Koichi Hasegawa

Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is the nematode responsible for a devastating epidemic of pine wilt disease in Asia and Europe, and represents a recent, independent origin of plant parasitism in nematodes, ecologically and taxonomically distinct from other nematodes for which genomic data is available. As well as being an important pathogen, the B. xylophilus genome thus provides a unique opportunity to study the evolution and mechanism of plant parasitism. Here, we present a high-quality draft genome sequence from an inbred line of B. xylophilus, and use this to investigate the biological basis of its complex ecology which combines fungal feeding, plant parasitic and insect-associated stages. We focus particularly on putative parasitism genes as well as those linked to other key biological processes and demonstrate that B. xylophilus is well endowed with RNA interference effectors, peptidergic neurotransmitters (including the first description of ins genes in a parasite) stress response and developmental genes and has a contracted set of chemosensory receptors. B. xylophilus has the largest number of digestive proteases known for any nematode and displays expanded families of lysosome pathway genes, ABC transporters and cytochrome P450 pathway genes. This expansion in digestive and detoxification proteins may reflect the unusual diversity in foods it exploits and environments it encounters during its life cycle. In addition, B. xylophilus possesses a unique complement of plant cell wall modifying proteins acquired by horizontal gene transfer, underscoring the impact of this process on the evolution of plant parasitism by nematodes. Together with the lack of proteins homologous to effectors from other plant parasitic nematodes, this confirms the distinctive molecular basis of plant parasitism in the Bursaphelenchus lineage. The genome sequence of B. xylophilus adds to the diversity of genomic data for nematodes, and will be an important resource in understanding the biology of this unusual parasite.

Bacterial role in pine wilt disease development – review and future perspectives

Mutualistic and beneficial relationships between nematodes and bacteria are highly present in nature, mostly occurring because of nutritional dependence and pathogen protection, and intrinsically related with the environment, the ecological conditions and the nematode life stages. Thirty-four years have passed since the first hypothesis suggesting a bacterial role in pine wilt disease (PWD), associated with the pinewood nematode (PWN), Bursaphelenchus xylophilus. In 1980, researchers reported that bacteria associated with the PWN could produce toxins that lead to PWD development in pine seedlings. It was also suggested a double vector system for PWD, where bacteria were vectored by the PWN and the PWN vectored by an insect from the Monochamus genus. Presently, the specific involvement of bacteria in such complex disease is still controversial, even though the increased number of studies focused on the potential bacteria role has increased considerably. This review is an up-to-date comprehensive perspective and brings new insights on the role of PWN-associated bacteria in PWD.

Characterization of bacterial communities associated with the pine sawyer beetle Monochamus galloprovincialis, the insect vector of the pinewood nematode Bursaphelenchus xylophilus

Pine wilt disease (PWD) has a tremendous impact on worldwide forestlands, both from the environmental and economical viewpoints. Monochamus sp., a xylophagous insect from the Cerambycidae family, plays an important role in dissemination of the pinewood nematode, Bursaphelenchus xylophilus, the primary pathogenic agent of PWD. This study investigates, for the first time, the bacterial communities of Monochamus galloprovincialis collected from Portuguese Pinus pinaster trees and B. xylophilus free, using a metagenomics approach. Overall, our results show that natural bacterial communities of M. galloprovincialis are mainly composed by γ-proteobacteria, Firmicutes and Bacteroidetes, which may be a reflection of insects feeding diet and habitat characteristics. We also report different bacterial communities composition in the thorax and abdomen of M. galloprovincialis, with high abundance of Serratia sp. in both. Our results encourage further studies in the possible relationship between bacteria from the insect vector and B. xylophilus.

Pinewood nematode-associated bacteria contribute to oxidative stress resistance of Bursaphelenchus xylophilus

