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[
International Worm Meeting,
2007]
To confer resistance against the root gall nematode Meloidogyne incognita, a study was performed to introduce Bacillus thuringiensis (Bt) crystal protein toxin genes into tomato plant (Lycopersicon esculentum var. Rutgers select) hairy roots. Crystal toxin gene sequences were altered to allow plant expression and in some cases also included unique intron sequences to aid in protein expression. Genes were inserted into the pBluscript in concert with a double 35S plant promoter and kanamycin resistance. The vector was used to transform the plant pathogenic bacterium Agrobacterium rhizogenes. The transformed Agrobacterium was used to induce the toxin genes into plants by co-cultivation with tomato cotyledons. Hairy root lines were selected via kanamycin resistance and once established, root extracts were tested for relative expression of crystal toxin by western blot analysis using a polyclonal detection sera specific for each toxin. Induced resistance against the root gall nematode was examined by hairy root challenge against a load of J2 stage parasitic nematodes. Subsequent enumeration of total egg masses (EM), total sites of infection (INF) and total calculated eggs (TE) per root plate were used to determine resistance against nematode infections. Results of these studies will be discussed. Data suggests that some Bt crystal proteins have excellent potential to control plant parasitic nematode (PPN) infections in transgenic plants.
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MacParland, Sonya, Gilleard, John, Cowen, Leah, Kitner, Megan, Roy, Peter, Redman, Elizabeth, Lautens, Mark, Xiao, Qi, Dowling, James, Volpatti, Jonathan, Zasada, Inga, Burns, Andrew, MacDonald, Margaret, Krause, Henry, Chung, Sai, Snider, Jamie, Palmeira, Bruna, Cutler, Sean, Stagljar, Igor, Castelli, Jack, Meyer, Susan, Finney, Constance, Hu, Chun, Marwah, Sagar, Vaidya, Aditya, Puumala, Emily, Ross, Rachel, Tiefenbach, Jens
[
International Worm Meeting,
2021]
Global food security is threatened as the world amasses 10 billion people amid limited arable land. While nematode pests are a major barrier to agricultural intensification, most traditional nematicides are now banned because of poor nematode-selectivity, leaving farmers with inadequate controls. Here, we describe a screen carried out in the model nematode Caenorhabditis elegans that enriches for selective nematicides by identifying molecules that are bioactivated by cytochrome P450s, which are phylogenetically diverse. We identify a family of structures, called nemactivins, that are robustly bioactivated to a toxic metabolite selectively in nematodes. At low parts-per-million concentrations, nemactivins perform comparably well with commercial nematicides at controlling infection by the world's most destructive plant-parasitic nematode Meloidogyne incognita. Hence, nemactivins are first-in-class bioactivated nematicides that provide much needed nematode-selectivity.
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[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2014]
Root knot nematodes (RKN) can infect most of the world's agricultural crop species and are among the most important of all plant pathogens. As yet however we have little understanding of their origins or the genomic basis of this extreme polyphagy. It has been suggested that the most damaging pathogens have originated from interspecific hybridizations between unknown parental taxa. We have sequenced the genome of Meloidogyne floridensis, and use comparative evolutionary genomics of RKN to test the hypothesis that this species was involved in the hybrid origin of the agriculturally important species Meloidogyne incognita. Phylogenomic analyses of gene families indicate that RKN species may have very complex origins involving the mixing of several parental genomes by hybridization. The extreme polyphagy of some RKN, and their success in agricultural environments, may be related to this hybridisation, producing transgressive variation on which natural selection can act. This comparative genomic analysis provides a compelling example of the importance and complexity of hybridization in generating animal species diversity more generally.
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[
International Worm Meeting,
2015]
Plant parasitic nematodes are one of the major pests affecting agricultural crops. Root-knot nematodes (RKNs) are among the most damaging group of plant parasitic nematodes. These nematodes are obligate parasites that infect their hosts in the root system. These parasites lead to a loss in excess of $100 billion for farmers due to reduced crop yield. Currently, the means of control for plant parasitic nematodes are chemical pesticides and resistant cultivars. However, many of the chemicals have been banned due to health concerns and certain populations of the nematodes have been able to overcome the resistance in selectively bred cultivars. As a result, a new method of control needs to be developed. We previously showed that when Caenorhabditis elegans are cultured on media containing specific polyunsaturated fatty acids (PUFAs) they become sterile. This led us to hypothesize that if plant parasitic nematodes are exposed to these particular PUFAs, they will display a reduction in the production of offspring. We obtained transgenic Arabidopsis plants expressing a specific PUFA and are currently conducting experiments to determine if this PUFA can reduce infection rates and reduce fertility of the root knot nematode Meloidogyne incognita. This represents a novel technique for controlling plant parasitic nematodes. .
