FUNCTION OF ROOT BORDER CELLS IN PLANT HEALTH: PIONEERS IN THE RHIZOSPHERE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0151683
• Project Start Date: Oct 1, 2009
• Project End Date: Sep 30, 2012
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF ARIZONA
888 N EUCLID AVE
TUCSON,AZ 85719-4824

Performing Department
Soil, Water & Environmental Science

Non Technical Summary
(1) Importance to agriculture and rural life of Arizona and the West: The process of border cell release provides a mechanism to equip the leading edge of the rhizosphere--the root tip region where growth, nutrient and water uptake, and initiation of microbial relationships occur--with chemicals that foster plant and/or human health. Soilborne diseases perennially remain among the primary disease problems in Arizona agriculture. The work especially will be of importance in controlled environment agriculture, which increasingly will be of commercial importance in arid lands. (2) Needs the project will fill: Border cells potentially can be used to deliver small doses of chemicals directly to the site where it is needed to control disease, without polluting vast acreages. The promoters of genes expressed in border cells can be linked to structural genes encoding products such as antibiotics, pesticides, and nutrient-solubilizing enzymes. The use of border cell specific promoters that are expressed only after the cells separate from the root minimizes problems inherent in overexpressing such products within plant tissue. (3) Ways the public welfare and scientific knowledge will be advanced: Potential applications include altering physical properties of the environment such as pH to facilitate nutrient uptake, "bioremediation" or detoxication of contaminated soils, and establishment or inhibition of specific microbial relationships. As more genes encoding metabolites that influence plant-microbe recognition are characterized, more opportunities to design border cells for specific environments will become available. Our primary model system has been the garden pea, P. sativum, with alfalfa used for comparative analysis. Pea remains among the principal food crops in the U.S. with significant increases in production in the Northwest in recent years (usda.gov), and alfalfa remains the nation's top hay crop, with 10-12 harvests annually in southwestern Arizona (epa.gov). The research applies generally to crop species including cereals, legumes, and cotton.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	1419	1050	100%

Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1419 - Leguminous vegetables, general/other;

Field Of Science
1050 - Developmental biology;

Keywords

root border cells

gene silencing

root cap secretome

plant-microbe interactions

rhizosphere

root rotting pathogens

soil fertility

plant extracellular matrix

Goals / Objectives
The long term goal of my program is to define the role of border cells in plant health and to explore their agronomic potential for a broad-based method to understand and control root-microbe interactions. The specific objectives are to (1) clone and characterize the structure and expression of the root cap gene(s) encoding specific classes of proteins; (2) use gene silencing under the control of promoters which drive expression specifically at the cap periphery to examine the hypothesis that their expression underlies secretome function. For each of the agronomically important interactions examined, we found specific behavioral changes suggesting a possible role for border cells in controlling the process. Our experimental approach to explore this hypothesis has been to create plants with specific changes in border cell properties, and use these as a tool to explore the effect of each root-microbe relationship. Translational integration with extension faculty in CEAC and commercial application(s) is a current focus.

Project Methods
PROCEDURE: The goal is to test the hypothesis that the root cap controls the capacity to control which microorganisms can become established on root systems, and to direct the type of relationships that can develop, by the release of border cells and an array of extracellular proteins. A. CLONING, CHARACTERIZATION, AND SILENCING OF THE SECRETOME ENCODING GENE(S). The identification of the genes encoding secretome proteins, and their sequence specific gene silencing will be carried out essentially according to procedures we have used routinely over the past decade (2,3,33-53). Candidate cDNA clones will be compared with DNA sequences deduced from peptides identified in the pea root tip secretome to identify the target genes. Those clones with exact matches will be used in gene silencing studies. To silence these genes, the region with unique sequence will be used in RNAi gene silencing (e.g. Agrawal et al 2003; Woo et al 2006). Promoters chosen to control the RNAi expression in this study will be the rcpme1 promoter (Wen et al 1999) that drives PME gene expression at the root cap periphery and the BRD13 promoter that drives gene expression in root border cells. Antibodies will also be raised against the unique cDNA region deduced peptides and in situ immunolocalization will be used to confirm the extracellular presence of the specific gene products and to examine their structure and distribution outside cells. B. MOLECULAR TOOLS AND TRANSFORMATION PROTOCOLS. Transgenic hairy roots, transformed with antisense mRNA or RNAi, is the system we find most effective in examining gene function with regard to roots and border cells. The experimental power of this system is reflected in the fact that within 2 weeks, a single root is amplified to several hundred roots representing genetic clones which can be amplified indefinitely for detailed quantitative analysis. After >20 years since the first plant transformations were accomplished, it has become clear that using transformation to study plant biology does not lend itself to high-throughput methods and that each biological question requires careful framing to achieve interpretable results. The proposed experiments focus on using the hairy root system to carry out a detailed analysis of each of the core proteins in response to each category of microorganisms, but whole plant expression will be a goal of future experiments and it is possible that such experiments could be initiated before the end of the funding period. For example, if initial results with a particular protein yield especially promising evidence for a critical role in preventing root tip infection, expression in whole plants would become a priority even before the remaining proteins are surveyed in hairy roots. The M. truncatula model is the plant of choice for whole plant studies, so M. truncatula hairy roots will be used in parallel with pea throughout the study. All the assays are described in published articles.

