Progress 10/01/02 to 09/30/07
Outputs
OUTPUTS: Parasitic nematodes are the largest source of uncontrollable biotic stress on plants and cause as much as $US100 billion in annual crop loss. The majority of the damage is inflicted by the tylenchid nematodes, especially members of the genus Meloidogyne (root-knot nematode: RKN). A main objective of this project was to understand how the parasite redirects normal plant functions, including initiation of developmental pathways and regulation of plant cell fate. From prior work, we have glimpsed how the regulatory networks are assembled, and how they function, but the real goal is to take this thinking to the next level: what are the regulators regulating? Identifying and understanding these biochemical effectors has been a major objective of this proposal. The specific objectives were: 1. Assemble a set of at least 4,000 genes expressed in tomato root and use this unigene set to print microarrays; 2. Challenge the root unigene microarrays with nematode infected tomato tissues
from various backgrounds such as presence/absence of R-loci; 3. Use computational tools to group genes by common expression profiles and assign genes to biochemical pathways; 4. Use antisense technology in tomato hairy roots to silence genes hypothesized to play key roles in regulating root biochemical networks; 5. Use in situ methods to attribute biochemical processes at cellular resolution. We have successfully completed each of these objectives. We chose to study the interaction between RKN and tomato (Solanum lycopersicum), partly because of the importance of tomato as a crop, but also because effective resistance exists to certain RKN species, including that conditioned by the tomato Mi gene. 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, M. incognita, and the Mi-resistant species, M. 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 Meloidogyne 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.
PARTICIPANTS: Dr. J. E. Schaff. Research Associate. Ms. P. Sumanasinghe. Research Associate.
TARGET AUDIENCES: "Mechanisms and evolution of symbiosis: Parasites and mutualists invade plants via a shared response pathway." Nematode-Bacteria Symbiosis Workshop, University of Arizona, Tucson, AZ April 21-23, 2007. "What does a worm want with 20,000 genes?" Center for Integrated Legume Research annual meeting, Kingscliffe, NSW, Australia, April 11-14, 2007. "Mechanisms and evolution of symbiosis: Parasites and mutualists invade plants via a shared response pathway." Dept. Crop Sciences, University of Illinois, Urbana, IL, March 28, 2007. "The M. hapla genome project" and "Mechanisms and evolution of symbiosis: Parasites and mutualists invade plants via a shared response pathway." Nematology workshop, Indian Agricultural Research Institute, New Delhi, India, March 2-9, 2007. "The Genomic Sciences graduate program at NCSU." Seoul National University, Seoul, Korea, January 3-8, 2007. "Mechanisms and evolution of symbiosis: Parasites and mutualists invade plants via a shared
response pathway." Dept. Biology Clemson University, Clemson SC, November 9, 2006. "What does a worm want with 20,000 genes? First answers from the Meloidogyne hapla genome sequencing project." Dept. Nematology, UC-Davis, CA, November 3, 2006. Discussion Leader, joint Cold Spring Harbor Laboratory/Welcome Trust conference on Genomic Perspectives to Host Pathogen Interactions, Hinxton, UK, September 7-10, 2006. "Comparative genomics among plant-parasitic nematodes." American Phytopathological Society annual meeting, Quebec City, Canada, July 29-August 2, 2006. "Mechanisms and evolution of symbiosis: Parasites and mutualists invade plants via a shared response pathway." Center for Integrated Legume Management, University of Western "Plants respond to rhizobia and root-knot nematodes via a common pathway." Third International Conference for Legume Genomics and Genetics, Brisbane, Australia, April 9-13, 2006. "Mechanisms and evolution of symbiosis: Parasites and mutualists invade plants
via a shared response pathway." AgResearch Grasslands, Palmerston North, NZ, April 6, 2006. "Mechanisms and evolution of symbiosis: Parasites and mutualists invade plants via a shared response pathway." Department of Energy Joint Genome Institute, Walnut Creek, CA, March 22, 2006. "Mechanisms and evolution of symbiosis: Parasites and mutualists invade plants via a shared response pathway." Department of Molecular and Structural Biochemistry, NCSU, January 26, 2006. "Plants respond to rhizobacterial Nod Factor and root-knot nematode Nem Factor via a shared response pathway" International Society for Molecular Plant-Microbe Interactions meeting, Cancun, Mexico, December 14-19, 2005. "Plants respond to rhizobacterial Nod Factor and root-knot nematode Nem Factor via a shared response pathway." Department of Plant Pathology & Microbiology, Texas A&M University, November 9, 2005. "Plants respond to mutualistic rhizobia and parasitic root-knot nematodes via a common pathway." Department of
Botany, University of British Columbia, Vancouver, Canada, November 1, 2005. "Confocal imaging to test genomic hypotheses." Imaging: Integrating Across Disciplines Symposium, NCSU, September 16, 2005.
