The genome sequence of Barbarea vulgaris facilitates the study of ecological biochemistry

peer-reviewedThe genus Barbarea has emerged as a model for evolution and ecology of plant defense compounds, due to its unusual glucosinolate profile and production of saponins, unique to the Brassicaceae. One species, B. vulgaris, includes two ‘types’, G-type and P-type that differ in trichome density, and their glucosinolate and saponin profiles. A key difference is the stereochemistry of hydroxylation of their common phenethylglucosinolate backbone, leading to epimeric glucobarbarins. Here we report a draft genome sequence of the G-type, and re-sequencing of the P-type for comparison. This enables us to identify candidate genes underlying glucosinolate diversity, trichome density, and study the genetics of biochemical variation for glucosinolate and saponins. B. vulgaris is resistant to the diamondback moth, and may be exploited for “dead-end” trap cropping where glucosinolates stimulate oviposition and saponins deter larvae to the extent that they die. The B. vulgaris genome will promote the study of mechanisms in ecological biochemistry to benefit crop resistance breeding

Insect bioactive capabilities of Epichloë festucae var lolii AR48 infected Lolium perenne : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand

Figures printed with permission from respective publishers.As the modern world expands and develops, new innovative methodologies for more efficient and environmentally friendly agricultural practices are required. Loss of crops through abiotic (e.g. drought) and biotic (e.g. herbivory) stresses has a major effect on the success of an agricultural industry. For animal production pasture crops are a key aspect of animal husbandry and directly affects yield and health. Symbiotic fungi belonging to the genus Epichloë form associations with cool season forage grasses and have been exploited as a new innovative method for insect pest management. Ryegrass infected with the asexual E. festucae var lolii strain AR48 has insect bioactivity against both the stem boring fly (SBF-Ceradontha australis) and cutworm moth caterpillar (CC -Agrotis ipsilion). The bioactive/s targeting both insects is currently unknown. The aim of this thesis was to identify the gene/s and/or bioactive/s present in AR48 infected ryegrass that have bioactivity against the SBF and/or CC. Two approaches were taken; the known insect bioactive secondary metabolite pathways in Epichloë were investigated in AR48 through bioinformatics and mass spectrometry, and the gene ‘makes caterpillars floppy’ (mcf), encoding an insect toxin like protein, was investigated through reverse genetics and insect bioactivity trials. A new indole diterpene compound (IDT) was identified in AR48 infected plant material and this compound was absent in other Epichloë strains that do not have SBF and CC bioactivity. The same mcf gene allele as that present in the E. typhina mcf model, previously identified as having CC bioactivity, is present and predicted to be functional in AR48. The other Epichloë strains also have mcf genes predicted to be functional, however the mcf allele is different to the bioactive E. typhina mcf model. Overall, this project was able to identify a new IDT compound with potential insect bioactivity as well as identify two Epichloë mcf gene alleles that potentially have differing insect bioactivities

Complex evolutionary dynamics of massively expanded chemosensory receptor families in an extreme generalist chelicerate herbivore

While mechanisms to detoxify plant produced, anti-herbivore compounds have been associated with plant host use by herbivores, less is known about the role of chemosensory perception in their life histories. This is especially true for generalists, including chelicerate herbivores that evolved herbivory independently from the more studied insect lineages. To shed light on chemosensory perception in a generalist herbivore, we characterized the chemosensory receptors (CRs) of the chelicerate two-spotted spider mite, Tetranychus urticae, an extreme generalist. Strikingly, T. urticae has more CRs than reported in any other arthropod to date. Including pseudogenes, 689 gustatory receptors were identified, as were 136 degenerin/Epithelial Na+ Channels (ENaCs) that have also been implicated as CRs in insects. The genomic distribution of T. urticae gustatory receptors indicates recurring bursts of lineage-specific proliferations, with the extent of receptor clusters reminiscent of those observed in the CR-rich genomes of vertebrates or C. elegans. Although pseudogenization of many gustatory receptors within clusters suggests relaxed selection, a subset of receptors is expressed. Consistent with functions as CRs, the genomic distribution and expression of ENaCs in lineage-specific T. urticae expansions mirrors that observed for gustatory receptors. The expansion of ENaCs in T. urticae to > 3-fold that reported in other animals was unexpected, raising the possibility that ENaCs in T. urticae have been co-opted to fulfill a major role performed by unrelated CRs in other animals. More broadly, our findings suggest an elaborate role for chemosensory perception in generalist herbivores that are of key ecological and agricultural importance

Postembryonic RNAi in Heterorhabditis bacteriophora: a nematode insect parasite and host for insect pathogenic symbionts

Background: Heterorhabditis bacteriophora is applied throughout the world for the biological control of insects and is an animal model to study interspecies interactions, e.g. mutualism, parasitism and vector-borne disease. H. bacteriophora nematodes are mutually associated with the insect pathogen, Photorhabdus luminescens. The developmentally arrested infective juvenile (IJ) stage nematode (vector) specifically transmits Photorhabdus luminescens bacteria (pathogen) in its gut mucosa to the haemocoel of insects (host). The nematode vector and pathogen alone are not known to cause insect disease. RNA interference is an excellent reverse genetic tool to study gene function in C. elegans, and it would be useful in H. bacteriophora to exploit the H. bacteriophora genome project, currently in progress. Results: Soaking L1 stage H. bacteriophora with seven dsRNAs of genes whose C. elegans orthologs had severe RNAi phenotypes resulted in highly penetrant and obvious developmental and reproductive abnormalities. The efficacy of postembryonic double strand RNA interference (RNAi) was evident by abnormal gonad morphology and sterility of adult H. bacteriophora and C. elegans presumable due to defects in germ cell proliferation and gonad development. The penetrance of RNAi phenotypes in H. bacteriophora was high for five genes (87-100%; Hba-cct-2, Hba-daf-21, Hba-icd-1; Hba-nol-5, and Hba-W01G7.3) and moderate for two genes (usually 30-50%; Hba-rack-1 and Hba-arf-1). RNAi of three additional C. elegans orthologs for which RNAi phenotypes were not previously detected in C. elegans, also did not result in any apparent phenotypes in H. bacteriophora. Specific and severe reduction in transcript levels in RNAi treated L1s was determined by quantitative real-time RT-PCR. These results suggest that postembryonic RNAi by soaking is potent and specific. Conclusion: Although RNAi is conserved in animals and plants, RNAi using long dsRNA is not. These results demonstrate that RNAi can be used effectively in H. bacteriophora and can be applied for analyses of nematode genes involved in symbiosis and parasitism. It is likely that RNAi will be an important tool for functional genomics utilizing the high quality draft H. bacteriophora genome sequence

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

Identification and characterization of a novel non-structural protein of bluetongue virus

Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77–79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell