Elizabeth H. Scholl

Sequence and genetic map of Meloidogyne hapla: A compact nematode genome for plant parasitism

We have established Meloidogyne hapla as a tractable model plant-parasitic nematode amenable to forward and reverse genetics, and we present a complete genome sequence. At 54 Mbp, M. hapla represents not only the smallest nematode genome yet completed, but also the smallest metazoan, and defines a platform to elucidate mechanisms of parasitism by what is the largest uncontrolled group of plant pathogens worldwide. The M. hapla genome encodes significantly fewer genes than does the free-living nematode Caenorhabditis elegans (most notably through a reduction of odorant receptors and other gene families), yet it has acquired horizontally from other kingdoms numerous genes suspected to be involved in adaptations to parasitism. In some cases, amplification and tandem duplication have occurred with genes suspected of being acquired horizontally and involved in parasitism of plants. Although M. hapla and C. elegans diverged >500 million years ago, many developmental and biochemical pathways, including those for dauer formation and RNAi, are conserved. Although overall genome organization is not conserved, there are areas of microsynteny that may suggest a primary biological function in nematodes for those genes in these areas. This sequence and map represent a wealth of biological information on both the nature of nematode parasitism of plants and its evolution.

Horizontally transferred genes in plant-parasitic nematodes: a high-throughput genomic approach

BackgroundPublished accounts of horizontally acquired genes in plant-parasitic nematodes have not been the result of a specific search for gene transfer per se, but rather have emerged from characterization of individual genes. We present a method for a high-throughput genome screen for horizontally acquired genes, illustrated using expressed sequence tag (EST) data from three species of root-knot nematode, Meloidogyne species.ResultsOur approach identified the previously postulated horizontally transferred genes and revealed six new candidates. Screening was partially dependent on sequence quality, with more candidates identified from clustered sequences than from raw EST data. Computational and experimental methods verified the horizontal gene transfer candidates as bona fide nematode genes. Phylogenetic analysis implicated rhizobial ancestors as donors of horizontally acquired genes in Meloidogyne.ConclusionsHigh-throughput genomic screening is an effective way to identify horizontal gene transfer candidates. Transferred genes that have undergone amelioration of nucleotide composition and codon bias have been identified using this approach. Analysis of these horizontally transferred gene candidates suggests a link between horizontally transferred genes in Meloidogyne and parasitism.

Comprehensive Transcriptome Profiling in Tomato Reveals a Role for Glycosyltransferase in Mi-Mediated Nematode Resistance

Root-knot nematode (RKN; Meloidogyne spp.) is a major crop pathogen worldwide. Effective resistance exists for a few plant species, including that conditioned by Mi in tomato (Solanum lycopersicum). We interrogated the root transcriptome of the resistant (Mi+) and susceptible (Mi–) cultivars ‘Motelle’ and ‘Moneymaker,’ respectively, during a time-course infection by the Mi-susceptible RKN species Meloidogyne incognita and the Mi-resistant species Meloidogyne hapla. In the absence of RKN infection, only a single significantly regulated gene, encoding a glycosyltransferase, was detected. However, RKN infection influenced the expression of broad suites of genes; more than half of the probes on the array identified differential gene regulation between infected and uninfected root tissue at some stage of RKN infection. We discovered 217 genes regulated during the time of RKN infection corresponding to establishment of feeding sites, and 58 genes that exhibited differential regulation in resistant roots compared to uninfected roots, including the glycosyltransferase. Using virus-induced gene silencing to silence the expression of this gene restored susceptibility to M. incognita in ‘Motelle,’ indicating that this gene is necessary for resistance to RKN. Collectively, our data provide a picture of global gene expression changes in roots during compatible and incompatible associations with RKN, and point to candidates for further investigation.

Mitochondrial Genome Sequences and Structures Aid in the Resolution of Piroplasmida phylogeny.

