 and male stages of H. schachtii. Proteins from H. schachtii were characterised using western blot analysis. An antibody developed to recognise a sequence within a vitellogenin gene was synthesised, and then validated by using dot blot analysis, demonstrating that the antibodies recognised proteins within cyst homogenates. The molecular weight of proteins likely to be vitellogenin were then obtained by western blot analysis. The molecular weight of these proteins in cyst homogenates of H. schachtii were 146 kDa, 95 kDa, 89 kDa, 47 kDa, 34 kDa and 19 kDa. Also, the molecular weight of the G. pallida proteins obtained from cyst homogenates were 157 kDa, 107 kDa, 81 kDa, 55 kDa, 52 kDa and 46 kDa. Finally, the five proteins from G. rostochiensis cyst homogenates had molecular weights of 110 kDa, 82 kDa, 77 kDa, 47 kDa and 13 kDa. The antibody designed to recognise vitellogenin, detected proteins in the females, eggs, and J2 of H. schachtii, with proteins recognised that were specific to each stage. Furthermore, in preliminary investigations using the anti-vitellogenin antibody to immunolocalisation the proteins in H. schachtii, G. pallida and G. rostochiensis, proteins were detected at various stages of the nematode life cycle, including the female, egg and J2. The genomic DNA sequence of the region of the H. schachtii containing the vit gene, Hsc_gene_6726 was determined using molecular methods of polymerase chain reaction, cloning and whole plasmid sequencing. Novel primers were developed to the region of the H. schachtii genome containing the vit gene. These primers were then used to amplify the sequence from DNA extracted from H. schachtii J2. This DNA sequence was inserted into a plasmid vector and sequenced by Oxford nanopore sequencing. The sequence was then analysed to obtain a partial vitellogenin genomic sequence for this H. schachtii population. Antibodies have previously been developed with specificity to cyst nematode egg proteins in G. pallida and G. rostochiensis. Two antibodies tested, MAC 356 and PC320, were confirmed by western blot analysis to be recognising a 34 kDa protein, when using the same cyst homogenate samples. These proteins were isolated and identified using liquid chromatography–mass spectrometry, giving a putative identification of annexin in both species. Therefore, the target of both antibodies is the protein annexin. Immunofluorescent study was again used for the preliminary investigation of the target tissue within nematode samples of these antibodies. The PC320 antibody recognised protein within the eggshell of G. pallida and G. rostochiensis, whereas fluorescence was observed in the whole egg when using MAC 356. Greater fluorescence was observed in G. pallida samples than G. rostochiensis when using the MAC 356 antibody. By determining the putative vit genes and their expression within PPN species, a greater understanding of vitellogenin and its role in PPN has been established. The development of an anti-vitellogenin antibody that successfully recognised proteins within cyst homogenates by western blotting, aided in the characterisation of the protein in cyst nematode species. Preliminary immunolocalisation studies have given an insight into the tissue location of vitellogenin in H. schachtii, G. rostochiensis and G. pallida. Furthermore, the determination of the vitellogenin DNA sequence from this population of H. schachtii can aid in further molecular study of the vitellogenin genes and proteins. Therefore, this work develops the understanding of vitellogenin in cyst nematodes, indicating a role in lipid transport and nutrient provision for the development of the nematode within the egg, and forms a basis for further study of the function of vitellogenin in H. schachtii and other cyst nematodes.