Progress 07/01/07 to 06/30/12
Outputs
OUTPUTS: My research program is aimed at understanding the molecular-level interaction between parasitic weeds and their hosts. Currently this work focuses on two specific areas, the sequencing of expressed genes in Egyptian broomrape (Phelipanche aegyptiaca, name recently changed from Orobanche aegyptiaca), and the characterization of host-to-parasite translocation of mRNA in dodder (Cuscuta pentagona). The P. aegyptiaca sequencing has been conducted as part of the NSF-funded Parasitic Plant Genome Project, which I have coordinated with colleagues from three other institutions. This work involved the generation of tissue from specific growth stages of the parasite, extraction of RNA, and synthesis of the cDNA required for sequencing. Because the project involved other institutions, it required a considerable amount of coordination, including monthly conference calls, annual project meetings, and participation in the NSF annual Plant Genome Awardees meeting in Arlington, VA. The project sequenced 41,000 megabases of cDNA from Egyptian broomrape, and an additional 83,000 megabases from related parasitic species witchweed (Striga) and Triphysaria, and the closely related non-parasite Lindenbergia philippensis. When assembled, these data yielded 555 megabases of sequences for the four species and represent the world's largest collection of parasitic plant expressed gene sequences. All sequences are available to the public on the project website (http://ppgp.huck.psu.edu/) and have been submitted to GenBank, the national repository of gene sequences. The project provided several opportunities for mentoring and training, and contributed to the training of three post-doctoral researchers. It also contributed to numerous publications and conference presentations. With respect to my research on RNA translocation, the work focused on messenger RNA (mRNA) mobility from host plants arabidopsis and tomato to the parasite lespedeza dodder (Cuscuta pentagona). We have characterized this phenomenon by combining studies of movement of individual mRNAs with a global approach to characterizing genome scale mRNA mobility using next-generation sequencing technology. This project supported three graduate students, a visiting international graduate student, several undergraduates, and summer research experiences for four high school students from the Roanoke Valley Governor's School. The project resulted in three publications, with several more in preparation, as well as numerous presentations at scientific conferences. PARTICIPANTS: Over the course of this project it has provided training opportunities for individuals at many different levels: James H. Westwood, Principle Investigator; designed and supervised research. Verlyn Stromberg and Hope Gruszewski, Technicians; conducted research and supervised students. Malay Das, Shanon Alford, and Monica Fernandez-Aparicio, Post-Doctoral Associates, conducted research on Phelipanche genomics. Rachid Mentag, Visiting Scholar (INRA, Morocco); worked on characterizing host response to Phelipanche. Beneeta Patel, Megan LeBlanc and Gunjune Kim, Graduate Students; conducted research on RNA mobility genomics analysis of mRNA trafficking, and nanobiology applications to macromolecule tracking. Rocio Pineda-Martos (University of Cordoba, Spain) and Guangda Liu, (University of Inner Mongolia, China), Visiting Graduate Students; worked on Phelipanche genomics and mobility of mitochondrial mRNA between dodder and hosts. Will Wadlington, Alex Paik, Christian Gauthier, Jessica Boden, Caitlyn Felkoski, Courtney Klotz, Erik Jeanes, and Bryan Whittington, Undergraduate students, worked on Phelipanche genomics and RNA mobility projects. Natalie Dilley, Alex Snodgrass, Troy von Beck, and Allie Greene, High School Students; worked on RNA mobility project. Stephen Smith, High School Teacher; worked on mobile RNA project. Collaborators: Michael Timko, University of Virginia, Collaborator on Parasitic Plant Genome Project. John Yoder, University of California, Davis, Collaborator on Parasitic Plant Genome Project. Claude dePamphilis, Penn State University, Collaborator on Parasitic Plant Genome Project. TARGET AUDIENCES: The target audience for training encompass students and researchers at all levels. During the reporting period the project has provided hands-on research training for 24 individuals (not counting PI from the list above). These include 12 women. All trainees were provided with direct experience in laboratory research, and were included in discussions of experimental design and analysis. They maintained laboratory notebooks and regularly presented their research in formal or informal settings. The target audience for research is the broader scientific community interested in parasitic plants and plant growth and development. This audience is reached through scholarly presentations at conferences and publications. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The Parasitic Plant Genome Project has changed the way research on parasitic plants is conducted. The public availability of abundant sequence information for parasites enables researchers anywhere in the world to easily obtain sequences for these organisms and conduct the type of genomics-driven science that is revolutionizing our understanding of crops and model plants. Another important output from our research has been the development of a method to genetically transform Phelipanche aegyptiaca, thereby enabling researchers to study function of any gene in a parasitic plant by inserting new copies of the gene or shutting down expression of existing genes. The project also has revealed insights into the evolution of parasitism in plants through characterization of gene gains or losses that are unique to the parasitic lineage. The research on mobile mRNAs is shifting our understanding of a potentially important mechanism by which plants coordinate development. Our data indicate that mRNA transfer between hosts and dodder includes thousands of different genes and is bidirectional in nature, opening up new possibilities for the function of mobile RNA in plants. These findings challenge the current understanding of cell-cell interactions and suggest that either mRNA trafficking between cells is more common than currently thought or that dodder has an unusual capacity for mRNA exchange with host cells. The phenomena of RNA-mediated gene silencing and micro-RNA control of gene expression suggest that translocation of mRNAs between species could be used to modify parasite growth via RNA signals transmitted from the host. The idea is that a host plant can be engineered to express a silencing version of an important parasite gene, the mRNA for this gene will translocate into the parasite and arrest its development. If successful, this approach could be used to produce new parasite-resistant crops.
