Progress 01/01/15 to 12/31/18
Outputs
Target Audience:State legislators, state leaders, and public: The significance of plant nutrient acquisition to sustainable agriculture makes our research relevant not only to the scientific community but others interested in plant nutrition. We believe that the work on plant preference for specific rhizobial strains could have practical implications for the development of crops that would associate better with high-performance rhizobial inoculants. Therefore, the target audience for this project includes farmers, gardeners, and all citizens concerned with the sustainability of our agriculture and its implications for ecosystem stability at the local and global level. Six undergraduate students working with Co-PD presented their research findings from this project to Wisconsin state legislators, state leaders and general public at the Research in the Rotunda events held at the Wisconsin State Capitol during 2016 and 2017. Research in the Rotunda is an opportunity for students and their faculty advisors across the University of Wisconsin system to present undergraduate research on essential topics to state legislators, state leaders, UW alumni and the public (https://www.uwplatt.edu/news/ag-students-present-research-state-capitol; https://www.uwplatt.edu/news/undergraduate-research-showcased ). We have worked to bridge the gap between our laboratory work and farmers by attending many outreach activities. We continue to make ongoing progress in our connections with the local community of gardeners and voters by participating in multiple community events year-round, including Family Horticulture Day, a springtime family event focused on bringing families together through gardening; Darwin Day, a fall event for graduate students, high school students and general public focused on instilling the participants with the joy and wonder of the discovery of biology; Agronomy/Soils Field Day and Grant County Family Fun Night, which highlight the research on emerging technologies and relevant crop production issues to farmers and those who are interested in sustainable agriculture. Our ongoing efforts to connect with local communities are essential not only for the alignment of our goals with the fulfillment of a societal need but a sustained public understanding of the importance of our research to societal well-being. Scientific community: The most relevant target audience is scientists in the field of plant-microbe interactions, as our work investigates one of the critical questions regarding the regulation of symbiotic cation channels in the symbiotic pathway, which are required for plant symbioses by both rhizobia and arbuscular mycorrhizal fungi. These target audiences have been served by our contributions to our field during the most recent funding period through our work with collaborators and our presentation of our work at multiple community-specific symposia/conferences (Plant Biology Meeting by American Society of Plant Biologists; International Conference on Arabidopsis Research; Research in Rotunda; UW System Symposium for Undergraduate Research and Creative Activity). Through these contributions, we have continued to participate in an ongoing effort within our field to understand the mechanisms of plant-microbe symbioses to improve plant nutrient acquisition for the benefit of society. Students (undergraduate, high- and middle-school students): The project has been served as a base for the development of a curriculum to teach principles and techniques of Plant Breeding, Plant Biotechnology, and Plant Physiology. Nearly 100 students from Plant Breeding class (SCSCI 4240) taught by Co-PD during 2015-2018, learned the crossing techniques and the availability of genetic mapping tools in M. truncatula which are currently pursued through this project. Similarly, students of Plant Physiology class (SCSCI 4340; more than hundreds of students during 2015-18) and Plant Development and Biotechnology class (SCSCI 3220; more than 100 students during 2015-2018) learned about gene expression analyses, promoter-GUS assays and Nod factor-induced ENOD11-GUS assays as plant-microbe symbiotic signaling events in demonstration conducted as a part this project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?At the PD's laboratory, a graduate student (Mr. Shane Bernard) is working on the project to accomplish the tasks towards achieving our goals on the regulation of DMI1 in Medicago truncatula and POLLUX and CASTOR in Lotus japonicus. Mr. Bernard also worked with the university's technology transfer office to help enhance networks within the UW-Madison community. At the Co-PD's laboratory, so far 20 undergraduate students (Mr. Kendell Welch, Ms. Emily Hefty, Ms. Katie Martin, Ms. Leanna Otz, Mr. Arlyn Ackerman, Mr. Tyler Nitz, Ms. Laura DeClercq, Mr. Michael Ely, Mr. Brett Pluemer, Mr. Sean Strueder, Mr. Nickolas Theisen, Ms. Katie Chipman, Ms. Sara Rubeck, Mr. Noah McVay, Mr. Tristan Martins, Mr. Theodore Hoffman, Mr. Charles Peterson, Mr. Ryan Larsen, Mr. Ryan Nadig and Ms. Jessica Helwig) and one high-school student (Mr. Adam Compton) have been trained in this project since the beginning of the project. These students have been trained in the fields of Plant Genetics and Breeding, Plant Molecular Biology, Microbiology, and in vitro culture techniques. Mr. Kendell Welch, Mr. Arlyn Ackerman, Mr. Brett Pluemer, and