Progress 10/01/09 to 09/30/12

Outputs
OUTPUTS: New findings on border cell function in 'extracellular trapping' processes were reported at meetings, at invited seminars given at the University of Wisconsin and the Botanical Society of America annual meeting in Columbus, OH. Continued efforts to explore applications of the work are reflected in collaborative work with cotton extension specialist Ayman Moustafa and with local grower Thomas Pew, both resulting in peer reviewed publications in American Journal of Botany (in press) and Phytopathology (March 2013), as well as collaborators in France (Azzedine Driouich, Rouen) and Italy (Giacomo Pietromellara, Firenze). Service on graduate student committees in Plant Pathology, SWES, and ABE is ongoing, as well as advisory work with undergraduates. PARTICIPANTS: Xiong Zhongguo, Co PI, Gilberto Curlango-Rivera, postdoc, David Huskey, MS student, Hans VanEtten, John Kessler, collaborators. TARGET AUDIENCES: Research scientists and growers with interest in sustainable approaches to root disease control. PROJECT MODIFICATIONS; The dual focus is elaboration of underlying mechanisms of root-microbe interactions, and application of the information in disease control approaches. PARTICIPANTS: PARTICIPANTS: Xiong Zhongguo, Co PI, Gilberto Curlango-Rivera, postdoc, David Huskey, MS student, Hans VanEtten, John Kessler, collaborators. TARGET AUDIENCES: TARGET AUDIENCES: Research scientists and growers with interest in sustainable approaches to root disease control. PROJECT MODIFICATIONS: PROJECT MODIFICATIONS; The dual focus is elaboration of underlying mechanisms of root-microbe interactions, and application of the information in disease control approaches.

Impacts
The implementation of new disease control strategies based on understanding how disease develops, continues to be the outcome of work accomplished in 2012. Discovery of a large change in carbon delivery into the rhizosphere of genetically modified cotton (in press) is expected to stimulate renewed research into this biological problem. Biochemical parallels of border cells extracellular trapping in plant defense, with newly identified aspects of the mammalian immune system suggest that the research may have clinical applications as well.

Publications

• Hawes MC, Curlango-Rivera G, Xiong Z, Kessler JO. 2012. Roles of root border cells in plant defense of rhizosphere microbial populations by extracellular DNA 'trapping'. Marschner review, Plant and Soil 355: 1-16.
• Hawes MC, Xiong Z, Curlango-Rivera G, Kessler JO. 2012. Discoveries in DNA defense. International Innovation 5: 15-17.
• Pietramellara G, Ascher J, Ceccerini M, Laveccia A, Jennotte R, Hawes MC. 2012. Fate and ecological relevance of extracellular DNA. Eurosoil meeting, proceedings.
• Hawes MC. 2012. Root border cells and extracellular 'trapping': new insight into an old conundrum. Botanical Society of America, Proceedings.
• Negelsbach M, Curlango-Rivera G, Hawes MC 2012. Enzymatic reversal of extracellular trapping of bacteria by plant cells. Biochemistry symposium, University of Arizona, proceedings.
• Negelsbach M, Greenberg J, Xiong Z, Hawes MC. 2012. Extracellular trapping of bacteria by plant cells. Undergraduate Biology Research Symposium, University of Arizona, proceedings.
• Cochran AM, Trinh SA, Lim B, Curlango-Rivera G, Kessler JO, Xiong Z, Hawes MC. 2012. Extracellular trapping of Bacillus Spp. by plant cells: biofilms or border patrol. Undergraduate Biology Research Symposium, University of Arizona, proceedings.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: OUTPUTS: Activities included presentation of new results on border cell function at meetings, and efforts to develop cooperative work on application of these findings with Arizona growers. This included assembly of a team for submission of a grant to the SARE program. Continued efforts to define the underlying molecular mechanisms were reflected in continued funding of a grant (ExDNA in Plant Defense) from the National Science Foundation Symbiosis panel. Gilberto Curlango-Rivera completed his Ph.D. degree in 2011. Services included the transition of the Winter Term Fellowships in Plant Biology to Bio5 education and outreach, graduate and undergraduate research monitoring. I also served on graduate committees of students in SWES, plant pathology and ecology and evolutionary biology. Target audiences include growers, especially those with an interest in exploiting technologies to facilitate root system regulation of whole plant growth. Controlled environment agriculture, in which fungal pathogens are a continuing problem, are a special focus given recent discoveries in my lab. PARTICIPANTS: Research team, ExDNA in Plant Defense: Xiong Zhongguo, co PI, Gilberto Curlango-Rivera, postdoc, David Huskey, MS student, Hans VanEtten, John Kessler, collaborators. Research team, SARE grant (Title: Compost for sustainable agriculture: developing a system for monitoring effectiveness and safety of specific compost batches on specific crops): Tom Pew, co-PI Steve Husman, Director, Tucson Area Agriculture Centers John Kessler, Ph.D, Professor of Physics, Physics Dept, Bldg 81, Tucson, AZ 85721 Chieri Kubota, Ph.D. (controlled greenhouse experiments (http://cals.arizona.edu/abe/page/chieri-kubota) Mary Olsen, Ph.D.: controlled field experiments (http://extension.arizona.edu/contacts/mary-olsen) David Pew, Terra Boa raspberry growers, Santa Cruz, California John Swanson, Fiesta Growers, Tucson AZ Troy Hollar, web coordinator, "Interactive Growing" TARGET AUDIENCES: Target audiences include research scientists and growers with an interest in developing sustainable approaches to root disease control. With the phaseout of methyl bromide underway, the need for new methods is more critical than ever. PROJECT MODIFICATIONS: Our dual focus is to continue to elaborate the underlying mechanisms of root-microbe infection, and exploit the information to develop new approaches to disease control. PARTICIPANTS: Professor Z. Xiong, co PI Professor John Kessler, collaborator Dr. Gilberto Curlango-Rivera, Postdoc David Huskey, MS student Alicia Cochran, undergraduate research Sylvia Trinh, undergraduate research Brian Lim, undergraduate research Sarah O'Connor, undergraduate research Melissa Negelspach, undergraduate research Jonathan Greenberg, undergraduate research Tom Pew, grower, collaborator TARGET AUDIENCES: Colleagues presented information at meetings of the American Society for Microbiology, American Phytopathological Society, American society for Plant Biology, and others with an interest in mechanisms of disease control in plants. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The implementation of new control strategies that employ sustainable approaches to disease control (e.g. compost and other soil additives) in the context of our new understanding of root-microbe assications continue to be the outome of the work accomplished in 2011. A paper reporting progress will be submitted to Plant Disease, documenting an integration of the border cell system knowledge with application in root disease control.