Publications
- LeBlanc, M., Kim, G. and Westwood J.H. 2012. RNA trafficking in parasitic plant systems. Frontiers in Plant Science 3:203. http://www.frontiersin.org/Plant_Physiology/10.3389/fpls.2012.00203/a bstract
- Westwood, J. H., C. W. dePamphilis, M. Das, M. Fernandez-Aparicio, L. Honaas, M. P. Timko, E. Wafula, N. Wickett and J. I. Yoder. 2012. The parasitic plant genome project: new tools for understanding the biology of Orobanche and Striga. Weed Science 60:295-306. http://wssajournals.org/doi/abs/10.1614/WS-D-11-00113.1
- Tasker, A. and J. H. Westwood. 2012. The US witchweed eradication effort turns 50: A retrospective and look-ahead on parasitic weed management. Weed Science 60:267-268. http://wssajournals.org/doi/full/10.1614/WS-D-12-00003.1
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: My research program is aimed at understanding the molecular-level interaction between parasitic weeds and their hosts. Currently this work focuses on two specific areas, the sequencing of expressed genes in Egyptian broomrape (Phelipanche aegyptiaca, name recently changed from Orobanche aegytiaca), and the characterization of host-to-parasite translocation of mRNA. The P. aegyptiaca sequencing is being conducted as part of the NSF-funded Parasitic Plant Genome Project, which I am coordinating with colleagues from three other institutions. This work involves the generation of tissue from specific growth stages of the parasite, extraction of RNA, and synthesis of the cDNA required for sequencing. Because the project involves other institutions, it requires a considerable amount of coordination, including monthly conference calls, an annual project meeting, and participation in the NSF annual Plant Genome Awardees meeting in Arlington, VA. To date the project has sequenced 41,000 megabases of cDNA from Egyptian broomrape, and an additional 92,000 megabases from related parasitic species witchweed (Striga) and Triphysaria, and the closely related non-parasite Lindenbergia philippensis. This project represents the world's largest collection of parasitic plant gene sequences, with several additional sequencing runs remaining before the end of the project. All sequences are available to the public on the project website (http://ppgp.huck.psu.edu/) and have been submitted to GenBank, the national repository of gene sequences. The project provides several opportunities for mentoring and training, and this year contributed to the training of post-doctoral researchers Monica Fernandez-Aparicio. I was invited to speak about this project at the Plant and Animal Genome XIX Conference, the Weed Science Society of America annual conference, and the American Phytopathological Society meeting. I also presented this work at the 11th World Congress on Parasitic Plants, in Martina Franca, Italy. With respect to my research on RNA translocation, the work focuses on mobility from host plants arabidopsis and tomato to the parasite lespedeza dodder (Cuscuta pentagona). We are combining studies of movement of individual mRNAs with a global approach to characterizing genome scale RNA mobility using next-generation sequencing technology. This year the project supported two graduate students, Megan LeBlanc and Gunjune Kim, undergraduate student Erik Jeanes, and summer research experiences for two high school students, Alex Snodgrass and Troy Von Beck (both from the Roanoke Valley Governor's School). A visiting graduate student from China, Guangda Liu, has spent six months in my laboratory working on the subject of RNA mobility as a potential mechanism for horizontal gene transfer in plants. We also presented results from this project at the 11th World Congress on Parasitic Plants. PARTICIPANTS: This project provided training opportunities for individuals at many different levels: James H. Westwood, Principle Investigator; designed