Impacts
This research already has begun to reveal unexpected molecular events responsible for the nematode-plant interaction, and will serve as the knowledge platform upon which new control strategies will be developed. In particular, it has identified new components of the host resistance pathway, suggesting an added and unexpected level of complexity that may be responsible for past failures to effectively move resistance traits across species boundaries. It also has revealed overlapping pathways in the host's response to diverse rhizosphere organisms, and better understanding these will be important in modifying the rhizosphere, either via farming practice (e.g., organic vs traditional) or plant transgenesis.
Publications
- Schaff, J. E., Scholl, E. H., Nielsen, D. M., Smith, C. P. and D. McK. Bird. 2007. Comprehensive transcriptome profiling in tomato reveals a role for glycosyltransferase in Mi-mediated nematode resistance. Plant Physiology, 144: 1079-1092.
- Bird, D. McK., Weerasinghe, R. R., Allen, N. S., Schaff, J. E. and D. P. Lohar. 2006. Legume symbiotic signal transduction pathways are co-opted for parasitism by root-knot nematodes. Biology of Plant-Microbe Interactions Vol. 5, pp 648-654. IS-MPMI Press, St. Paul, MN.
- Elling, A. E., Mitreva, M., Recknor, J., Gai, X., Martin, J., Maier, T. R., McDermott, J. P., Hewezi, T., Bird, D. McK., Davis, E. L., Hussey, R. S., Nettleton, D. S., McCarter, J. P., and T. J. Baum. 2007. Divergent evolution of arrested development in the dauer stage of Caenorhabditis elegans and the infective stage of Heterodera glycines. Genome Biology, 8(10): 211.1-211.19.
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Progress 10/01/05 to 09/30/06
Outputs
Parasitic nematodes are the largest source of essentially uncontrollable biotic stress on plants and cause as much as $US100 billion in annual crop loss. They are cosmopolitan pests that impact most crops. The majority of the damage is inflicted by the tylenchid nematodes, especially members of the genus Meloidogyne (root-knot nematode: RKN). RKN induce stereotypical giant cells at the heart of a gall in the root vasculature. I had previously hypothesized that giant cells are a type of induced meristem with similarity to rhizobial nodules, and recent work has focussed on characterizing early events in the RKN-plant interaction, with a particular focus on exploring further parallels between mutualistic symbionts (such as rhizobia) and the parasitic symbiont, RKN. The earliest described events in the rhizobial-plant interaction occur on the root surface and involve rapid cytoskeletal reorganization in root-hair cells. To dynamically monitor the cytoskeleton during
nematode and rhizobial invasion, I developed composite Lotus plants transgenic for GFP-fusions of the microtubule associated protein, MAP-4, and the actin binding protein, Talin. Gfp::MAP4, gfp::Fim ABD2 and gfp::Talin. Collectively, my findings suggested that: 1) RKN produce a diffusible signaling molecule we named NemF, 2) RKN and rhizobia elicit remarkably similar responses during their initial encounters with the plant, 3) the primary Nod factor (NF) receptors, NFR1 and NFR5, play a role in perception of NemF. To undertake a genetic analysis of NemF perception I performed an analysis of the influence of multiple alleles of the Lotus nfr1, nfr5, symRK and nin2 genes on perception of RKN using a gall-rating scheme which directly measures parasitic ability. These experiments confirm that mutations in genes encoding what are presumed to be the primary NF receptors (NRF1 and NFR5) also cause a reduction in the number of root galls induced by RKN. The tested alleles of nfr1 were all
statistically equivalent and had more effect than alleles of nfr5. Mutation in the second gene in the NF perception pathway (symRK) had a greater influence on gall formation than either of the primary receptors. Although I had previously shown a role for symRK, its influence was not as strong as current results indicate. This experiment also revealed an important role for in gall formation for the nin2 gene, which encodes a presumed transcription factor downstream in the NF perception pathway. Intriguingly, although their response to NF is altered, nin2 plants show the same response to NemF as do wild type plants. Further, in an nfr1-2 genetic background, actin and microtubule reorganization is delayed compared to wild-type cells. Collectively, these results point to the hypothesis that the cellular machinery for the initial perception of NF also is utilized for the perception of NemF, but the transduction of that signal via Ca++ spiking is bypassed for NemF perception, even though
later transcriptional responses, involving nin2 and genes such as ENOD40, PHAN, KNOX and ccs-52, is common to NF and NemF perception. Understanding how and where these pathways diverge and converge is a goal of my future research.