BackgroundPine wilt disease (PWD) caused by the pinewood nematode Bursaphelenchus xylophilus is one of the most serious forest diseases in the world. The role of B. xylophilus-associated bacteria in PWD and their interaction with the nematode, have recently been under substantial investigation. Several studies report a potential contribution of the bacteria for the PWD development, either as a helper to enhance the pathogenicity of the nematode or as a pathogenic agent expressing interesting traits related to lifestyle host-adaptation.ResultsWe investigated the nematode-bacteria interaction under a severe oxidative stress (OS) condition using a pro-oxidant hydrogen peroxide and explored the adhesion ability of these bacteria to the cuticle surface of the nematodes. Our results clearly demonstrated a beneficial effect of the Serratia spp. (isolates LCN-4, LCN-16 and PWN-146) to B. xylophilus under the OS condition. Serratia spp. was found to be extremely OS-resistant, and promote survival of B. xylophilus and down-regulate two B. xylophilus catalase genes (Bxy-ctl-1 and Bxy-ctl-2). In addition, we show that the virulent isolate (Ka4) of B. xylophilus survives better than the avirulent (C14-5) isolate under the OS condition. The bacterial effect was transverse for both B. xylophilus isolates. We could not observe a strong and specific adhesion of these bacteria on the B. xylophilus cuticle surface.ConclusionsWe report, for the first time, that B. xylophilus associated bacteria may assist the nematode opportunistically in the disease, and that a virulent B. xylophilus isolate displayed a higher tolerance towards the OS conditions than an avirulent isolate.

Catalases Induction in High Virulence Pinewood Nematode Bursaphelenchus xylophilus under Hydrogen Peroxide-Induced Stress

Considered an EPPO A2 quarantine pest, Bursaphelenchus xylophilus is the causal agent of the pine wilt disease and the most devastating plant parasitic nematode attacking coniferous trees in the world. In the early stages of invasion, this nematode has to manage host defence mechanisms, such as strong oxidative stress. Only successful, virulent nematodes are able to tolerate the basal plant defences, and furthermore migrate and proliferate inside of the host tree. In this work, our main objective was to understand to what extent B. xylophilus catalases are involved in their tolerance to oxidative stress and virulence, using as oxidant agent the reactive oxygen species hydrogen peroxide (H2O2). After 24 hours of exposure, high virulence isolates of B. xylophilus could withstand higher H2O2 concentrations in comparison with low virulence B. xylophilus and B. mucronatus, corroborating our observation of Bxy-ctl-1 and Bxy-ctl-2 catalase up-regulation under the same experimental conditions. Both catalases are expressed throughout the nematode intestine. In addition, transgenic strains of Caenorhabditis elegans overexpressing B. xylophilus catalases were constructed and evaluated for survival under similar conditions as previously. Our results suggest that catalases of high virulence B. xylophilus were crucial for nematode survival under prolonged exposure to in vitro oxidative stress, highlighting their adaptive response, which could contribute to their success in host conditions.

Activated and inactivated immune responses in Caenorhabditis elegans against Photorhabdus luminescens TT01

The Gram-negative bacterium Photorhabdus luminescens which symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora, has a broad insecticidal and nematicidal activity. The virulence of P. luminescens toward the non-mutualistic nematode Caenorhabditis elegans has not been described. We showed that when fed on P. luminescens, the intestinal cells of C. elegans worms become delicate and some crystal-like structure was developed within the intestinal lumen. Next, we examined the requirement of the p38 mitogen-activated protein kinase (MAPK) and insulin/IGF-1 signaling pathway against P. luminescens. Depletion of pmk-1 by RNAi enhances susceptibility to P. luminescens, and numerous downstream targets regulated by the p38 MAPK pathway were induced when fed on P. luminescens. On the other hand, knockdown of daf-16 has no effects on C. elegans lifespan, but knockdown of daf-2 dramatically increased resistance to P. luminescens in a daf-16-dependent manner. We also revealed one of the daf-2 ligands ins-7 was induced and ins-7 deletion mutant survived longer when fed on P. luminescens. These results suggest the p38 MAPK pathway is activated and required for the host defense against P. luminescens. Insulin/IGF-1 signaling pathway is inactivated by P. luminescens through the overexpression of insulin-like gene.

Chromosome structure and behaviour in Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) germ cells and early embryo

Chromosome structure and behaviour in both meiosis of the germ cells and mitosis of the embryo from fertilisation to the two-cell stage in Bursaphelenchus xylophilus were examined by DAPI staining and three-dimensional reconstruction of serial-section images from confocal laser-scanning microscopy. By this method, each chromosomes shape and behaviour were clearly visible in early embryogenesis from fertilisation through the formation and fusion of the male and female pronuclei to the first mitotic division. The male pronucleus was bigger than that of the female, although the oocyte is larger and richer in nutrients than the sperm. From the shape of the separating chromosomes at anaphase, the mitotic chromosomes appeared to be polycentric or holocentric rather than monocentric. Each chromosome was clearly distinguishable in the male and female germ cells, pronuclei of the one-cell stage embryo, and the early embryonic nuclei. The haploid number of chromosomes (N) was six (2n = 12), and all chromosomes appeared similar. The chromosome pair containing the ribosomal RNA-coding site was visualised by fluorescence in situ hybridisation. Unlike the sex determination system in Caenorhabditis elegans (XX in hermaphrodite and XO in male), the system for B. xylophilus may consist of an XX female and an XY male.