Mr. Michael Ely served as Research Specialists in this project and performed high throughput screening to identify genetic suppressors, confirmation of suppressor phenotypes and Medicago crossings. Many of these students obtained their research training through SCSCI 4390: Undergraduate Research in Crop Science, ENVHORT: 3370 Undergraduate Research in Horticulture, and SCSCI 3390: Special Problems in Crop Science. These students learned to work as a team, coordinate various project activities to accommodate research in their class schedules and in mentoring new students in their area of expertise. During the project period, Co-PD traveled to the PD's Laboratory at UW-Madison to train an undergraduate student from UW-Platteville on the use of confocal microscopy for calcium imaging and sub-cellular localization of candidate proteins in M. truncatula root hairs. Two UW-Platteville students (Mr. Brett Pluemer and Mr. Ryan Larsen) worked during Winterim 2018 and Summer 2018 at the PD's laboratory at UW-Madison on this project. Many undergraduate students who worked on this project at Co-PD's lab are pursuing graduate studies (Clemson University, Iowa State University, North Dakota State University) and research career in private industries (WinField United: Agriculture Solutions. The Co-PD attended and presented research findings from this project at many National and international conferences and symposiums. These include, International Conference on Arabidopsis Research held at Hyatt Regency, St. Louis, MO, USA (June 19-23, 2017), 2017 Plant Biology Meeting organized by the American Society of Plant Biologists (ASPB), Honolulu, HI, USA (June 24-28, 2017), 2018 Microbes organized by American Microbiological Society (June 20-24, 2018), 2018 Wisconsin Science and Technology Symposium (July 30-31, 2018). These professional meetings provided the Co-PD opportunities to learn recent developments in the field of molecular plant-microbe interactions and plant genomics, to meet our collaborators and for networking with eminent plant biologists. How have the results been disseminated to communities of interest?The results obtained from this research project have been disseminated as publications, poster and oral presentations at various conferences and symposia. What do you plan to do during the next reporting period to accomplish the goals?Overall Impact A.Cereals like rice and corn, develop symbiotic associations with arbuscular mycorrhizal fungi that help in the acquisition ofnutrients and water from the soil. Legumes associate with these beneficial fungi like cereals but also with nitrogen-fixing bacteria called rhizobia that convert atmospheric nitrogen into ammonium assimilable by the plant. Cereals require a lot of nitrogen to reach maximal yields. For economic and environmentalreasons, it would be advantageous to develop cereal cropsthat associate better with nitrogen-fixing bacteria like legumes and require less synthetic nitrogen fertilizers. Interestingly, the PD, co-PD, and others found that legumes use very similar molecular mechanisms to accommodate rhizobia and mycorrhizal fungi. Given that cereals can associatewith arbuscular mycorrhizal fungi, it seems possible to use these pre-existing mycorrhizal mechanisms in cereals to engineer rhizobia-cereal symbioses. B. Plant receptors perceive signals produced by rhizobia and arbuscular mycorrhizal fungi at the plasma membrane level. Activation of these receptorselicits a signaling cascade leading tooscillations of the calcium concentration in and around the nucleus. This phenomenon, called "calcium spiking", is decoded and regulates the expression of genes controlling symbiotic associations. Calcium spiking is a central event in this signaling cascade and is mediated by ion channels on the nuclear envelope.This USDA-funded project was aiming at (1) better understanding, in legumes,the regulation of these ion channels by second messengers coming from the plasma membrane and by calcium and (2) determining ifthese molecular mechanisms are conserved between legumes and cereals. C. Funding provided through this project allowed us to understand better how these nuclear ion channels are regulated by metabolites derived from the mevalonate biosynthetic pathway and calcium. In particular, we characterized specific sites on the regulatory region of these ion channels essential for the initiation of calcium spiking. This project also allowed us to demonstrate that these mechanisms are conserved between legumes and cereals. It is excellent news since this suggests that it may not be necessary to modify these mechanisms to engineers rhizobia-cereal associations. D. In the short term, this project allowed us to train graduate and undergraduate students in plant genetics, new imaging techniques, and microbiology. It also allowed us to inform growers and the public about the current benefits of associations between crops and microbes. We also had interesting discussions with the public about the prospect of developing better nitrogen-fixing associations in cereals. In the long term, there is no doubt that knowledge acquired from this project will be useful for the engineering of rhizobia-cereal associations. Understanding better the mechanisms of rhizobia-legume associations and the extent to which these mechanisms are conservedin cereals is an essential step towards the exciting prospectof nitrogen-fixing cereals.