Impacts
This research already has begun to reveal unexpected molecular events responsible for the nematode-plant interaction, and will serve as the knowledge platform upon which new control strategies will be developed. It also has revealed overlapping pathways in the host's response to diverse rhizosphere organisms, and better understanding these will be important in modifying the rhizosphere, either via farming practice (e.g., organic vs traditional) or plant transgenesis.
Publications
- Snyder, D. W., Opperman, C. H. and Bird, D. McK. 2006. A method for generating Meloidogyne incognita males. Journal of Nematology, 38: 192-194.
- Waterman, J. T., Bird, D. McK. and Opperman, C. H. 2006. A rapid method for isolating Pasteuria penetrans endospores, Journal of Nematology, 38: 165-167.
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Progress 10/01/04 to 09/30/05
Outputs
Parasitic nematodes are the largest source of essentially uncontrollable biotic stress on plants and cause as much as $US100 billion in annual crop loss. They are cosmopolitan pests that impact most crops. The majority of the damage is inflicted by the tylenchid nematodes, especially members of the genus Meloidogyne (root-knot nematode: RKN). RKN induce stereotypical giant cells at the heart of a gall in the root vasculature. I had previously hypothesized that giant cells are a type of induced meristem with similarity to rhizobial nodules, and recent work has focussed on characterizing early events in the RKN-plant interaction, with a particular focus on exploring further parallels between mutualistic symbionts (such as rhizobia) and the parasitic symbiont, RKN. The earliest described events in the rhizobial-plant interaction occur on the root surface and involve rapid cytoskeletal reorganization in root-hair cells. To dynamically monitor the cytoskeleton during
nematode and rhizobial invasion, I developed composite Lotus plants transgenic for GFP-fusions of the microtubule associated protein, MAP-4, and the actin binding protein, Talin. Gfp::MAP4, gfp::Fim ABD2 and gfp::Talin. Collectively, my findings suggested that: 1) RKN produce a diffusible signaling molecule we named NemF, 2) RKN and rhizobia elicit remarkably similar responses during their initial encounters with the plant, 3) the primary Nod factor (NF) receptors, NFR1 and NFR5, play a role in perception of NemF. To undertake a genetic analysis of NemF perception I performed an analysis of the influence of multiple alleles of the Lotus nfr1, nfr5, symRK and nin2 genes on perception of RKN using a gall-rating scheme which directly measures parasitic ability. These experiments confirm that mutations in genes encoding what are presumed to be the primary NF receptors (NRF1 and NFR5) also cause a reduction in the number of root galls induced by RKN. The tested alleles of nfr1 were all
statistically equivalent and had more effect than alleles of nfr5. Mutation in the second gene in the NF perception pathway (symRK) had a greater influence on gall formation than either of the PRIMARY receptors. Although I had previously shown a role for symRK, its influence was not as strong as current results indicate. This experiment also revealed an important role for in gall formation for the nin2 gene, which encodes a presumed transcription factor downstream in the NF perception pathway. Intriguingly, although their response to NF is altered, nin2 plants show the same response to NemF as do wild type plants. Further, in an nfr1-2 genetic background, actin and microtubule reorganization is delayed compared to wild-type cells. Collectively, these results point to the hypothesis that the cellular machinery for the initial perception of NF also is utilized for the perception of NemF, but the transduction of that signal via Ca++ spiking is bypassed for NemF perception, even though
later transcriptional responses, involving nin2 and genes such as ENOD40, PHAN, KNOX and ccs-52, is common to NF and NemF perception. Understanding how and where these pathways diverge and converge is a goal of my future research.
Impacts
This research already has begun to reveal unexpected molecular events responsible for the nematode-plant interaction, and will serve as the knowledge platform upon which new control strategies will be developed. It also has revealed overlapping pathways in the host's response to diverse rhizosphere organisms, and better understanding these will be important in modifying the rhizosphere, either via farming practice (e.g., organic vs traditional) or plant transgenesis.
Publications
- Bird, D. McK., Blaxter, M. L., McCarter, J. P., Mitreva, M., Sternberg, P. W., and W. K.Thomas. 2005. A white paper on nematode comparative genomics. Journal of Nematology, 37: in press.
- McCarter, J. P., Bird, D. McK., and M. Mitreva. 2005. Nematode gene sequences: Update for December 2005. Journal of Nematology, 37: in press.
- Vanholme, B., Mitreva, M., van Criekinge, W., Logghe, M., Bird, D. McK., McCarter, J. P. and G. Gheysen. 2005. Detection of putative secreted proteins in the plant parasitic nematode Heterodera schachtii. Parasitology Research, in press.