Publications

• M. C. Hawes, G. Curlango-Rivera, F. Wen, G.J. White, H.D. VanEtten, Z. Xiong. 2011. Extracellular DNA: the tip of root defenses Plant Science 180: 741-745.
• G. Curlango-Rivera, M. C. Hawes. 2011. Plant Signalling and Behavior 6 (5): 1-2. Root tips moving through soil: an intrinsic vulnerability.
• Undergraduate Biology Research Program Conference 2011 (January 22, 2011) Extracellular DNA vs extracellular DNase: Trapping and release at the interface of root-microbe interactions. B. Lim, S. O'Connor, G. Curlango-Rivera, M.C. Hawes
• American Society for Microbiology, 50th Anniversary meeting, (April 16, 2011) Extracellular plant defense responses: Bacterial trapping in real time. G. Curlango-Rivera, A.M. Cochran, S.A. Trinh, J.O. Kessler, Z. Xiong, M.C. Hawes
• American Phytopathological Society 2011 (July 2011) Extracellular Trapping of Bacteria in Plant Defense Responses: Dynamics and Specificity. G.Curlango-Rivera, Z. Xiong, J.O. Kessler, M.C. Hawes
• American Society for Plant Biology 2011 (August 2011) Extracellular DNA: an overlooked component of eucaryotic defense. M. C. Hawes, G. Curlango-Rivera, Z. Xiong

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Activities included presentation of new results on border cell function at the Bio5 Frontiers meeting (February 2010), and cooperative work on application of these findings with Arizona growers. This included assembly of a team for submission of a grant to the SARE program; resubmission was encouraged. Continued efforts to define the underlying molecular mechanisms were reflected in funding of a grant (ExDNA in Plant Defense) submitted (2010) to the National Science Foundation Symbiosis panel. Services included the annual meeting of the American Society of Plant Biologists Committee on Public Affairs (March 2010), transition of the Winter Term Fellowships in Plant Biology to Bio5 education and outreach, graduate and undergraduate research monitoring. I also served on graduate committees of students in plant pathology and ecology and evolutionary biology. Target audiences include growers, especially those with an interest in exploiting technologies to facilitate root system regulation of whole plant growth. Controlled environment agriculture, in which fungal pathogens are a continuing problem, are a special focus given recent discoveries in my lab. PARTICIPANTS: Research team, ExDNA in Plant Defense: Xiong Zhongguo, co PI, Gilberto Curlango-Rivera, student, Gerard White, postdoc, Hans VanEtten, John Kessler, collaborators. Research team, SARE grant (Title: Compost for sustainable agriculture: developing a system for monitoring effectiveness and safety of specific compost batches on specific crops) submitted 2010, will resubmit: Tom Pew, co-PI Steve Husman, Director, Tucson Area Agriculture Centers John Kessler, Ph.D, Professor of Physics, Physics Dept, Bldg 81, Tucson, AZ 85721 Chieri Kubota, Ph.D. (controlled greenhouse experiments (http://cals.arizona.edu/abe/page/chieri-kubota) Mary Olsen, Ph.D.: controlled field experiments (http://extension.arizona.edu/contacts/mary-olsen) David Pew, Terra Boa raspberry growers, Santa Cruz, California John Swanson, Fiesta Growers, Tucson AZ Troy Hollar, web coordinator, "Interactive Growing" TARGET AUDIENCES: Target audiences include research scientists and growers with an interest in developing sustainable approaches to root disease control. With the phaseout of methyl bromide underway, the need for new methods is more critical than ever. PROJECT MODIFICATIONS: Our dual focus is to continue to elaborate the underlying mechanisms of root-microbe infection, and exploit the information to develop new approaches to disease control.

Impacts
The primary focus of last year's work (including a 6-month sabbatical and transition into SWES) was translational research to exploit research on root disease resistance in commercial application, especially in controlled environment agriculture. The recognition of a new level of innate immunity, and publication of research findings describing this new target for disease control, was the focus of implementation. This has now been reflected in two invited reviews (in press, February 2011), a funded NSF grant (January 2011 start date), and ongoing application-oriented research with local growers. The implementation of new control strategies that employ sustainable approaches to disease control (e.g. compost and other soil additives) in the context of our new understanding of root-microbe assications is the outome of the work accomplished in 2010.

Publications

• Hawes MC, Wen F, Curlango-Rivera G (2010) Extracellular DNA in eucaryotic defense. Invited lecture, Fifth Annual Frontiers in Immunobiology and Immunopathogenesis SymposiumProceedings.
• Curlango-Rivera R, Duclos DV, Ebolo JJ, Hawes MC (2010) Transient exposure of root tips to primary and secondary metabolites: Impact on root growth and production of border cells. Plant and Soil 306: 206-216.
• Curlango-Rivera G, White GJ, Cisneros LA, Kessler JO (2010) Extracellular trapping of bacteria in plant defense responses: dynamics and specificity. Proceedings, Fifth Annual Frontiers in Immunobiology and Immunopathogenesis Symposium, March 2010.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Activities for 2009 included fundamental experimental research into mechanisms of plant defense, and included teaching at the undergraduate and graduate level. Results were presented at several national and internations conferences (listed below). Products included peer reviewed publications, and ongoing efforts to develop technology to improve crop productivity using the fundamental research on plant defense. PARTICIPANTS: Fushi Wen completed her Ph.D. Dissertation in August 2009, and presented talks at APS and ASPB meetings. Gilberto Curlango-Rivera continued progress on his Ph.D. project, and made presentations of his work at the regional ASPB meeting in Tucson, and at a controlled environment agriculture internations meeting in Holland. Two winter term undergraduate students, Ana Santin and Ashley Colagross, completed research internships in January 2009. Efforts to expand and broaden the impact of the program with a stronger focus on translational research included expansion to the Bio5 Institute of life sciences, with four students recruited for 2010. Gabby Albala completed her undergraduate research program in my lab and graduated in 2009. Collaborators were Hans VanEtten, Gerard White, Zhongguo Xiong. TARGET AUDIENCES: Target audiences include growers, especially those with an interest in exploiting technologies to facilitate root system regulation of whole plant growth. Controlled environment agriculture, in which fungal pathogens are a continuing problem, are a special focus given recent discoveries in my lab. PROJECT MODIFICATIONS: A planned transition from basic research to its application is ongoing, and was a strong focus of the past year's work. Completion of the primary goal of the project--definiting the function of border cells--has fostered progress on this direction.