Impacts
What was accomplished under these goals?
Objective 1: Characterize the regulation of symbiotic cation channels by candidate second messengers (100% completed)- 1- Pharmacological screen of mevalonate-derived metabolites as activators of calcium spiking. As we followed up our pharmacological screen in planta, we found this approach to be limited by inconsistencies between our assays (calcium spiking vs. gene expression). Defining the relevant concentrations was also challenging. These issues may be due to the poor diffusion of some metabolites through cell walls and membranes. We are thinking about using microinjection techniques to resolve some of these discrepancies. 2- Regulation of the DMI1, CASTOR and POLLUX ion channels by calcium- We have continued to develop our understanding of the role of calcium as a second messenger regulating the activity of DMI1 in Medicago truncatula, and POLLUX and CASTOR in Lotus japonicus. X-ray crystallography data from our collaborator, Dr. Youxing Jiang at the University of Texas Southwestern at Dallas suggest that calcium binds to 3 distinct sites in the RCK (regulator of K conductance) domain of L. japonicus CASTOR. Similar data have been recently obtained for the RCK domain of L. japonicus POLLUX. Our laboratory utilized a site-directed-mutagenesis and mutant rescue approach on these two proteins to test the role of these sites on the activity of these channels in symbiotic signaling. In L. japonicas, we have observed a loss of the formation of symbioses with rhizobial bacteria when mutating calcium-binding residues of CASTOR. In M. truncatula, we have again found a loss of symbioses when mutating calcium-binding residues of the POLLUX ortholog DMI1 and further observed that some mutants displayed a decreased symbiosis phenotype. These results are consistent with the hypothesis that calcium binding to DMI1 and CASTOR regulates symbiotic calcium spiking. We are currently testing the effect of these mutations on early nodulin genes in response to purified Nod factors, and have observed that several mutants are incapable of inducing early symbiotic gene expression. 3- Relationships between partner (rhizobia) choice and DMI1 regulation in M. truncatula.Dr. Katy Heath at the University of Illinois at Urbana-Champaign recently published a population ecology study in which they found single nucleotide polymorphisms (SNPs) that correlate with the preference of M. truncatula accession for various rhizobia strains. Interestingly, one of these SNPs falls into the DMI1 gene suggesting that mutations in this nuclear ion channel could control the preference of M. truncatula for specific strains of rhizobia. Their initial study was purely correlative, but we initiated a collaboration between our laboratories to test this hypothesis using a site-directed-mutagenesis and mutant rescue approach similar the one we follow for calcium binding sites. Our initial data suggested that the mutations, identified by genome-wide approaches, do regulate the preference for rhizobial strains even when we make single mutations in the DMI1 ion channel. Further analyses demonstrated that this result was not replicable, and this project has been brought to a conclusion. 4- Role of mevalonate in symbiotic signaling pathway in cereal crops- During the early phase of the project, we had demonstrated a new role for mevalonate as a signaling molecule in legume-rhizobia symbiosis. However, the role of the mevalonate pathway in arbuscular mycorrhizal symbiosis, another important symbiosis is not well understood Utilizing both pharmacological and genetic approaches, we investigated the role of the mevalonate pathway in symbiotic signaling in cereal crops and its effect on growth and development, using rice as a model cereal crop. Rice plants (wild-type and mutant lines) were grown on half-strength Hoagland medium for ten days, and the roots were treated with various signaling molecules such as mevalonate, germinating spore exudates (GSE) of AM fungi and chitin oligomers (COs, that mimic the AM symbiotic signals) or water (negative