and supervised research. Verlyn Stromberg and Hope Gruszewski, Technicians; conducted research and supervised students. Monica Fernandez-Aparicio, Post-doctoral Associate, conducted research on Phelipanche genomics. Megan LeBlanc, Graduate student, RNA mobility and nanobiology applications to macromolecule tracking. Gunjune Kim, Graduate student, genomics analysis of mRNA trafficking. Guangda Liu, Visiting graduate student (China), worked on mobility of mitochondrial mRNA between dodder and hosts. Courtney Klotz and Erik Jeanes, Undergraduate students, worked on Phelipanche genomics and RNA mobility projects. Alex Snodgrass and Troy Von Beck, High school students, worked on RNA mobility project. Collaborators: Michael Timko, University of Virginia, Collaborator on Parasitic Plant Genome Project. John Yoder, University of California, Davis, Collaborator on Parasitic Plant Genome Project. Claude dePamphilis, Penn State University, Collaborator on Parasitic Plant Genome Project. TARGET AUDIENCES: The target audience for training encompass students and researchers at all levels. During the reporting period the project has provided hands-on research training for ten individuals (not counting PI from the list above). These include five women. All trainees were provided with direct experience in laboratory research, and were included in discussions of experimental design and analysis. They maintained laboratory notebooks and regularly presented their research in formal or informal settings. The target audience for research is the broader scientific community interested in parasitic plants and plant growth and development. This audience is reached through scholarly presentations at conferences and publications. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The Parasitic Plant Genome Project is changing the way research on parasitic plants is conducted. The public availability of abundant sequence information for parasites enables researchers anywhere in the world to easily obtain sequences for these organisms and conduct the type of genomics-driven science that is revolutionizing our understanding of crops and model plants. As more sequences emerge, it will further accelerate research on parasitic weeds and enhance to prospects for their control. Another important output from our research has been the development of a method to genetically transform Phelipanche aegyptiaca, thereby enabling researchers to study function of any gene in a parasitic plant by inserting new copies of the gene or shutting down expression of existing genes. The project has also begun to reveal insight into the evolution of parasitism in plants through characterization of gene gain or loss that is unique to the parasitic lineage. The research on mobile mRNAs is shifting our understanding of a potentially import mechanism by which plants coordinate development. Our data indicate that mRNA transfer between hosts and dodder includes potentially thousands of different genes and is bidirectional in nature, opening up new possibilities for the function of mobile RNA in plants. The phenomena of RNA-mediated gene silencing and micro-RNA control of gene expression further suggest that translocation of mRNAs between species could be used to modify parasite growth via RNA signals transmitted from the host. The idea is that a host plant can be engineered to express a silencing version of an important parasite gene, the mRNA for this gene will translocate into the parasite and arrest its development. If successful, this approach could be used to produce new parasite-resistant crops.
Publications
- Fernandez-Aparicio, M., D. Rubiales, P.C.G. Bandaranayake, J. I. Yoder and J. H. Westwood. 2011. Transformation and regeneration of the holoparasitic plant Phelipanche aegyptiaca. Plant Methods 7:36. http://www.plantmethods.com/content/7/1/36.