Impacts
A primary focus of this year's work (including a planned sabbatical) has been translational research to exploit research on root disease resistance in commercial application, especially in controlled environment agriculture. The recognition of a new level of innate immunity is the focus of implementation.

Publications

• G. Curlango-Rivera, D.V. Duclos, J.J. Ebolo & M.C. Hawes Transient exposure of root tips to primary and secondary metabolites: Impact on root growth and production of border cells. Plant and Soil (accepted January 10, 2010).
• F. Wen, G. White, H. D. VanEtten, Z. Xiong, M. C. Hawes Extracellular DNA is required for root tip resistance to fungal infection. Plant Physiology 151: 820-829 (published online October 2009)
• M. C. Hawes, P. Ronald (co-editors) 2009 Focus: Plant Interactions with Bacterial Pathogens. Editorial. Plant Physiology Focus issue
• F. Wen, G. White, H. D. VanEtten, Z. Xiong, M. C.Hawes (2009) Another 'extracellular polysaccharide' functioning in plant defense: Role of structural DNA in border cell-mediated defense of the legume root tip. Tsune Kosuge award; APS meeting in Portland, OR, August 2009.
• A. Colagross, A. Santin, G. Curlango-Rivera, H.D. VanEtten, M.C. Hawes. 2009. Factors controlling cell cycle regulation: More shocking news from plant biology. Poster presented at UBRP annual meeting, Tucson AZ, January 2009.
• G. Albala, G. Curlango-Rivera, MC Hawes 2009. The Lectin Protein's Role in the Evasion of Pathogens and in the Structure of the Growing Root Tip". Poster presented at UBRP annual meeting, Tucson AZ, January 2009.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Transcriptional profiling of root cap development in gymnosperms and legumes(publication, in collaboration with the Norwegian Forestry Institute). Collaborative research, development of Medicago truncatula mutants, with altered production of border cells and root cap secretome (invited talk, Noble Foundation, Ardmore, OK, February 2008; and participation in screening workshop, Fall 2008). Teaching at undergraduate (Winter Term fellowships, with 12 students) and high school level (Poster session, local high school students). PARTICIPANTS: Fush Wen, research assistant and Ph.D. candidate Gilberto Curlango-Rivera, Ph.D. Candidate Gabby Albala, undergraduate Jim Kemp, Undergraduate David Olsen, undergraduate Emily Hillebrand, undergraduate Denise Duclos, Postdoctoral Noble Foundation, collaboration Norwegian Forestry Research Institute TARGET AUDIENCES: Target audiences: growers with increasing problems with root diseases, as products like methyl bromide are removed from the market; especially sectors developing controlled environment agriculture. I continue to work with students at all levels, including women and minorities. My sabbatical leave this year will focus on working with industry groups to explore the potential for utilizing root cap-derived metabolites for disease control. PROJECT MODIFICATIONS: Major changes will focus on the discovery that exDNA is required for disease resistance. Technologies will need to be developed and adapted, because this is a novel discovery that has never been recognized or studies in plants.

Impacts
The goal of the work has been to define the mechanism of how root caps are protected against infection and injury by nematodes, bacteria, fungi, and toxic metabolites like aluminum. During the last year the underlying mechanism has been defined for the first time, by documenting a role for extracellular DNA in the process. The work provides new insight into how plant cell defense occurs, and reveals unrecognized parallels with human white blood cells. The potential applications are the establishment of new strategies to control root diseases, especially under conditions of controlled environment agriculture.

Publications

• Wen F, Celoy R, Price I, Ebolo JJ, Woo HH, Hawes MC (2008) Identification and characterization of a rhizosphere b-galactosidase from Pisum sativum L. Plant and Soil 304: 133-144.
• Wen F, Celoy RM, Nguyen T, Zeng W, Keegstra K, Woo HH, Pauly M, Immerzee P, Hawes MC (2008) Altered cell wall structure and function in pea hairy roots expressing Pisum sativum xyloglucan fucosyltransferase (Psfut1) antisense mRNA. Plant Cell Reports 27: 1125-1135
• Wen F, Woo HH, Pierson EA, Eldhuset TD Fossdal CG, Nagy NE, Hawes MC (2008) Synchronous elicitation of development in root caps induces transient gene expression changes common to legume and gymnosperm species. Plant Mol Biol Rep (online August 31,2008)
• Curlango-Rivera G, Albala G, Kemp JP, Duclos DV, Hawes MC. 2008. Contribution of the root cap to soil fertility: Extracellular plant lectins (in press)
• Wen F, VanEtten HD, Xiong Z, Hawes MC 2008 Fighting a fungal pathogen by secreting extracellular DNA at pea root tips. Phytopathology 98(6): 169.
• Wen F, VanEtten HD, Xiong Z, Hawes MC 2008 Extracellular DNA Plays a Key Role in Pea Root Tip Resistance to Fungal Pathogenesis. American Society for Plant Biologists, Proceedings, Merida, Mexico.
• Hillebrand E, Kemp J, Olsen D, Curlando-Rivera G, Wen F, Pierson LS, Hawes MC 2008. Role of extracellular lectins in eucaryotic defense: root border cells as a model. Nineteenth Annual Undergraduate Biology Research Conference, University of Arizona.