control). AM-specific gene expression was monitored in wild-type and mutant plants upon treatment with signaling molecules. We also tested the effects of mevalonate, GSE, and COs on plant growth and development, such as the growth of shoots and roots, and lateral root formation. Mevalonate induced the expression of OsAM1, OsAM3 and OsAM11 genes in rice wild-type similar to GSE and COs. However, the mevalonate-induced OsAM3 and OsAM11 gene expressions were absent in rice pollux mutant suggesting that the expression of these genes are dependent on symbiotic signaling pathway. mevalonate induced the shoot growth and lateral root development in wild-type, but not in rice dmi3 mutant suggesting the role of a signaling pathway in the mevalonate-induced growth and development. Objective 2: Identify new regulators of these symbiotic cation channels using a genetic suppressor screen (100% completed)- During the entire project period, we screened over 32,500 plants mutagenized (M2) in the presence of a knockout of cation channels required for the activation of the symbiotic pathway. So far, we had identified 38 putative genetic suppressors by their ability to restore the expression of symbiotic genes in our colorimetric assays. Nine of these lines formed functional nodules. In all our successful screening, we identified multiple suppressors, suggesting that those suppressor seeds may have been collected from the same pod, hence they are identical. So, these suppressor lines were grouped based on the successful screen dates for comparison purpose. The suppressor's lines identified in the M2 generation were confirmed at the M3 generation for suppressor phenotypes. Of the 38 putative suppressor lines, so far 14 had been successfully confirmed at M3 generation. Of these, the three suppressors (one each from different successful suppressor screening) have been from the M3 progeny were crossed twice to the parental mutant to remove other unwanted mutations. The remaining 11 suppressors have been backcrossed once to the parental mutant, and the progenies with suppressor phenotype are being selected. The progenies of the three suppressors that have been backcrossed twice with the original mutant have been tested for the expression of nodulation-specific genes, such as ERN1, ENOD11, ENOD40, MtLEC4, MtCtg_82104, and MtCtg_72999. We also examined the restoration of AM symbiosis phenotype in these three suppressor lines, and all the three suppressors showed incidences of AM colonization. However, quantification of AM colonization through grid-line intersection method showed a lower level of AM colonization for both the suppressor lines as well as the wild-type control. Experiments are in progress to ensure that no known mutations that lead to auto-activation of symbiotic signaling pathways resulting in spontaneous nodule are responsible for the suppressor phenotype. Once at least two rounds of backcrossing for all the 14 suppressors are complete, PD and co-PD plan to pursue whole genome sequencing of the suppressor lines to identify the suppressor loci that are potential regulators of DMI1. PD and co-PD will seek additional grant funding to accomplish this work.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Vald�s-L�pez O, Jayaraman D, Maeda J, Delaux PM, Venkateshwaran M, Isidra-Arellano MC, del Roc�o Reyero-Saavedra M, del Socorro S�nchez-Correa M, Verastegui-Vidal MA, Delgado-Buenrostro N, Van Ness L, Mysore KS, Wen J, Sussman MR, An� JM. A Novel Positive Regulator of the Early Stages of the Root Nodule Symbiosis Identified by Phosphoproteomics. Plant and Cell Physiology pcy228, DOI:10.1093/pcp/pcy228.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Wiley-Kalil, A., Binder, A., Han, L., Li, W., Venkateshwaran, M., Maeda, J., Bernard, S., Delaux, P.M., Mysore, K., Wen, J., Imaizumi-Anraku, H., Parniske, M., Otegui, M. and An�, J.M. NUP107-160 nuclear pore sub-complex members are required for the proper localization of symbiotic ion channels in Lotus japonicus and Medicago truncatula. The manuscript is submitted to Plant Physiology (Under review).