- Aly, R., N. Hamamouch, J. Abu-Nassar, S. Wolf, D. M. Joel, H. Eizenberg, E. Kaisler, C. Cramer, A. Gal-On and J. H. Westwood. 2011. Movement of protein and macromolecules between host plants and the parasitic weed Phelipanche aegyptiaca Pers. Plant Cell Reports 30:2233-2241. http://www.springerlink.com/content/5564134523m6n44p/.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: My research program is aimed at understanding the molecular-level interaction between parasitic weeds and their hosts. Currently this work focuses on two specific areas, the sequencing of expressed genes in Egyptian broomrape (Orobanche aegyptiaca), and the characterization of host-to-parasite translocation of mRNA. The O. aegyptiaca sequencing is being conducted as part of the NSF-funded Parasitic Plant Genome Project, which I am coordinating with colleagues from three other institutions. This work involves the generation of tissue from specific growth stages of the parasite, extraction of RNA, and synthesis of the cDNA required for sequencing. Because the project involves other institutions, it requires a considerable amount of coordination, including monthly conference calls, an annual project meeting, and participation in the NSF annual Plant Genome Awardees meeting in Arlington, VA. To date the project has sequenced 41,000 megabases of cDNA from Egyptian broomrape, and an additional 92,000 megabases from related parasitic species witchweed (Striga) and Triphysaria, and the closely related non-parasite Lindenbergia phillipensis. This project represents the world's largest collection of parasitic plant gene sequences, with several additional sequencing runs remaining before the end of the project. All sequences are available to the public on the project website (http://ppgp.huck.psu.edu/) and have been submitted to GenBank, the national repository of gene sequences. The project provides several opportunities for mentoring and training, and this year contributed to the training of post-doctoral researchers Shannon Alford and Monica Fernandez-Aparicio (the latter being supported primarily by an award from the Spanish government to work in my laboratory). A visiting graduate student from Spain, Rocio Pineda-Martos, has spent six months working on this project in my laboratory. Undergraduate students Jessica Boden and Courtney Klotz were also trained in my program this year. I was invited to speak about this project at Inner Mongolia University in China. With respect to my research on RNA translocation, the work focuses on mobility from host plants arabidopsis and tomato to the parasite lespedeza dodder (Cuscuta pentagona). This year we have taken a gene sequencing approach to expand our understanding of the types of mRNAs that move between plants and have also quantified mRNA amounts. The project supports two graduate students Megan McCullough and Gunjune Kim, as well as summer research experiences for high school teacher Steve Smith and high school student Alex Snodgrass (both from the Roanoke Valley Governor's School). We presented results from this work at the International Conference on Plant Vascular Biology 2010. PARTICIPANTS: This project provided training opportunities for individuals at many different levels: James H. Westwood, Principle Investigator; designed and supervised research. Verlyn Stromberg, Technician; conducted research and supervised students. Shannon Alford, Post-doctoral Associate; conducted research on Orobanche genomics. Monica Fernandez-Aparicio, Post-doctoral Associate; conducted research on Orobanche genomics. Megan McCullough, Graduate student; RNA mobility and quantum dot localization. Gunjune Kim, Graduate student; worked on a short-term research project on mRNA trafficking. Rocio Pineda-Martos, Visiting graduate student (Spain); worked on Orobanche genomics project. Jessica Boden, Undergraduate student; Worked on Orobanche genomics project. Courtney Klotz, Undergraduate student; worked on Orobanche genomics and RNA mobility projects. Steve Smith, High school teacher; Worked on RNA mobility project Alex Snodgrass, High school student; Worked on RNA mobility project. Collaborators: Michael Timko, University of Virginia, Collaborator on Parasitic Plant Genome Project. John Yoder, University of California, Davis, Collaborator on Parasitic Plant Genome Project. C. dePamphilis, Penn State University, Collaborator on Parasitic Plant Genome Project and mobile RNA project. Harro Bouwmeester, University of Wageningen, The Netherlands, Collaborator on implementing knowledge of parasite genomics to understand host-parasite signaling. TARGET AUDIENCES: The target audience for training encompass students and researchers at all levels. During the reporting period the project has provided hands-on research training for nine individuals (not counting PI and technician from the list above). These include six women. All trainees were provided with direct experience in laboratory research, and were included in discussions of experimental design and analysis. They maintained laboratory notebooks and regularly presented their research in formal or informal settings. The target audience for research is the broader scientific community interested in parasitic plants and plant growth and development. This audience is reached through scholarly presentations at conferences and publications. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The Parasitic Plant Genome Project is changing the way research on parasitic plants is conducted. The public availability of abundant sequence information for parasites enables researchers anywhere in the world to easily obtain sequences for these organisms and conduct the type of genomics-driven science that is revolutionizing our understanding of crops and model plants. As more sequences emerge, it will further accelerate research on parasitic weeds and enhance to prospects for their control. The research on mobile mRNA is shifting our understanding of a potentially import mechanism by which plants coordinate development. The phenomena of RNA-mediated gene silencing and micro-RNA control of gene expression are transforming our understanding of gene regulation, and our research on plant-plant mobile RNA adds a new dimension to these topics. Although the full implications of our research are not yet realized, the translocation of mRNAs between species will facilitate investigations of the role of mobile RNAs in plant biology, and represents a new hope for developing parasite-resistant crops. Other researchers are already seizing on this concept to explore the potential use of gene silencing signals transmitted from host to parasite for ability to modify expression of parasite genes. The idea is that a host plant can be engineered to express silencing version of an important parasite gene, the mRNA for this gene will translocate into the parasite and arrest its development.