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Activities: Analysis and presentation of data from transgenic analysis of altered expression of root cap genes (xyloglucan fucosysltransferase; galactosidase); two papers in press. Director, Winter Term Fellowships in Plant Microbiology and Molecular Biology, with Oberlin,Macalester, Luther, Centre, Gustavus Adolphus, Mt. Holyoke, Colorado, Carleton Colleges Mentor for 8 graduate students (Major professor for 2). Mentor for 8 undergraduate or high school students. Events: Wen F, Rivera-Curlango G, VanEtten HD, Hawes MC (2007) Extracellular 14-3-3 protein: role in root tip resistance to fungal infection. Proceedings, Botany 2007/Plant Biology 2007, Chicago IL. July 7-11, 2007. Curlango-Rivera G, Del Olmo-Ruiz M, Wen F, Hawes MC (2007) Influence of endogenous bacteria on rhizosphere secretome: Implications in hydroponic culture. Proceedings, American Society for Plant Physiology, Davis CA. Celoy R, Price I, Wen F, Ebolo JJ, Hawes MC (2007) Galactose from the legume root cap: structure, signal, toxin, trigger? Society for Experimental Botany, Scotland, May 2007. Services: Presentation of case for highlighting the UA CEAC in root-rhizosphere research, in meetings with Arizona senators and members of Congress as a member of the American Society for Plant Biology (May 9-13, 2007). Plant Physiology Editorial Board, Monitoring Editor for Root-Microbe Interactions. PARTICIPANTS: Fushi Wen, Graduate Student Gilberto Curlango-Rivera, Graduate Student Rhodesia Celoy, Graduate Student Trang Nguyen, Graduate student Iris Price, Graduate student Mariana Del Olmo-Ruiz, graduate student JJ. Ebolo, Scholar Rescue HH Woo, postdoctoral fellow Hans VanEtten, collaborator Kenneth Keegstra, collaborator Markus Pauly, collaborator Ann Hirsch, collaborator Training and professional development opportunities were instrinsic to work done by undergraduate and graduate students who participated in the project. TARGET AUDIENCES: Growers, controlled environment systems, researchers in plant pathology and physiology. PROJECT MODIFICATIONS: There have been no major changes in approach to date, and the focus remains on defining how border cells protect plant health by protecting the root stem cells that give rise to root systems.

Impacts
Fundamental insights into how plant cells function has been obtained. This includes establishing the functional impact of cell wall synthesizing and solubilizing enzymes already presumed to play a role in development. In the last year additional information regarding the role of an array of extracellular enzymes in disease resistance has been obtained. Most surprising has been the discovery that border cells exhibit functional parallels with human white blood cells called neutrophils, which are produced in response to infection and act specifically to neutralize threats in the form of pathogenic bacteria and fungi. Four additional publications summarizing these results are in preparation or in press.

Publications

• Plant Physiology 2007, 143 (2): 773-783
• Plant Signaling & Behavior 2007, 2:5, 410-412.
• Genomics 2007, 90: 143-153
• Physiologia Plantarum 2007, 130: 250-260

Progress 01/01/06 to 12/31/06

Outputs
Gene families controlling the delivery of border cells into the rhizosphere, apparently under the control of secondary metabolites in the root tip, have been characterized (Woo et al). In addition, we have documented, for the first time, the delivery of a discrete extracellular array of signalling and defense-related proteins from border cells of Pisum sativum, Medicago truncatula, and Zea mays (Wen et al 2006). Moreover, we have discovered that the system is active in long-term culture of roots, with massive increases in the presence of a physical layer that can be visualized using histochemical assays and can be shown to respond dynamically and specifically to benefical and pathogenic bacteria and fungi. The barrier layer is solubilized by enzymes that solubilize proteins. The implcations for control of root microbe disease and injury are substantial, as they open a previously unrecognized direction for disease control targets.

Impacts
The expected impact of this work will allow a new approach to the control of plant health through manipulation of root-rhizosphere biology. Our recent work validates the premise that border cells are a major controlling factor in the establishment of beneficial and pathogenic associations between roots and soilborne organisms.

Publications

• Wen F, VanEtten HD, Tsaprailis G, Hawes MC 2006 Extracellular proteins in Pisum sativum L. poot tip and border cell exudates. Plant Physiology December 1 2006 preview online.
• Woo HH, Jeong BR, Hirsch AM, Hawes MC 2007 Characterization of Arabidopsis gene families related to pea UDP-glycosyltransferase and Scutellaria glucuronidase (Physiologia Plantarum, in press)
• 52. Woo HH, Jeong BR, Choi JW, Hirsch AM, Hawes MC 2007 Altered expression of closely related UDP-glycosyltransferases from pea and Arabidopsis result in altered root development and function (Genomics, in press)

Progress 01/01/05 to 12/31/05

Outputs
Roots of most species have the capacity to shed thousands of living cells, called 'border' cells, from their root tips into the external environment. This unusual plant 'tissue' exhibits distinct patterns of gene expression and responds in a genotype-specific manner to challenge with soilborne bacteria, fungi, nematodes, and aluminum. Border cells appear to modulate growth of pathogenic fungi and thereby prevent invasion of growth centers within the root tip. The mechanism for this behavior is not clear, but previous studies revealed the rapid export of an array of extracellular proteins of unknown function. Treatment of pea roots with protease to degrade the extracellular proteins had no effect on growth and development, but the normal resistance of the root tip to fungal infection was lost. We have documented, for the first time, of a discrete extracellular array of signalling and defense-related proteins from border cells of Pisum sativum, Medicago truncatula, and Zea mays.

Impacts
Control of plant health through manipulation of root-rhizosphere biology

Publications

• Wen F, Laskowski M, Hawes MC 2006 Cell separation in roots. Annual Plant Reviews: Cell Separation and Adhesion in Plants, Blackwell Publishing
• Gunawardena U, Zhao X, Hawes MC 2005 Update on Roots: Contribution to the Rhizosphere. Encyclopedia of Life Sciences
• Hamamato L, Hawes MC, Rost TL 2006 The production and release of living root cap border cells is a function of root apical meristem type. Annals of Botany, in press
• Hawes MC 2006 Interactions between roots and the soil. Chapter 6, pp 107-119. Co-Editors Richard W. Zobel, Sara F. Wright, American Society of Agronomy, Madison Wisconsin
• Wen F, Woo HH, Pierson EA, Hawes, MC 2005 Transcriptional profiling to identify pathways controlling rhizosphere development. Asilomar, June 5-9
• Hawes MC, Celoy RM, Nguyen T, Wen F, Zeng W, Keegstra K, Pauly M, Immerzee P 2005 Altered cell wall structure and function in border cells of pea hairy roots expressing (Psfut1) antisense mRNA. Cell Wall meeting, August 2005.