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[
International C. elegans Meeting,
2001]
To build upon knowledge gained from the genome of C. elegans , we have begun generating Expressed Sequence Tags (ESTs) from parasitic (and free-living) nematodes. This project will generate >225,000 5' ESTs from 14 species by 2003. Additionally, the Sanger Centre and Edinburgh Univ. will complete 80,000 ESTs from 7 species. Through these combined efforts, we anticipate the identification of >80,000 new nematode genes. At the GSC, approximately 35,000 ESTs have been generated to date including sequences from Ancylostoma caninum, Heterodera glycines, Meloidogyne incognita and javanica, Parastrongyloides trichosuri, Pristionchus pacificus, Strongyloides stercoralis and ratti, Trichinella spiralis, and Zeldia punctata . We will report on our progress in sequence analysis, including the creation of the NemaGene gene index for each species by EST clustering and consensus sequence generation, identification of common and rare transcripts, and identification of genes with orthologues in C. elegans and other nematodes. All sequences are publicly available at www.ncbi.nlm.nih.gov/dbEST. NemaGene sequences and project details are available at WWW.NEMATODE.NET. We would like to thank collaborators who have provided materials and ideas for this project including Prema Arasu, David Bird, Rick Davis, Warwick Grant, John Hawdon, Doug Jasmer, Andrew Kloek, Thomas Nutman, Charlie Opperman, Alan Scott, Ralf Sommer, and Mark Viney. This work is funded by NIH-AI-46593, NSF-0077503, and a Merck / Helen Hay Whitney Foundation fellowship.
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[
International C. elegans Meeting,
2001]
Bacillus thuringiensis (Bt) delta-endotoxins, a family of crystal (Cry) proteins, are widely used as insecticides in agriculture. Recently, we described two Bt toxins (Cry5B and Cry6A) that are effective at killing C. elegans . Here, we test whether Bt toxins are generally nematicidal. We analyzed the toxicities of eight Cry proteins (Cry 5A, 5B, 6A, 6B, 12A, 13A, 14A, and 21A) on five free-living nematodes species ( C. elegans, Pristionchus pacificus, Panagrellus redivivus, Acrobeloides sp. , and Distolabrellus veechi ). The results of health assays, morphology, and brood size indicated that four out of the eight proteins were nematicidal and were able to kill multiple nematodes species. One nematode species was resistant to all toxins. Bt toxin was toxic to all four of the other free-living nematodes tested, including one species closely related to plant-parasitic nematodes (PPNs). Structure function studies indicate it can be trimmed to a small 42 kD active core that retains full toxicity. Given its effects on many free-living nematodes and that PPNs are able to exclude large proteins from their diet, this small toxin holds promise for controlling plant-parasitic nematodes. To test this directly, we are transforming Arabidopsis plants and tomato hairy roots with the Bt toxin under control of three different promoters. Currently, successfully transformed plant lines are being isolated and our nematode infection assays are being perfected. We are growing Arabidopsis plants in modified Knop medium and are infecting them with active Meloidogyne incognita J2's under sterile conditions. We hope to soon assay whether expression of the toxin adversely affects infection rates and survival of M. incognita .
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[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2014]
Horizontal gene transfer (HGT) is the transmission of genes by means other than direct inheritance from an ancestor to the offspring. HGT is accepted as a biologically and evolutionary significant phenomenon in bacteria but generally overlooked in animals. However, HGTs of biological importance have been suggested as soon as 1998 in nematodes with the identification of functional cellulases of bacterial origin in cyst nematodes. These secreted cellulases degrade cellulose, the main component of the plant cell wall and play an important role in these plant parasites. Initial analysis of the genome of the root-knot nematode Meloidogyne incognita revealed that as much as 60 genes from six different families encode plant cell wall-degrading enzymes and show highest similarity to bacterial genes. A similar repertoire was found in the genome of M. hapla, indicating that these genes have been acquired at least in a common ancestor of the two species. Using systematic phylogenetic analyses, we have shown that the most likely hypothesis for the presence of these gene families, otherwise generally absent in animals, was acquisition from bacteria via HGT followed by duplications. A survey of the literature allowed us to identify at least 12 different reported cases of HGT event in plant parasitic nematodes. Besides degradation of the plant cell wall, the corresponding gene products play important roles in other aspects of the parasitism such as modulation of plant defense, establishment of a feeding structure or nutrient metabolism. Recently, we have systematically scanned the genomes of the root-knot nematodes to identify genes potentially acquired via HGT. Using a combination of homology-based and phylogeny-based approaches, we have shown that at least 3.3 % of protein-coding genes are of foreign origin and were probably acquired via HGT. Hence, not only do HGT appear to have played an important role in the emergence of plant parasitism in nematodes but they also significantly contributed to the composition of the gene set itself. Thus, far from being negligible HGT appear as major evolutionary events in animals too. Interestingly, recent reports indicate that this phenomenon is not limited to nematode but also present in other animal lineages. Bdelloid rotifers currently hold the record with more than 8% of protein-coding genes possibly acquired via HGT of non animal origin.