Publications
- Westwood JH, Yoder JI, Timko MP, DePamphilis CW (2010) The evolution of parasitism in plants. Trends Plant Sci. 15:227-235.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: My research program is aimed at understanding the molecular-level interaction between parasitic weeds and their hosts. Currently this work focuses on two specific areas, the sequencing of expressed genes in Egyptian broomrape (Orobanche aegyptiaca), and the characterization of host-parasite translocation of mRNA. The O. aegyptiaca sequencing is being conducted as part of the NSF-funded Parasitic Plant Genome Project, which I am coordinating with colleagues from three other institutions. This work involves the generation of tissue from specific growth stages of the parasite, extraction of RNA, and synthesis of the cDNA required for sequencing. Because the project involves other institutions, it requires a considerable amount of coordination, including monthly conference calls, an annual project meeting, and participation in the NSF annual Plant Genome Awardees meeting in Arlington, VA. To date the project has sequenced nearly 200,000 megabases of cDNA from Egyptian broomrape, which has assembled into more than 43,000 genes or gene fragments. Similar numbers have been produced from related parasitic species witchweed (Striga) and Triphysaria, and this project now represents the world's largest collection of parasitic plant gene sequences. I gave a presentation on the parasitic plant genome project at the 10th World Congress on Parasitic Plants, in Kusadasi, Turkey. As president of the International Parasitic Plant Society, I also played a major role in organizing this conference, which attracted participants from 37 different countries and resulted in the publishing of an article about the meeting in New Phytologist. The project provides several opportunities for mentoring and training, and this year contributed to the training of post-doctoral researchers Malay Das, Shannon Alford, and Monica Fernandez-Aparicio (the latter being supported primarily by an award from the Spanish government to work in my laboratory). Undergraduate students Jessica Boden, Caitlyn Felkoski and highschool student Natalie Dilley, have also received training in my program this year. With respect to my research on RNA translocation, the work continues to focus on mobility from host (tomato) to parasite (lespedeza dodder, Cuscuta pentagona). Efforts aimed at characterizing the movement and fate of host mRNAs in the parasite received a boost from an NSF award that has supported graduate students Megan McCullough, Sy Traore, and Gunjune Kim. Research has focused on using nanotechnology (quantum dots) to localize mRNAs in host a parasite, but is shifting to include genomics and bioinformatic approaches as well. A manuscript from this work has been published in the journal Pest Management Science. This year I have been invited to give presentations on my research at Penn State University and the University of Wageningen, Wageningen, The Netherlands. PARTICIPANTS: This project provided training opportunities for individuals at many different levels: James H. Westwood, Principle Investigator, Designed and supervised research. Verlyn Stromberg, Technician, Conducted research and supervised students. Malay Das, Post-doctoral Associate, Conducted research on Orobanche genomics. Shannon Alford, Post-doctoral Associate, Conducted research on Orobanche genomics. Monica Fernandez-Aparicio, Post-doctoral Associate, Conducted research on Orobanche genomics. Beneeta Patel, Graduate Student, Conducted research on RNA translocation. Megan McCullough, Graduate Student, RNA mobility and quantum dot localization. Sy Traore, Graduate Student, Worked on a short-term research project on parasite transformation. Gunjune Kim, Graduate Student, Worked on a short-term research project on mRNA trafficking. Jessica Boden, Undergraduate student, Worked on Orobanche genomics project. Caitlyn Felkoski, Undergraduate student, Worked on Orobanche genomics project. Natalie Dilley high school student, Worked on Orobanche genomics project. Collaborators: John Jelesko, Virginia Tech, Provided technical expertise and equipment for RNA translocation work. Michael Timko, University of Virginia, Collaborator on Parasitic Plant Genome Project. John Yoder, University of California, Davis, Collaborator on Parasitic Plant Genome Project. Claude dePamphilis, Penn State University, Collaborator on Parasitic Plant Genome Project. Harro Bouwmeester, University of Wageningen, The Netherlands, Collaborator on implementing knowledge of parasite genomics to understand host-parasite signaling. TARGET AUDIENCES: The target audience for training encompass students and researchers at all levels. During the reporting period the project has provided hands-on research training for ten individuals (not counting PI and technician from the list above). These include seven women. All trainees were provided with direct experience in laboratory research, and were included in discussions of experimental design and analysis. They maintained laboratory notebooks and regularly presented their research in formal or informal settings. The target audience for research is the broader scientific community interested in parasitic plants and plant growth and development. This audience is reached through scholarly presentations at conferences and publications. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The Parasitic Plant Genome Project is changing the way research on parasitic plants is conducted. The public availability of abundant sequence information for parasites enables researchers anywhere in the world to easily obtain sequences for these organisms and conduct the type of genomics-driven science that is revolutionizing our understanding of crops and model plants. As more sequences emerge, it will further accelerate research on parasitic weeds and enhance to prospects for their control. The research on mobile mRNA is shifting our understanding of a potentially import mechanism by which plants coordinate development. The phenomena of RNA-mediated gene silencing and micro-RNA control of gene expression are transforming our understanding of gene regulation, and our research on plant-plant mobile RNA adds a new dimension to these topics. Although the full implications of our research are not yet realized, the translocation of mRNAs between species will facilitate investigations of the role of mobile RNAs in plant biology, and represents a new hope for developing parasite-resistant crops. Other researchers are already seizing on this concept to explore the potential use of gene silencing signals transmitted from host to parasite for ability to modify expression of parasite genes. The idea is that a host plant can be engineered to express silencing version of an important parasite gene, the mRNA for this gene will translocate into the parasite and arrest its development.
Publications
- Westwood, J.H., Roney, J.K. Khatibi, P.A. and Stromberg, V.K. 2009. RNA translocation between parasitic plants and their hosts. Pest Management Science. 65: 533-539.
- Davis, A. S., Hall, J. C., Jasieniuk, M., Locke, M. A., Luschei, E. C., Mortensen, D. A., Riechers, D. E., Smith, R. G., Sterling, T. M. and Westwood, J. H. 2009. Weed Science Research and Funding: A Call to Action. Weed Science 57:442-448.
- Stewart Jr, C. N., Tranel, P. J., Horvath, D. P., Anderson, J. V., Rieseberg, L. H., Westwood, J. H., Mallory-Smith, C. A., Zapiola, M. L. and Dlugosch, K. M. 2009. Evolution of Weediness and Invasiveness: Charting the Course for Weed Genomics. Weed Science 57: 451-462.
- Westwood, J.H., and Bouwmeester, H. 2009. Parasitic plants tap into the main stream. New Phytologist 184: 284-287.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Research outputs for this year derive from ongoing work aimed at understanding the molecular-level interaction between parasitic weeds and their hosts. Currently this work is focuses on two specific areas, the sequencing of Egyptian broomrape (Orobanche aegyptiaca) expressed genes, and the characterization of host-parasite translocation of mRNA. The O. aegyptiaca sequencing is being conducted as part of the NSF-funded Parasitic Plant Genome Project, which I am coordinating with colleagues from three other institutions. This work involves the generation of tissue from specific growth stages of the parasite, extraction of RNA, and synthesis of the cDNA required for sequencing. Because the project involves other institutions, it requires a considerable amount of coordination. This year I held the first annual project meeting, a two-day workshop that provided training and interaction opportunities for 13 people associated with the project. In addition, I represented the project at the NSF annual Plant Genome Awardees meeting in Arlington, VA. The project provides several opportunities for mentoring and training, and this year contributed to the training of post-doctoral researcher Malay Das, graduate student Megan McCullough, undergraduate students Jessica Boden and Caitlyn Felkoski, high school student Natalie Dilley, and visiting scientist Rachid Mentag (A Moroccan scientist supported on the Norman E. Borlaug International Agricultural Science and Technology Fellows Program). A presentation was given on this project at the Weed Science Society of America annual meeting and resulted in my being invited to contribute to a workshop and forthcoming