Progress 01/01/04 to 12/31/04

Outputs
Recent studies have confirmed that an array of 70-80 proteins secreted by the root cap and/or border cells function to carry out 'cellular' activities in the extracellar environment. The functional analysis of this root cap 'secretome' is the subject of continuing work. The goal is to test the hypothesis that secretome-based signaling between plant and microbial cells, modulated through the action of extracellular 14-3-3 proteins, enables the plant to control its relationships with soilborne pathogens and symbionts. In the manner of mammalian white blood cells, which produce extracellular signals that serve to localize infection and prevent systemic disease, our data suggest that border cells and their associated products serve to localize root infection and thereby prevent destruction of the stem cells of the root meristem.

Impacts
Control of plant health through manipulation of root-rhizosphere biology

Publications

• Gunawardena U, VanEtten H, Hawes MC 2005 Tissue specific localization of pea (Pisum sativum L.) root infection by Nectria haematococca: Mechanisms and consequences. Plant Physiology 137: 1363-1374.
• Hubbard JE, Schmitt N, McClure M, Stock SP, Hawes MC. 2005. Characterization of root exudate induced quiescence in parasitic, entomophathogenic, and free-living nematode species. Nematology (in press)
• Woo HH, Hirsch AM, Hawes MC 2004 Altered susceptibility to infection by Sinorhizobium meliloti and Nectria haematococca in alfalfa roots with altered cell cycle. Plant Cell Reports 22: 967-973. Zhu Y, Wen F, Zhao X, Hawes MC 2004 Isolation of the promoter of a root cap expressed pectinmethylesterase gene from Pisum sativum L. Plant and Soil 265: 47-59.

Progress 01/01/03 to 12/31/03

Outputs
We have developed and characterized the impact of border cells on development, pathogenesis, and symbiosis and presented it in peer reviewed publications listed below. In addition, a collaborative relationship to develop materials commercially has been developed with Eli Lilly.

Impacts
Control of plant health through manipulation of root-rhizosphere biology

Publications

• J. E. Hubbard, M. Schmitt, M. McClure, P. Stock, and M. C. Hawes. 2004. Characterization of root exudate induced quiescence in nematode species. Nematology (submitted
• Woo HH, Hirsch AM, Hawes MC 2004 Altered susceptibility to infection by Sinorhizobium meliloti and Nectria haematococca in alfalfa roots with altered cell cycle. Plant Cell Reports (in press
• Zhu Y, Wen F, Zhao X, Hawes MC 2004 Correlation between localized expression of pectinmethylesterase (PME) and release of root exudates from the root tip. Plant and Soil (in press
• Hawes MC, Woo HH, Wen F 2004 Root border cells: A delivery system for chemicals controlling plant health. Soil Science (in press
• Farrar J, Hawes MC, Jones D, Lindow S 2003 How roots control the flux of carbon to the rhizosphere. Ecology 84: 827

Progress 01/01/02 to 12/31/02

Outputs
Characterization of major soilborne stresses--fungi, bacteria, nematodes, and toxic chemicals--and their impact on root tip function as it relates to border cells has been characterized, and is either published and in preparation.

Impacts
A major initiative to develop plants with altered border cell properties, using genomics approaches, has now been developed and is in review. These tools will facilitate transfer of the technologies into commercial production of improved cultivars with enhanced growth, development, and disease resistance.

Publications

• Hubbard J, Hawes MC 2002 Characterization of a root cap secreted product that induces transient paralysis in nematodes. M. S. thesis, University of Arizona
• Price I 2002 Characterization of galactosidase in pea. M. S. thesis, University of Arizona.
• Hawes MC, Woo HH, Wen F 2003 Root border cells: a delivery system for chemicals controlling plant health. Soil Science (in press).
• Hawes MC, Bengough GA, Cassab G 2003 Root caps and rhizosphere. J Plant Growth Regulation (in press)
• Farrar J, Hawes MC, Jones D, Lindow S Root exudates. Ecology (in press) Gunawardena U, Hawes MC 2002 Role of border cells in localized root infection by pathogenic fungi. Molecular Plant Microbe Interactions 15: 1128-1136..

Progress 01/01/01 to 12/31/01

Outputs
Many plants release large numbers of metabolically active root 'border' cells into the rhizosphere. The goal of this project is to define the mechanism(s) by which this uniquely specialized root 'tissue' facilitates plant health and development by controlling the ecology of the rhizosphere. We have described a major new regulatory network that controls the production of border cells, characterized external and endogenous signals that activate border cell production, and have identified genes needed for their synthesis in the meristem and their separation from the root cap periphery. Transgenic hairy roots of pea are being used to systematically test the role of specific genes in the process of border cell production and release, in conjunction with the use of transgenic alfalfa to define the impact of the process on health and development.