- Charles, L., Carbone, I., Davies, K. G., Bird, D. McK., Burke, M., Kerry, B. R. and C. H. Opperman. 2005. Phylogenetic analysis of Pasteuria penetrans using multiple genetic loci. Journal of Bacteriology, 187:5700-5708.
- Mitreva, M., Blaxter, M. L., Bird, D. McK. and J. P. McCarter. 2005. Comparative Genomics in Nematodes. Trends in Genetics, 21:573-581.
- Scholl, E. H. and D. McK. Bird. 2005. Resolving tylenchid evolutionary relationships through multiple gene analysis. Molecular Phylogenetics and Evolution, 36:536-545.
- Bird, D. McK. 2005. Model systems in agriculture: Lessons from a worm. Annals of Applied Biology, 146:147-154.
- Weerasinghe, R. R., Bird, D. McK. and N. S. Allen. 2005. Root-knot nematodes and bacterial Nod factors elicit common signal transduction events in Lotus japonicus root hair cells. Proceedings of the National Academy of Sciences (USA), 102:3147-3152.
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Progress 10/01/03 to 09/30/04
Outputs
Our approach addresses basic aspects of plant biology and the interactions between plants and other organisms, particularly nematodes and bacteria. Importantly, because it can be experimentally manipulated, the nematode can be exploited as a tool to address fundamental questions of plant development, physiology and biochemistry that are otherwise difficult to approach. The biology of root-knot nematode implies that it interacts in a fundamental way with its host plant, and its broad host-range implies that the complex changes the parasite elaborates in its host must involve some universal aspect of plant biology. We have selected 3 host plants: tomato (major crop plant with extensive nematode-plant biology and moderate genomic resources); Medicago truncatula (crop legume with some nematode-plant biology and extensive genomic resources); Lotus japonicus (model legume with no nematode-plant biology, but extensive genomic resources). A first step we established Lotus as
a robust model for studying nematode-plant interactions. We have screened a number of Lotus mutants, and found that one (har-1) has a striking nematode phenotype. This is significant, because har-1 encodes a transmembrane receptor kinase in the CLAVATA family and may be directly involved in perception of a nematode signal. To further understand nematode signaling we have used confocal microscopy to examine plant cytoskeletal responses to nematodes. Initial findings are consistent with a model in which root-knot nematode produces a diffusible signal with functional equivalence to rhizobial nod factor. To gain insight into more global changes in plant responses to invading nematodes, we have initiated a microarray approach to directly and simultaneously examine thousands of root genes. Central to array experiments is an integrated method for managing array data in accord with the MIAME standards, and we have built a database based on the BioArray Software Environment. This platform is
open-source, and integrates with our MySQL tools. Also essential are computational tools for array analysis, and we have established an experimental design based on interwoven loops, which can be analyzed using a Mixed Model approach.
Impacts
This research already has begun to reveal unexpected molecular events responsible for the nematode-plant interaction, and will serve as the knowledge platform upon which new control strategies will be developed. It also has revealed overlapping pathways in the host's response to diverse rhizosphere organisms, and better understanding these will be important in modifying the rhizosphere, either via farming practice (e.g., organic vs traditional) or plant transgenesis.
Publications
- Bird, D.McK. 2004. High Society (of Nematologists): Report of the forty-third annual meeting of the Society of Nematologists, Estes Park, CO, August 7-11, 2004. Genome Biology 5: 353.1-353.3.
- Mitreva, M.D., Elling, A.A., Dante, M., Kloek, A.P., Kalyanaraman, A., Aluru, S., Clifton, S.W., Bird, D.McK., Baum, T.J. and McCarter, J.P. 2004. A survey of SL1-spliced transcripts from the root-lesion nematode Pratylenchus penetrans. Molecular and General Genomics 272: 138-148.
- Bird, D.McK. 2004. Signaling between nematodes and plants. Current Opinion in Plant Biology 7: 372-376.
- Lohar, D.P., Schaff, J.E., Laskey, J.G., Kieber, J.J., Bilyeu, K.D. and Bird, D.McK. 2004. Cytokinins play opposite roles in lateral root formation, and nematode and rhizobial symbioses. The Plant Journal, 38: 203-214.
- Bird, D.McK., Weerasinghe, R.R., Allen, N.S., Schaff, J.E. and Lohar, D.P. 2004. Lotus japonicus and Medicago truncatula as platforms to understand primary signaling events with root-knot nematode. Legumes for the Benefit of Agriculture, Nutrition and the Environment. pp 117-119. AEP Press, Dijon, France.
