Johji Miwa

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.

C. elegans osm-3 gene mediating osmotic avoidance behaviour encodes a kinesin-like protein.

In the nematode Caenorhabditis elegans, mutants in osm-3 gene are known to be defective in osmotic avoidance, chemotaxis and dauer formation behaviours. To study the molecular basis of these pleiotropic defects we have cloned the osm-3 gene by germline transformation of osm-3 (p802) mutants through microinjection of the wild type genomic DNA. Northern analysis reveals a 3.0 kb transcript corresponding to osm-3. DNA sequencing of the transforming 4.3 kb fragment revealed a kinesin heavy chain-like protein, which contains conserved ATPase and microtubule binding domains. Our results are consistent with the previous EM data on osm-3 (p802) mutants that show an accumulation of dense matrix material in the amphid sheath cytoplasm and a shortened distal segment of the amphid channel cilium. These data suggest a kinesin-like role of the osm-3 product in axonal transport.

Cell cycle control by daf-21/Hsp90 at the first meiotic prophase/metaphase boundary during oogenesis in Caenorhabditis elegans

DAF‐21, a Caenorhabditis elegans homologue of Hsp90, is expressed primarily in germline cells. Although mutations in the daf‐21 gene affect animal fertility, its cellular roles have remained elusive. To phenocopy daf‐21 mutations, we impaired the daf‐21 function by RNA interference (RNAi), and found that oocytes skipped the diakinesis arrest and displayed a defective diakinesis arrest, which led to the production of endomitotic oocytes with polyploid chromosomes (Emo phenotype). The same Emo phenotype was also observed with RNAi against wee‐1.3. To identify a cause for Emo, we examined the CDK‐1 (Cdc2) phosphorylation status in Emo animals, since CDK‐1 is a key regulator of the prophase/metaphase transition and is kept inactivated by WEE‐1.3 kinase during prophase. We immunostained both daf‐21(RNAi) and wee‐1.3(RNAi) animals with anti‐phosphorylated‐CDK‐1 antibody and observed no detectable phosphates on CDK‐1 in either of the animals. We also examined WEE‐1.3 expression in daf‐21(RNAi) and found a significant reduction of WEE‐1.3. These results indicate that CDK‐1 was not phosphorylated in either daf‐21(RNAi) or wee‐1.3(RNAi) animals, and suggest that daf‐21 was necessary for producing functional WEE‐1.3. Thus, all together, we propose that DAF‐21 indirectly regulates the meiotic prophase/metaphase transition during oocyte development by ensuring the normal function of WEE‐1.3.

Genetic and cellular characterization of Caenorhabditis elegans mutants abnormal in the regulation of many phase II enzymes.

Background The phase II detoxification enzymes execute a major protective role against xenobiotics as well as endogenous toxicants. To understand how xenobiotics regulate phase II enzyme expression, acrylamide was selected as a model xenobiotic chemical, as it induces a large number and a variety of phase II enzymes, including numerous glutathione S-transferases (GSTs) in Caenorhabditis elegans. Methodology/Principal Findings To begin dissecting genetically xenobiotics response pathways (xrep), 24 independent mutants of C. elegans that exhibited abnormal GST expression or regulation against acrylamide were isolated by screening about 3.5×105 genomes of gst::gfp transgenic strains mutagenized with ethyl methanesulfonate (EMS). Complementation testing assigned the mutants to four different genes, named xrep-1, -2, -3, and -4. One of the genes, xrep-1, encodes WDR-23, a nematode homologue of WD repeat-containing protein WDR23. Loss-of-function mutations in xrep-1 mutants resulted in constitutive expression of many GSTs and other phase II enzymes in the absence of acrylamide, and the wild-type xrep-1 allele carried on a DNA construct successfully cured the mutant phenotype of the constitutive enzyme expression. Conclusions/Significance Genetic and cellular characterization of xrep-1 mutants suggest that a large number of GSTs and other phase II enzymes induced by acrylamide are under negative regulation by XREP-1 (WDR-23), which is likely to be a functional equivalent of mammalian Keap1 and a regulator of SKN-1, a C. elegans analogue of cap-n-collar Nrf2 (nuclear factor erythroid 2-related factor 2).

Behavioral genetics of caenorhabditis elegans unc-103-encoded erg-like K(+) channel.