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[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2014]
Plant parasitic nematodes are a remarkably varied and adapted set of pathogens, and the order Tylenchida represents the most diverse and successful group. In addition to the Meloidogyne hapla genome we previously sequenced, we have recently completed the genomes of Pratylenchus coffeae, and Radopholus similis, and are in progress on 5 additional species: M. chitwoodi, Helicotylenchus multicinctus, Scutellonema bradys, Belonolaimus longicaudatus, and P. goodeyii. These species represent a diverse spectrum of migratory ecto- and endo-parasites. In combination with the complete M. hapla and M. incognita genomes and the recently completed Globodera pallida genome, these species provide a core to perform comparative genomic studies across the Tylenchida. The M. hapla genome contains 14,754 protein-coding genes, 5,800 fewer than the free living nematode, Caenorhabditis elegans. The genomes of P. coffeae and R. similis have approximately 6,500 and 9,000 gene models, respectively. Extensive annotation has revealed that these species do share many genes in common, but there are also genes that appear to be Meloidogyne specific. We are building on that observation to perform pan-order comparisons. We hypothesize that there may be a core Tylenchid genome, and acquiring these sequences provides solid data to identify it. We are examining the role of HGT, gene family expansion/contraction, and chromosomal organization in the progression from migratory to sedentary endo-parasitism. In combination with other recently completed genomes including Pristionchis pacificus, and numerous animal parasitic species, these data provide an important platform for comparative genomics across the phylum Nematoda.
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[
International Worm Meeting,
2013]
Dispersal is an important nematode behavior for survival. Upon crowding or food depletion, the free living bacteriovorus nematode Caenorhabditis elegans produces stress resistant dispersal larvae, known as dauer. Other nematodes also have dispersal larvae. In plant parasitic Meloidogyne spp., it is called J2 and in insect parasites (entomopathogenic nematodes, EPN), it is known as infective juveniles (IJs). Even though pheromones regulate entry into dispersal larvae in C. elegans and insect parasites, it is not known whether pheromones regulate dispersal. We hypothesized that pheromones may regulate dispersal behavior in C. elegans and in other nematodes. Liquid chromatography-mass spectrometry analysis of C. elegans dauer/dispersal supernatant with dispersal activity revealed four known ascarosides (ascr#2, ascr#3, ascr#8, icas#9). A synthetic pheromone blend at physiologically relevant concentrations dispersed C. elegans in the presence of food and also caused dispersion in insect parasite (Steinernema feltiae) and plant parasitic (Meloidogyne spp). Assay guided fractionation revealed structural analogs as major active components of the S. feltiae (ascr#9) and C. elegans (ascr#2) dispersal blends. Further analysis revealed that all Steinernema spp. and Heterorhabditis spp. infected insect host cadavers share a common pheromone, ascr#9, suggesting one species can recognize another's blend. Pheromones are fundamentally important for nematode communication across diverse habitats, and thus may provide sustainable means for control of parasitic nematodes.
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[
International Worm Meeting,
2013]
Root-knot nematodes (Meloidogyne spp.; RKN) are sedentary endoparasites that infect many plants and cause substantial crop loses worldwide. L2 hatch from eggs in the soil and must locate and invade host roots to complete their life cycle. They are attracted to the root zone of elongation, but what attracts them to this region is unknown. After invasion, nematodes migrate to the vascular cylinder where they cause differentiation of host cells into multinucleated giant cells that serve as the source of nutrients and cause the formation of a characteristic "root-knot." We have developed an assay system using a gel of Pluronic F-127 and demonstrated L2 attraction to the elongation zone as well as to volatile and soluble components of root tip exudates. Our goal is to analyze these extracts to identify chemicals responsible for modifying nematode behavior. We are also pursuing a genetic/genomic approach using the northern RKN Meloidogyne hapla. The genome of this species has been sequenced and annotated, and a set of F2 lines, which, due to the unusual reproductive mode, resemble recombinant inbred lines, has been produced and used to develop a sequence-anchored molecular map. These lines are maintained as cultures on tomato plants and have been used to assess behaviors including clumping and host attraction. The two parental strains differ in relative attraction to Arabidopsis and to the model legume Medicago truncatula. We assessed attraction of each of 90 F2 lines to each host and preliminary analysis has identified QTL contributing significantly to each phenotype, but different QTL were identified for the two host species suggesting that different nematode genes modulate attraction to each host. The clumping and aggregation behavior of RKN as well as their accumulation near root tips, suggests that chemical communication occurs among L2. We have identified ascarosides previously identified from C. elegans in exudates from RKN L2. Interestingly, several peaks were seen that had elution properties suggesting that they could be ascarosides not present in C. elegans. Potential roles of these ascarosides in RKN communication are under investigation.