manuscript on the topic of genomics for weed science. With respect to research on RNA translocation, the work continues to focus on mobility from host (tomato) to parasite (lespedeza dodder, Cuscuta pentagona). Current efforts are aimed at characterizing the movement and fate of host mRNAs in the parasite. The research involves the use of quantitative RT-PCR to determine specific numbers of mRNA copies in the parasite, and also employs transgenic plants expressing reporter gene fusions to mobile transcripts in order to assess mRNA translation. Recent work uses nanotechnology (quantum dots) to localize mRNAs in host a parasite. This research has contributed to the training of graduate students Beneeta Patel, Devdutta Deb, and undergraduate student Christian Gauthier. The work was invited for presentation at a conference on parasitic weed management in Italy, with a resulting manuscript accepted and in press in the journal Pest Management Science. The research was also presented to the National Institute of Agronomic Research, Morocco. PARTICIPANTS: This project provided training opportunities for individuals at many different levels (ranging from visiting scientist to high school student): James H. Westwood, Principle Investigator, Designed and supervised research. Verlyn Stromberg, Technician, Conducted research and supervised students. Malay Das, Post-doctoral Associate, Conducted research on Orobanche genomics. Beneeta Patel, Graduate Student, Conducted research on RNA translocation. Megan McCullough, Graduate Student, Worked on a short-term research project on Orobanche induced host gene expression. Devdutta Deb, Graduate Student, Worked on a short-term research project on localizing host-parasite mRNA. Rachid Mentag, Visiting Scholar (INRA, Morocco), Worked on a short-term research project on Orobanche induced host gene expression. Christian Gauthier, Undergraduate student, Conducted research on RNA translocation. Jessica Boden, Undergraduate student, Worked on Orobanche genomics project. Caitlyn Felkoski, Undergraduate student, Worked on Orobanche genomics project. Natalie Dilley high school student, Worked on Orobanche genomics project. Collaborators: John Jelesko, Virginia Tech, Provided technical expertise and equipment for RNA translocation work. Michael Timko, University of Virginia, Collaborator on Parasitic Plant Genome Project. John Yoder, University of California, Davis, Collaborator on Parasitic Plant Genome Project. Claude dePamphilis, Penn State University, Collaborator on Parasitic Plant Genome Project. TARGET AUDIENCES: Target audiences: The target audience for training encompass students and researchers at all levels. During the reporting period the project has provided hands-on research training for nine individuals (not counting PI and technician from the list above). These include one international visitor and six women. All trainees were provided with direct experience in laboratory research, and were included in discussions of experimental design and analysis. They maintained laboratory notebooks and regularly presented their research in formal or informal settings. The target audience for research is the broader scientific community interested in parasitic plants and plant growth and development. This audience is reached through scholarly presentations at conferences and publications. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The research on mobile mRNA is shifting our understanding of a potentially import mechanism by which plants coordinate development. The phenomena of RNA-mediated gene silencing and micro-RNA control of gene expression are transforming our understanding of gene regulation, and our research on plant-plant mobile RNA adds a new dimension to these topics. Although the full implications of our research are not yet realized, the translocation of mRNAs between species will facilitate investigations of the role of mobile RNAs in plant biology, and may represent a novel mechanism for developing parasite-resistant crops. Other researchers are already seizing on this concept to explore the potential use of gene silencing signals transmitted from host to parasite for ability to modify expression of parasite genes. The idea is that a host plant can be engineered to express silencing version of an important parasite gene, the mRNA for this gene will translocate into the parasite and arrest its development.
Publications
- Martin S., J. Westwood, M. N'Diaye, A. Goble, D. Mullins, R. Fell, B. Dembele, K. Gamby. 2008. Characterization of foliar-applied potash solution as a non-selective herbicide in Malian agriculture. Journal of Agriculture, Food, and Environmental Sciences 2(1) http://www.scientificjournals.org/journals2008/articles/1356.pdf.