The Caenorhabditis elegans unc-103 gene encodes a potassium channel whose sequence is most similar to the ether-a-go-go related gene (erg) type of K+ channels. We find that the n500 and e1597 gain-of-function (gf) mutations in unc-103 cause reduced excitation in most muscles, while loss-of-function (lf) mutations cause mild muscle hyper-excitability. Both gf alleles change the same residue near the cytoplasmic end of S6, consistent with this region regulating channel activation. We also report additional dominant-negative and lf alleles of unc-103 that can antagonize or reduce the function of both gf and wild-type alleles. The unc-103 locus contains 6 promoter regions that express unc-103 in different combinations of body-wall and sex-specific muscles, motor-, inter- and sensory-neurons. Each promoter drives transcripts containing a unique first exon, conferring sequence variability to the N-terminus of the UNC-103 protein, while three splice variants introduce variability into the UNC-103 C-terminus. unc-103(0) hermaphrodites prematurely lay embryos that would normally be retained in the uterus and lay eggs under conditions that inhibit egg-laying behavior. In the egg-laying circuit, unc-103 is expressed in vulval muscles and the HSN neurons from different promoters. Supplying the proper UNC-103 isoform to the vulval muscles is sufficient to restore regulation to egg-laying behavior.

Caenorhabditis elegans DAF-21 (HSP90) is characteristically and predominantly expressed in germline cells: Spatial and temporal analysis

Three monoclonal antibodies against antigens that exist in the Caenorhabditis elegans germ line have previously been described. In the present study, a full‐length mRNA for one of these antigens was isolated, and by sequencing its corresponding cDNA, it was predicted that the protein would show a high homology with the 90 kDa heat shock protein (HSP90) in other species, and with the protein of daf‐21, a previously identified hsp90 homologue. The spatial and temporal distribution of the antigen (DAF‐21) was analyzed in C. elegans, and the localization of daf‐21 mRNA, as detected by in situ hybridization, agreed with that detected by the monoclonal antibody. Under normal conditions, daf‐21 mRNA is characteristically distributed in postembryonic germ cells derived from Z2 and Z3 cells in both hermaphrodites and males. Under heat stress conditions, however, daf‐21 mRNA was not only detected in germ cells, but also apparently expressed all over the body. In addition, the DAF‐21 protein seemed to be localized in the perinuclear region of somatic cells.

Early embryogenesis of the pinewood nematode Bursaphelenchus xylophilus.

The early embryogenesis and cell lineage of the pinewood nematode Bursaphelenchus xylophilus was followed from a single‐cell zygote to a 46‐cell embryo under Nomarski optics, and elongation of the microtubules was studied by immunostaining. As a B. xylophilus oocyte matures, it passes through a passage connecting the oviduct with the quadricolumella, the distal part of the uterus, and reaches the quadricolumella where it stays for a few minutes and is fertilized. After fertilization, the germinal vesicle disappears, an eggshell is formed, and the male and female pronuclei appear. The pronuclei move toward each other and fuse at the center of the egg. Around this time, the microtubule‐organizing center appears. The presumptive region of sperm entry into the oocyte becomes the future anterior portion of the embryo. This anterior–posterior axis determination is opposite to that of Caenorhabditis elegans, where the sperm entry site becomes the posterior portion of the embryo. The optimal growth temperatures of these two nematodes also differ in that temperatures of about 30°C afford the fastest growth rate and highest hatching frequency in B. xylophilus. Otherwise, the lineage resembles that of C. elegans with respect to timing, positioning and the axis orientation of each cell division.

Tumor promoters specifically and reversibly disturb development and behavior of Caenorhabditis elegans.

SummaryThe effect of phorbol ester tumor promoters on the development and behavior of a free-living soil nematode, Caenorhabditis elegans, was studied. When young developing C. elegans were grown on E. coli-seeded agar with low concentrations (0.1 μg/ml) of 12-0-tetradecanoyl-phorbol-13-acetate or phorbol-12,13-didecanoate, their growth was arrested. These tumor promoters reduced the brood size when gravid adults were treated and caused uncoordinated movement in animals treated at any stage of development. The effects of these tumor promoters on nematode development and behavior were partially reversible. The nonpromoting derivatives phorbol and 4α-phorbol-12,13-didecanoate showed no effect on the animals.

Germline‐specific Antigens Identified by Monoclonal Antibodies in the Nematode Caenorhabditis elegans1

Of 27 monoclonal antibodies identified to react, by indirect immunofluorescent antibody staining, with specific cells and tissues of the nematode Caenorhabditis elegans, we report here three monoclonal antibodies pertaining to the gonadal tissues. One antibody defines an antigen that is distributed over the entire embryo at earlier development and later becomes unique to the gonad, including mature oocytes. The antigens recognized by the other two are distributed asymmetrically in the posterior region of the fertilized eggs cytoplasm destined to become the germline precursor cell. Each antigen is successively segregated only to the germline precursor cells of the developing embryo and, postembryonically, is uniquely localized around the germline cell nuclei of the larvae and adults.

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.