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2018
Citation:
Jayaraman, D., Venkateshwaran, M. and An�, J.M. 2018. Leveraging proteome and phosphoproteome to unravel the molecular mechanisms of legume-rhizobia symbiosis. In The Model Legume Medicago truncatula. Frans J. de Bruijn (Editor). Wiley/Blackwell publications.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
Pluemer, B. *, Hoffman, T. *, An�, J.M. and Venkateshwaran, M. 2018. Role of mevalonate pathway in the symbiotic signaling in cereal crops. 2018 Midwest American Society of Plant Biologists Annual Meeting, Iowa State University, Ames, IA. March 3-4, 2018. (Poster presentation).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
Ely, M. *, An�, J.M., Venkateshwaran, M. 2018. Role of Medicago truncatula LYR1 in recognizing fungal symbionts. 2018 Pioneer Creative Activities and Research Day, UW-Platteville, WI. April 25, 2018. (Poster Presentation).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
Peterson, C. *, Larsen, R. *, Ely. M.*, Ackerman, A.*, Theisen, N.*, An�, J.M. and Venkateshwaran, M. 2017. Genetic suppressor screening to identify new regulators of symbiotic signaling in the model legume Medicago truncatula. 2018 Microbes-American Society of Microbiology Annual Meeting, Atlanta, GA. June 7-11, 2018 (Poster presentation).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
Peterson, C. *, Ely, M. *, Larsen, R. *, An�, J.M. and Venkateshwaran, M. 2018. Unraveling the molecular mechanisms of how plants recognize their fungal partners. 2018 Wisconsin Science and Technology Symposium, UW-Parkside, WI. July 30-31, 2018. (Poster presentation).
|
Progress 01/01/17 to 12/31/17
Outputs
Target Audience:State legislators, state leaders, and public The significance of plant nutrient acquisition to sustainable agriculture makes our research relevant not only to the scientific community but others interested in plant nutrition. We believe that the work on plant preference for specific rhizobial strains could have practical implications for the development of crops that would associate better with high-performance rhizobial inoculants. Therefore, the target audience for this project includes farmers, gardeners, and all citizens concerned with the sustainability of our agriculture and its implications for ecosystem stability at the local and global level. Four undergraduate students working with Co-PD presented their research findings from this project to Wisconsin state legislators, state leaders and general public at the Posters in the Rotunda event held at the Wisconsin State Capitol on April 12, 2017. Research in the Rotunda is an opportunity for students and their faculty advisors across the University of Wisconsin system to present undergraduate research on important topics to state legislators, state leaders, UW alumni and the public (https://www.uwplatt.edu/news/undergraduate-research-showcased ). We have worked to bridge the gap between our laboratory work and farmers by attending many outreach activities. We continue to make ongoing progress in our connections with the local community of gardeners and voters by participating in multiple community events year-round, including Family Horticulture Day, a springtime family event focused on bringing families together through gardening; Darwin Day, a fall event for graduate students, high school students and general public focused on instilling the participants with the joy and wonder of the discovery of biology; Agronomy/Soils Field Day and Grant County Family Fun Night, which highlight the research on emerging technologies and relevant crop production issues to farmers and those who are interested in sustainable agriculture. Our ongoing efforts to connect with local communities are essential not only for an alignment of our goals with the fulfillment of a societal need but a sustained public understanding of the importance of our research to societal well-being. Scientific community: The most relevant target audience is scientists in the field of plant-microbe interactions, as our work investigates one of the critical questions regarding the regulation of symbiotic cation channels in the symbiotic pathway, which are required for plant symbioses by both rhizobia and arbuscular mycorrhizal fungi. These target audiences have been served by our contributions to our field during the most recent funding period through our work with collaborators and our presentation of our work at multiple community-specific symposia/conferences (Plant Biology Meeting by American Society of Plant Biologists; International Conference on Arabidopsis Research; Research in Rotunda; UW System Symposium for Undergraduate Research and Creative Activity). Through these contributions, we have continued to participate in an ongoing effort within our field to understand the mechanisms of plant-microbe symbioses to improve plant nutrient acquisition for the benefit of