The taxonomy of the order Piroplasmida, which includes a number of clinically and economically relevant organisms, is a hotly debated topic amongst parasitologists. Three genera (Babesia, Theileria, and Cytauxzoon) are recognized based on parasite life cycle characteristics, but molecular phylogenetic analyses of 18S sequences have suggested the presence of five or more distinct Piroplasmida lineages. Despite these important advancements, a few studies have been unable to define the taxonomic relationships of some organisms (e.g. C. felis and T. equi) with respect to other Piroplasmida. Additional evidence from mitochondrial genome sequences and synteny should aid in the inference of Piroplasmida phylogeny and resolution of taxonomic uncertainties. In this study, we have amplified, sequenced, and annotated seven previously uncharacterized mitochondrial genomes (Babesia canis, Babesia vogeli, Babesia rossi, Babesia sp. Coco, Babesia conradae, Babesia microti-like sp., and Cytauxzoon felis) and identified additional ribosomal fragments in ten previously characterized mitochondrial genomes. Phylogenetic analysis of concatenated mitochondrial and 18S sequences as well as cox1 amino acid sequence identified five distinct Piroplasmida groups, each of which possesses a unique mitochondrial genome structure. Specifically, our results confirm the existence of four previously identified clades (B. microti group, Babesia sensu stricto, Theileria equi, and a Babesia sensu latu group that includes B. conradae) while supporting the integration of Theileria and Cytauxzoon species into a single fifth taxon. Although known biological characteristics of Piroplasmida corroborate the proposed phylogeny, more investigation into parasite life cycles is warranted to further understand the evolution of the Piroplasmida. Our results provide an evolutionary framework for comparative biology of these important animal and human pathogens and help focus renewed efforts toward understanding the phylogenetic relationships within the group.

Solution NMR studies of the plant peptide hormone CEP inform function

The C‐terminally Encoded Peptide (CEP) family of regulatory peptides controls root development in vascular plants. Here, we present the first NMR structures of CEP. We show that root‐knot nematode (RKN: Meloidogyne spp.) also encodes CEP, presumably to mimic plant CEP as part of their stereotypic, parasitic interaction with vascular plants. Molecular dynamics simulations of plant‐ and nematode‐encoded CEP displaying known posttranslational modifications (PTM) provided insight into the structural effects of PTM and the conformational plasticity and rigidity of CEP. Potential mechanisms of action are discussed with respect to the structure and sampling of conformational space.

The plant parasite Pratylenchus coffeaecarries a minimal nematode genome

Here we report the genome sequence of the lesion nematode, Pratylenchus coffeae, a significant pest of banana and other staple crops in tropical and sub-tropical regions worldwide. Initial analysis of the 19.67 Mb genome reveals 6712 protein encoding genes, the smallest number found in a metazoan, although sufficient to make a nematode. Significantly, no developmental or physiological pathways are obviously missing when compared to the model free-living nematode Caenorhabditis elegans, which possesses approximately 21 000 genes. The highly streamlined P. coffeaegenome may reveal a remarkable functional plasticity in nematode genomes and may also indicate evolutionary routes to increased specialisation in other nematode genera. In addition, the P. coffeaegenome may begin to reveal the core set of genes necessary to make a multicellular animal. Nematodes exhibit striking diversity in the niches they occupy, and the sequence of P. coffeaeis a tool to begin to unravel the mechanisms that enable the extraordinary success of this phylum as both free-living and parasitic forms. Unlike the sedentary endoparasitic root-knot nematodes ( Meloidogynespp.), P. coffeaeis a root-lesion nematode that does not establish a feeding site within the root. Because the P. coffeaenematode genome encodes fewer than half the number of genes found in the genomes of root-knot nematodes, comparative analysis to determine genes P. coffeaedoes not carry may help to define development of more sophisticated forms of nematode-plant interactions. The P. coffeaegenome sequence may help to define timelines related to evolution of parasitism amongst nematodes. The genome of P. coffeaeis a significant new tool to understand not only nematode evolution but animal biology in general.

Proteomic and Bioinformatic Analysis of the Root-Knot Nematode Meloidogyne hapla: The Basis for Plant Parasitism

On the basis of the complete genome sequence of the root-knot nematode Melodogyne hapla, we have deduced and annotated the entire proteome of this plant-parasite to create a database of 14,420 proteins. We have made this database, termed HapPep3, available from the Superfamily repository of model organism proteomes (http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY). To experimentally confirm the HapPep3 assignments using proteomics, we applied a data-independent LC/MS(E) analysis to M. hapla protein extracts fractionated by SDS-PAGE. A total of 516 nonredundant proteins were identified with an average of 9 unique peptides detected per protein. Some proteins, including examples with complex gene organization, were defined by more than 20 unique peptide matches, thus, providing experimental confirmation of computational predictions of intron/exon structures. On the basis of comparisons of the broad physicochemical properties of the experimental and computational proteomes, we conclude that the identified proteins reflect a true and unbiased sampling of HapPep3. Conversely, HapPep3 appears to broadly cover the protein space able to be experimentally sampled. To estimate the false discovery rate, we queried human, plant, and bacterial databases for matches to the LC/MS(E)-derived peptides, revealing fewer than 1% of matches, most of which were to highly conserved proteins. To provide a functional comparison of the acquired and deduced proteomes, each was subjected to higher order annotation, including comparisons of Gene Ontology, protein domains, signaling, and localization predictions, further indicating concordance, although those proteins that did deviate seem to be highly significant. Approximately 20% of the experimentally sampled proteome was predicted to be secreted, and thus potentially play a role at the host-parasite interface. We examined reference pathways to determine the extent of proteome similarity of M. hapla to that of the free-living nematode, Caenorhabditis elegans, revealing significant similarities and differences. Collectively, the analyzed protein set provides an initial foundation to experimentally dissect the basis of plant parasitism by M. hapla.