Impacts
(N/A)

Publications

• Woo HH, Kuleck G, Hirsch AM, Hawes CM 2001 Flavonoids: Signals in plant development. In "Experimental Medicine and Biology."
• Miyasaka S, Hawes MC. 2001. Possible role of root border cells in detection and avoidance of aluminum toxicity. Plant Physiol 125: 1978-87
• Brigham LA, Hawes MC, Miyasaka SC 2001 Avoidance of aluminum toxicity: Role of root border cells. pp 452-456 in Plant Nutrition--Food security and sustainability of agroecosystems, WJ Horst et al (Eds).
• Gunawardena U, Zhao X, Hawes MC 2001. Roots: Contribution to the Rhizosphere. Encyclopedia of Life Sciences 1. http://www.els/net

Progress 01/01/00 to 12/31/00

Outputs
Many plants release large numbers of metabolically active root 'border' cells into the rhizosphere. The goal of this project is to define the mechanism(s) by which this uniquely specialized root 'tissue' facilitates plant health and development by controlling the ecology of the rhizosphere. We have described a major new regulatory network that controls the production of border cells, characterized external and endogenous signals that activate border cell production, and have identified genes needed for their synthesis in the meristem and their separation from the root cap periphery. Transgenic hairy roots of pea are being used to systematically test the role of specific genes in the process of border cell production and release, in conjunction with the use of transgenic alfalfa to define the impact of the process on health and development.

Impacts
(N/A)

Publications

• Miyasaka, S., Hawes, M.C. 2000. Possible role of root border cells in aluminum tolerance in legumes. Plant Physiol 123
• Zhao, X., Misaghi, I., Hawes, M.C. 2000. Stimulation of border cell production in response to increased carbon dioxide levels. Plant Physiol 122: 1-8.
• Woo, H.H., Kuleck, G., Hirsch, A.M., Hawes, M.C. 2000 Flavonoids: Signals in plant development. In Experimental Medicine and Biology, UCLA press.
• Gunawardena, U., Zhao, X., Hawes, M.C. 2000. Roots: Contribution to the Rhizosphere. Encyclopedia of Life Sciences 1.

Progress 01/01/99 to 12/31/99

Outputs
Many plants release large numbers of metabolically active root border cells into the rhizosphere. The function of these cells and the process of releasing these cells is unknown, but properties of the cells are consistent with the hypothesis that they protect plant health by enabling the root to dictate which microorganisms can share its ecological niche. We have described a major new regulatory network that controls the production of border cells, identified several genes that play a role in the process, and defined endogenous and environmental signals that control their expression. We have also identified several border cell specific genes which have no homology with any known genes. These tools have been used to demonstrate that the properties of the rhizosphere can be altered drastically in response to normal stimuli facing roots, and that such changes can significantly alter the susceptibility of roots to infection by at least one soilborne fungal pathogen.

Impacts
(N/A)

Publications

• Gunawardena, U, Zhao, Z, Hawes, M.C. 1999. Roots: Contribution to the Rhizosphere. Encyclopedia of Life Sciences
• Wen, F, Zhu, Y, Brigham, L.A., Hawes, M.C. 199. Expression of an inducible pectinmethylesterase gene is required for border cell separation from roots of pea. Plant Cell 11:1129-1140.
• Woo, H.H., Brigham, L.A., Hawes. 1999. Detection of low abundance messages by a combination of PCR and ribonuclease protection. Expression Genetics: Differential Display. Pardee A, McClelland M, eds.
• Woo, H.H., Orbach, M.J., Hirsch, A.M., Hawes, M.C. 1999. Meristerm localized inducible expression of a UDP-glycosyltransferase gene is essential for growth and development. Plant Cell 11: 1-14

Progress 01/01/98 to 12/31/98

Outputs
Many plants release large numbers of metabolically active root border cells into the rhizosphere. The function of these cells and the process of releasing these cells is unknown, but properties of the cells are consistent with the hypothesis that they protect plant health by enabling the root to dictate which microorganisms can share its ecological niche. We have described a major new regulatory network that controls the production of border cells, identified several genes that play a role in the process, and defined endogenous and environmental signals that control their expression. One of the genes we have identified is rcpme1 encoding pectinmethylesterase. Expression of this gene was found to be tightly correlated, both spatially and temporally, with border cell separation from pea root caps. Partial inhibition of the gene's expression by antisense mRNA in transgenic pea hairy roots prevented the normal separation of root border cells from the root tip into the external environment. We have also identified several border cell specific genes which have no homology with any known genes. These tools have been used to demonstrate that the properties of the rhizosphere can be altered drastically in response to normal stimuli facing roots, and that such changes can significantly alter the susceptibility of roots to infection by at least one soilborne fungal pathogen.

Impacts
(N/A)

Publications

• Brigham LA, Woo HH, Wen F, Hawes MC. 1998. Meristem specific suppression of mitosis and a global switch in gene expression in the root cap of pea by endogenous signals. Plant Physiol 118: 1223-1231.
• Hawes MC, Brigham LA, Wen F, Woo HH, Zhu Y. 1998. Function of root border cells in plant health: pioneers in the rhizosphere. Ann Rev Phytopathol 36: 311-327.

Progress 01/01/97 to 12/31/97

Outputs
Our goal is to test the function of pectolytic enzymes in border cell separation. Genes encoding polysaccharide synthesizing and degrading enzymes have been cloned and their expression patterns have been evaluated using Northern blot analysis. A full length cDNA encoding a pectinmethylesterase has been cloned and sequenced. Transgenic roots expressing PME antisense mRNA have been developed and analysed. Expression of PME in peripheral cells of the root cap is required for border cell separation.

Impacts
(N/A)

Publications

• Woo, H.H., Hawes, M.C. 1997. Rapid mapping and subcloning of genomic clones in bacteriophage lambda by PCR. Biotechniques 22:822-824.
• Woo, H.H., Brigham, L.A., Hawes. 1997. Differential expression of mRNAs during early stages of inducible gene expression in the root cap. Plant Molec Biol 35:1045-1049.
• Hawes, M.C., Brigham, L.A., Woo, H.H., Zhu,Y., Wen, F. 1997. Root border cells: Phenomenology of signal exchange. In Advances and Perspectives on the Function of Plant Roots, Ed. H.E. Flores, Am Soc Plant Physiol.

Progress 01/01/96 to 12/30/96

Outputs
Our goal is to test the function of pectolytic enzymes in border cell separation. Genes encoding polysaccharide synthesizing and degrading enzymes have been cloned and their expression patterns have been evaluated using Northern blot analysis. A full length cDNA encoding a pectinmethylesterase has been cloned and sequenced. Transgenic roots expressing PME antisense mRNA and their analysis will be published in 1997.