- Scholl, E.H., Thorne, J.L., McCarter, J.P. and Bird, D.McK. 2004. Horizontal gene transfer in Meloidogyne: A computational approach. Journal of Nematology 36: 344-345.
- Bird, D.McK., Weerasinghe, R.R., Allen, N.S. and Lohar, D.P. 2004. The first 24 hours: Primary signaling events between root-knot nematode and its host. Journal of Nematology 36: 306-307.
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Progress 10/01/02 to 09/30/03
Outputs
Our approach addresses basic aspects of plant biology and the interactions between plants and other organisms, particularly nematodes and bacteria. Importantly, because it can be experimentally manipulated, the nematode can be exploited as a tool to address fundamental questions of plant development, physiology and biochemistry that are otherwise difficult to approach. The biology of root-knot nematode implies that it interacts in a fundamental way with its host plant, and its broad host-range implies that the complex changes the parasite elaborates in its host must involve some universal aspect of plant biology. We have initiated a microarray approach to directly and simultaneously examine thousands of root genes which might respond to invading nematodes. We have selected 3 host plants: tomato (major crop plant with extensive nematode-plant biology, and moderate genomic resources); Medicago truncatula (minor crop legume, with some nematode-plant biology, and
extensive genomic resources); Lotus japonicus (model legume, with no nematode-plant biology, but extensive genomic resources). A first step has been to establish Lotus as a robust model for studying nematode-plant interactions, and indeed, we have found this species to be superior even to tomato. We have screened a number of Lotus mutants, and found that one (har-1) has a striking nematode phenotype. An important future direction will be to incorporate plant mutants into our microarray screens. Central to array experiments is an integrated method for managing array data in accord with the MIAME standards, and we have built a database based on the BioArray Software Environment. This platform is open-source, and integrates with our MySQL tools. Also essential are computational tools for array analysis, and we have established an experimental design based on interwoven loops, which can be analyzed using a Mixed Model approach.
Impacts
This research already has begun to reveal unexpected molecular events responsible for the nematode-plant interaction, and will serve as the knowledge platform upon which new control strategies will be developed. It also has revealed overlapping pathways in the host's response to diverse rhizosphere organisms, and better understanding these will be important in modifying the rhizosphere, either via farming practice (e.g., organic vs traditional) or plant transgenesis.
Publications
- McCarter, J.P., Mitreva, M.D., Martin, J., Dante, M., Wylie, T., Rao, U., Pape, D., Bowers, Y., Theising, B., Murphy, C., Kloek, A.P., Chiapelli, B., Clifton, S.W., Bird, D.McK. and Waterston, R. 2003. Analysis and functional classification of transcripts from the root-knot nematode Meloidogyne incognita. Genome Biol., 4: R26.1-R26.19.
- Hirsch, A.M., Bauer, W.D., Bird, D.McK., Cullimore, J., Tyler, B. and Yoder, J.I. 2003. Molecular signals and receptors--controlling rhizosphere interactions between plants and other organisms. Ecology 84: 858-868.
- Bird, D.McK., J. Schaff, Scholl, E.S. and Lohar, D.H. 2003. Bacterial pathogens and symbionts of nematodes, and their role in the evolution of parasitism. Phytopathology 93: S108.
- Schaff, J.,E. Scholl E.S. and Bird, D.McK. 2003. Dissecting host responses to root-knot nematode infection. Phytopathology 93: S76.
- Bird, D.McK., Opperman, C.H. and Davies, K.G. 2003. Interactions between bacteria and plant-parasitic nematodes: Now and then. Int. J. Parasitol. 33: 1269-1276.
- Bird, D.McK. and Kaloshian, I. 2003. Are roots special?: Nematodes have their say. Physiol. Molec. Plant Pathol. 62: 115-123.
- Scholl, E.H., Thorne, J.L., McCarter, J.P. and Bird, D.McK. 2003. Horizontally transferred genes in plant-parasitic nematodes: A high-throughput genomic approach. Genome Biol., 4: R39.1-R39.12.
- Lohar, D.P. and Bird, D.McK. 2003. Lotus japonicus: A new model to study root-parasitic nematodes Plant Cell Physiol., 44: in press.
- McCarter, J.P., Mitreva, M., Clifton, S.W., Bird, D.McK and Waterston, R. 2003. Nematode gene sequences: Update for December 2003. J. Nematol. 35: in press.
- Weerasinghe, R.R., Bird, D.McK. and Allen, N.S. 2003. A comparative study of the cytoskeleton during nematode infection and nodulation in Lotus japonicus. Molec. Biol. Cell, Abst., in press.
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