Comparative Genomic Analysis Identifies Divergent Genomic Features of Pathogenic Enterococcus cecorum Including a Type IC CRISPR-Cas System, a Capsule Locus, an epa-Like Locus, and Putative Host Tissue Binding Proteins

Enterococcus cecorum (EC) is the dominant enteric commensal of adult chickens and contributes to the gut consortia of many avian and mammalian species. While EC infection is an uncommon zoonosis, like other enterococcal species it can cause life-threating nosocomial infection in people. In contrast to other enterococci which are considered opportunistic pathogens, emerging pathogenic strains of EC cause outbreaks of musculoskeletal disease in broiler chickens. Typical morbidity and mortality is comparable to other important infectious diseases of poultry. In molecular epidemiologic studies, pathogenic EC strains were found to be genetically clonal. These findings suggested acquisition of specific virulence determinants by pathogenic EC. To identify divergent genomic features and acquired virulence determinants in pathogenic EC; comparative genomic analysis was performed on genomes of 3 pathogenic and 3 commensal strains of EC. Pathogenic isolates had smaller genomes with a higher GC content, and they demonstrated large regions of synteny compared to commensal isolates. A molecular phylogenetic analysis demonstrated sequence divergence in pathogenic EC genomes. At a threshold of 98% identity, 414 predicted proteins were identified that were highly conserved in pathogenic EC but not in commensal EC. Among these, divergent CRISPR-cas defense loci were observed. In commensal EC, the type IIA arrangement typical for enterococci was present; however, pathogenic EC had a type IC locus, which is novel in enterococci but commonly observed in streptococci. Potential mediators of virulence identified in this analysis included a polysaccharide capsular locus similar to that recently described for E. faecium, an epa-like locus, and cell wall associated proteins which may bind host extracellular matrix. This analysis identified specific genomic regions, coding sequences, and predicted proteins which may be related to the divergent evolution and increased virulence of emerging pathogenic strains of EC.

New approach for the study of mite reproduction: The first transcriptome analysis of a mite, Phytoseiulus persimilis (Acari: Phytoseiidae)

Many species of mites and ticks are of agricultural and medical importance. Much can be learned from the study of transcriptomes of acarines which can generate DNA-sequence information of potential target genes for the control of acarine pests. High throughput transcriptome sequencing can also yield sequences of genes critical during physiological processes poorly understood in acarines, i.e., the regulation of female reproduction in mites. The predatory mite, Phytoseiulus persimilis, was selected to conduct a transcriptome analysis using 454 pyrosequencing. The objective of this project was to obtain DNA-sequence information of expressed genes from P. persimilis with special interest in sequences corresponding to vitellogenin (Vg) and the vitellogenin receptor (VgR). These genes are critical to the understanding of vitellogenesis, and they will facilitate the study of the regulation of mite female reproduction. A total of 12,556 contiguous sequences (contigs) were assembled with an average size of 935bp. From these sequences, the putative translated peptides of 11 contigs were similar in amino acid sequences to other arthropod Vgs, while 6 were similar to VgRs. We selected some of these sequences to conduct stage-specific expression studies to further determine their function.

Computational and phylogenetic validation of nematode horizontal gene transfer

Sequencing of expressed genes has shown that nematodes, particularly the plant-parasitic nematodes, have genes purportedly acquired from other kingdoms by horizontal gene transfer. The prevailing orthodoxy is that such transfer has been a driving force in the evolution of niche specificity, and a recent paper in BMC Evolutionary Biology that presents a detailed phylogenetic analysis of cellulase genes in the free-living nematode Pristionchus pacificus at the species, genus and family levels substantiates this hypothesis.See research article: http://www.biomedcentral.com/1471-2148/11/13