- Owens, D.K., A.B. Alerding, K.C. Crosby, A. Bandara, J.H. Westwood and B.S.J. Winkel. 2008. Functional analysis of a predicted flavonol synthase gene family in Arabidopsis thaliana. Plant Physiology 147:1046-1061.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: The research and outputs for this year have focused on the recently discovered phenomenon of host-to-parasite transport of messenger RNA (mRNA). Following up on our initial work, we have now demonstrated mobility from host (tomato) to parasite (lespedeza dodder) of 21 mRNAs, including 20 mRNAs not previously reported to be phloem mobile. This more than doubles the number of long-distance translocated mRNAs known in plants. Experiments have also been conducted to further characterize the mRNA after it moves into the parasite with the specific objective of determining the fate of host mRNA in the parasite system. To this end, mRNA from one gene, gibberellic acid insensitive (GAI), has been quantified in the parasite system and the tools have been developed to determine whether the mRNA is converted into protein. Because the protein is likely to be present in quantities too low to detect, the GAI gene has been linked to a gene for luciferase, which produces light in the
presence of the appropriate substrate and can be detected easily with a sensitive camera. The GAI:Luciferase gene fusion, as well as an appropriate control fusion involving the non-translocated small subunit of Rubisco have been expressed in tobacco plants. To economize time and resources, the genes have been studied using the technique of transient gene expression, which has been adapted to serve the current experimental requirements. For transient expression, a solution of Agrobacterium containing the gene fusion is injected into specific leaves, which then produce the mRNA and protein over a period of a few days. Because the gene expression is localized, the system allows for detection of mRNA as it moves out of the treated leaf, and since the protein itself is not mobile, light will only be produced outside the treated leaf where protein has been encoded by mobile mRNA. This research has been presented at two international conferences. First, it was presented as an invited lecture
at Plant Vascular Biology 2007 in Taipei, Taiwan, and then at the 9th World Congress on Parasitic Plants in Charlottesville, Virginia. Aside from host-parasite transport of mRNA, the project aims to understand the genomic-level changes associated with parasitism in the family Orobanchaceae. In September 2007, the National Science Foundation (NSF) funded a project to sequence expressed genes (ESTs) in branched broomrape (Orobanche ramosa). This is part of a larger project that I am coordinating that involves collaboration with other leading parasitic plant researchers and will include two related parasitic plants. This will result in a molecular-evolutionary analysis of parasitic plants representing a continuum from partial to complete parasitism.
PARTICIPANTS: Individuals: James H. Westwood, Principle Investigator. Designed and supervised research. Verlyn Stromberg, Technician. Conducted research and supervised students. Beneeta Patel, Graduate Student. Conducted research on RNA translocation. Hannah Holsinger, Undergraduate student. Conducted research on RNA translocation. Patrick Bales, Undergraduate student. Conducted research on RNA translocation. Will Wadlington, Undergraduate student. Conducted research on RNA translocation. Collaborators: John Jelesko, Virginia Tech. Provided technical expertise and equipment for RNA translocation work. Michael Timko, University of Virginia. Collaborator on Parasitic Plant Genome Project. John Yoder, University of California, Davis. Collaborator on Parasitic Plant Genome Project. Claude dePamphilis, Penn State University. Collaborator on Parasitic Plant Genome Project.
TARGET AUDIENCES: The target audience for training is students at both the undergraduate and graduate levels. During the reporting period the project has provided hands-on research training for two economically disadvantaged students, one minority, and one student from a small college with little access to major research equipment. All students were provided with direct experience in laboratory research, and were included in discussions of experimental design and analysis. They maintained laboratory notebooks and regularly presented their research in small group discussions.
Impacts
The results of the research on mobile mRNA is shifting our understanding of a potentially import mechanism by which plants coordinate development. The movement of mRNAs over long distances in plants is assumed to have a role in plant development, but at present, neither the impact nor the mechanisms by which they act is understood for translocated mRNAs. Our research has provided new insight into the identities of mobile mRNAs, which has both confirmed and challenged the currently held ideas of how mobile mRNAs communicate information. In addition, the finding that mRNA moves from host to parasite has profound implications for control of parasitic weeds. This work has provided new impetus for efforts to use gene silencing (an RNA-mediated approach to shut off gene expression) as a novel mechanism for resistance to parasitic weeds. The idea is that a host plant can be engineered to express silencing version of an important parasite gene, the mRNA for this gene will
translocate into the parasite and arrest its development. The feasibility of this approach is now being tested in several laboratories throughout the world.
Publications
- Flagg, J. K., P. A. Khatibi and J. H. Westwood. 2007. Cross-species translocation of mRNA from host plants into the parasitic plant dodder. Plant Physiology. 143:1037-1043.
- Denev, I., B. Deneva, R. Buchvarova and J. Westwood. 2007. Use of T-DNA activation tag Arabidopsis mutants in studying formation of germination stimulants for broomrapes (Orobanche spp.). Biotechnology and Biotechnological Equipment 21(4):267-271.
- Joel D., H. Bar, A. Mayer, V. Verdoucq, G. Welbaum and J. Westwood. 2007. Characterization of a dioxygenase gene with a potential role in steps leading to germination of the root parasite Orobanche aegyptiaca. Pp. 296-305 in: S. Navie, S. Adkins and S. Ashmore (eds.) Seeds: Biology, Development and Ecology. CAB International. Wallingford UK.
- Whaley, C. M., H. P. Wilson and J. H. Westwood. 2007. A new mutation in plant ALS confers resistance to five classes of ALS-inhibiting herbicides. Weed Science. 55:83-90.
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