Impacts
(N/A)

Publications

• Hawes MC, Brigham LA, Wen F, Woo HH, & Zhu Y 1996. Root border cells. Biology of Plant-Microbe Interactions 8:509-514.

Progress 01/01/95 to 12/30/95

Outputs
ARZT-136388-H-05-107 We tested predictions of the hypothesis that pectin methylesterase in the root cap plays a role in cell wall solubilization leading to separation of root border cells from the root tip. Root cap pectin methylesterase activity was detected only in species that release large numbers of border cells daily. In pea (Pisum sativum) root caps, enzyme activity is correlated with border cell separation during development: 6-fold more activity occurs during border cell separation than after cell separation is complete. Higher levels of enzyme activity are restored by experimental induction of border cell separation. A corresponding increase in transcription of a gene encoding root cap pectin methylesterase precedes the increase in enzyme activity. A dramatic increase in the level of soluble, de-esterified pectin in the root tip also is correlated with pectin methylesterase activity during border cell development. This increase in acidic, de-esterified pectin during development occurs in parallel with a decrease in cell wall/apoplastic pH of cells in the periphery of the root cap.

Impacts
(N/A)

Publications

• Brigham L.A., Woo H.H., & Hawes M.C. 1995 Differential expression of proteins and mRNA's from border cells and root tips of pea. Plant Physiol 109:457-463.
• Woo H.H., Brigham L.A., & Hawes M.C. 1995 Detection of low abundance messages by a combination of PCR and ribonuclease protection. BioTechniques 18:778-779.
• Nicoll S.M., Brigham L.A., Wen F., & Hawes M.C. 1995 Expression of transferred genes during hairy root development in pea. Plant Cell Tissue Organ Culture 42:57-66.
• Brigham L.A., Woo H.H., & Hawes M.C. 1995 Root border cells as tools in plant cell studies. Methods in Cell Biology 49:377-387.
• Woo H.H., Brigham L.A., & Hawes M.C. 1995 Molecular cloning and expression of mRNAs encoding H1 histone and H1 histone like sequences in root tips of pea. Plant Mol Biol 28:1143-1147.

Progress 01/01/94 to 12/30/94

Outputs
The goal of the project is to understand the impact of root border cells on plant-microbe interactions. This will be accomplished by creating transgenic plants with defined changes in the biology of root border cells and the chemicals that are released during the process of border cell separation. Studies this year have focused on studying gene expression during border cell development and on cloning genes that are specifically expressed in border cells. A marked switch in gene expression occurs upon differentiation of root cap cells into border cells. Thirty genes that are expressed in border cells but not in root specificity of gene expression has been initiated, and was used to confirm that mRNA from several genes expressed at high levels in border cells are not detectable in progenitor cells in the root cap.

Impacts
(N/A)

Publications

• NO PUBLICATIONS REPORTED THIS PERIOD.

Progress 01/01/93 to 12/30/93

Outputs
CSRS Hatch 0151683 A root cap pectinmethylesterase (PME) has been partially purified and shown to be present at high levels in pea root caps. Enzyme activity is correlated with border cell separation, with changes in degree of esterification of pectin in root caps, and with a decrease in cell wall pH. A putative PME encoding cDNA has been isolated from a pea root cap cDNA library. The cDNA clone exhibits 70% to 80% homology to known PME encoding genes from plants. Northern analysis has revealed that activity is transcriptionally regulated in correlation with border cell separation.

Impacts
(N/A)

Publications

• No publications reported this period.

Progress 01/01/92 to 12/30/92

Outputs
A method for studying expression of genes required for border cell separation inPisum sativum was developed using Agrobacterium rhizogenes together with a binary vector. Expression of chimaeric genes in transgenic hairy roots was comparable to expression of the same genes in whole plants. Characterization of a pectinmethylesterase in the root cap of pea reveals a heat stable molecule of MW approximately 37,000. Activity of the enzyme is correlated with border cell separation. A cDNA library of root caps induced to produce PME has been made, and is being screened using heterologous probes. Preliminary results suggest that a gene with homology to know pectinmethylesterase encoding genes is expressed in pea root caps.

Impacts
(N/A)

Publications

Progress 01/01/91 to 12/30/91

Outputs
Separation of root "border" cells from the root cap of pea is developmentally regulated. The activity of two different types of pectolytic enzymes is correlated with cell separation. An enzyme with properties consistent with those of a polygalacturonase is detectable while cell separation is active, but activity disappears after cell separation is complete. Similarly, activity consistent with that of a pectinmethylesterase is high in the root cap during cell separation, but activity is low after cell separation ceases. Both enzymes are ionically bound to the cell wall in the root cap, but are not present in border cells. Activity of the pectinmethylesterase may be regulated by a second inhibitory protein that increases in activity during cell separation.

Impacts
(N/A)

Publications

• No publications reported this period.

Progress 07/01/90 to 12/30/90

Outputs
A method of synchronization of root border cells that are released from the rootcap had been developed (Hawes & Lin 1990). We used the method to demonstrate that activity of a root cap localized pectolytic enzyme is corrlated with the developmentally regulated release of the cells from the cap. The enzyme has been partially purified. Biochemical characteristics of the enzyme (pH, substrate preference, heat stability, size etc) are consistent with the possibility that the enzyme is an endopolygalacturonase. Preliminary experiments suggest that the enzyme is encoded by a gene with structural similarity to a polygalacturonase from tomato fruit. Experiments to identify genes that are specifically expressed in root border cell of pea have been initiated. Protein profiles of border cells and root caps are very similar, but several proteins exist in border cells that appear to be absent in root caps.

Impacts
(N/A)

Publications

• HAWES, M.C. and LIN, H.J. 1990. Correlation of pectolytic enzyme activity with the programmed release of cells from the root cap of Pisum sativum. Plant Physiology 4:1855-1859.