Bacterial community associated to the pine wilt disease insect vectors Monochamus galloprovincialis and Monochamus alternatus.

Monochamus beetles are the dispersing vectors of the nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease (PWD). PWD inflicts significant damages in Eurasian pine forests. Symbiotic microorganisms have a large influence in insect survival. The aim of this study was to characterize the bacterial community associated to PWD vectors in Europe and East Asia using a culture-independent approach. Twenty-three Monochamus galloprovincialis were collected in Portugal (two different locations); twelve Monochamus alternatus were collected in Japan. DNA was extracted from the insects’ tracheas for 16S rDNA analysis through denaturing gradient gel electrophoresis and barcoded pyrosequencing. Enterobacteriales, Pseudomonadales, Vibrionales and Oceanospirilales were present in all samples. Enterobacteriaceae was represented by 52.2% of the total number of reads. Twenty-three OTUs were present in all locations. Significant differences existed between the microbiomes of the two insect species while for M. galloprovincialis there were no significant differences between samples from different Portuguese locations. This study presents a detailed description of the bacterial community colonizing the Monochamus insects’ tracheas. Several of the identified bacterial groups were described previously in association with pine trees and B. xylophilus, and their previously described functions suggest that they may play a relevant role in PWD.

Direct Interaction between the WD40 Repeat Protein WDR-23 and SKN-1/Nrf Inhibits Binding to Target DNA

ABSTRACT SKN-1/Nrf transcription factors activate cytoprotective genes in response to reactive small molecules and strongly influence stress resistance, longevity, and development. The molecular mechanisms of SKN-1/Nrf regulation are poorly defined. We previously identified the WD40 repeat protein WDR-23 as a repressor of Caenorhabditis elegans SKN-1 that functions with a ubiquitin ligase to presumably target the factor for degradation. However, SKN-1 activity and nuclear accumulation are not always correlated, suggesting that there could be additional regulatory mechanisms. Here, we integrate forward genetics and biochemistry to gain insights into how WDR-23 interacts with and regulates SKN-1. We provide evidence that WDR-23 preferentially regulates one of three SKN-1 variants through a direct interaction that is required for normal stress resistance and development. Homology modeling predicts that WDR-23 folds into a β-propeller, and we identify the top of this structure and four motifs at the termini of SKN-1c as essential for the interaction. Two of these SKN-1 motifs are highly conserved in human Nrf1 and Nrf2 and two directly interact with target DNA. Lastly, we demonstrate that WDR-23 can block the ability of SKN-1c to interact with DNA sequences of target promoters identifying a new mechanism of regulation that is independent of the ubiquitin proteasome system, which can become occupied with damaged proteins during stress.

Evidence of hermaphroditism and sex ratio distortion in the fungal feeding nematode Bursaphelenchus okinawaensis

Nematodes have many different reproductive strategies along with their divergent life histories; the ability of hermaphrodite to self- and cross-fertilize is useful for genetic manipulation. Here, we demonstrate the hermaphroditism of the fungal feeding nematode Bursaphelenchus okinawaensis, which was formerly described as a parthenogenetic nematode, and we show its other unique sexual characteristics. To determine that it is hermaphroditic, we performed the following experiments: observation of the pronuclear and chromosome behavior during oogenesis and early embryogenesis; observation of spermatogenesis during the fourth larval stage; investigation of sperm utilization; and investigation of phenotypic segregation after cross-mating using a chemically induced visible mutant. We then investigated the mating preferences and spermatid size difference between males and hermaphrodites. B. okinawaensis males successfully mated only with sperm-depleted old hermaphrodites, and the spermatid sizes of males were almost the same as those of hermaphrodites. Moreover, the sex ratio of cross-fertilized progeny was highly skewed toward hermaphrodites. B. okinawaensis is phylogenetically distant from established model nematodes such as C. elegans and is more closely related to some economically relevant parasitic nematodes. This newly discovered hermaphroditic nematode has great potential for evolutionary and parasitological research.