society. Students (undergraduate, high- and middle-school students): The project has been served as a base for the development of curriculum to teach principles and techniques about Plant Breeding, Plant Biotechnology, and Plant Physiology. Twenty-four students from Plant Breeding class (SCSCI 4240) taught by Co-PD during Fall 2017, learned the crossing techniques and the availability of genetic mapping tools in M. truncatula which are currently pursued through this project. Similarly, students of Plant Physiology class (SCSCI 4340; 40 students from Spring and Fall 2017) and Plant Development and Biotechnology class (SCSCI 3220; 24 students in Spring 2017) learned about gene expression analyses, promoter-GUS assays and Nod factor-induced ENOD11-GUS assays as plant-microbe symbiotic signaling events in demonstration conducted as a part this project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?At the PD's laboratory, a graduate student (Mr. Shane Bernard) is working on the project to accomplish the tasks towards achieving our goals on the regulation of DMI1 in Medicago truncatula and POLLUX and CASTOR in Lotus japonicus. Mr. Bernard gained experience mentoring undergraduate researchers in the UW-Madison community and worked with the university's technology transfer office to help enhance networks within the UW-Madison community. During 2017, Mr. Bernard was assisted in this research by an undergraduate mentee at the University of Wisconsin-Madison, Erin Sweeney. She has participated in making DNA constructs, transforming plant roots and assaying nodulation in Medicago truncatula. During Mr. Bernard's studies, he has been leveraging his developing scientific expertise by interning with UW-Madison's technology transfer office to promote networking within the UW community. He works to connect researchers across research departments to foster interdisciplinary collaborations and discovery within the scientific community. He also works to connect the university's researchers with technology transfer experts to help return the financial gains of scientific research to the UW-Madison research community. This participation in the UW community was made possible by the research experiences garnered during Mr. Bernard's scientific training. At the Co-PD's laboratory, eight undergraduate students (Mr. Arlyn Ackerman, Mr. Michael Ely, Mr. Brett Pluemer, Mr. Sean Strueder, Mr. Nickolas Theisen, Ms. Katie Chipman, Ms. Sara Rubeck, Mr. Noah McVay) and one high-school student (Mr. Adam Compton) have been trained in this project since January 2017. These students have been trained in the fields of Plant Genetics and Breeding, Plant Molecular Biology, Microbiology, and in vitro culture techniques. Mr. Arlyn Ackerman (September 2016-May 2017), Mr. Brett Pluemer (June 2017-August 2017) and Mr. Michael Ely (September 2017-current) served as Research Specialist in this project and performed high throughput screening to identify genetic suppressors, confirmation of suppressor phenotypes and Medicago crossings. Mr. Sean Strueder, Mr. Nickolas Thiesen, Ms. Katie Chipman, Ms. Sara Rubeck, Mr. Noah McVay obtained their research training through SCSCI 4390: Undergraduate Research in Crop Science, ENVHORT: 3370 Undergraduate Research in Horticulture, and SCSCI 3390: Special Problems in Crop Science. These students learned to work as a team, coordinate various project activities to accommodate research in their class schedules and in mentoring new students in their area of expertise. During Summer 2017, the Co-PD traveled to the PD's Laboratory at UW-Madison to train an undergraduate student from UW-Platteville (Mr. Brett Pluemer) on the use of confocal microscopy for calcium imaging and sub-cellular localization of candidate proteins in M. truncatula root hairs. An undergraduate student (Mr. Arlyn Ackerman) who served as research specialist in this project graduated in May 2017 with a major in Soil and Crop Science with Plant Breeding and Genetics Emphasis and a minor in Biotechnology from the University of Wisconsin-Platteville, who is currently pursuing doctoral studies in the Department of Genetics and Biochemistry program at Clemson University. Mr. Nickolas Theisen, another May 2017 graduate who obtained his undergraduate research experience through this project, is pursuing graduate studies (M.S. degree) in the Department of Plant Sciences at North Dakota State University. Two other students who worked in this project obtained research positions at private companies (WinField United: Agriculture Solutions). The Co-PD attended 2017 International Conference on Arabidopsis Research held at Hyatt Regency, St. Louis, MO, USA (June 19-23, 2017) and presented the data collected from this project as oral presentation titled "Role of Volatile Organic Compounds in Plant Symbiotic and Defense Signaling Pathways". The Co-PD attended 2017 Plant Biology Meeting organized by the American Society of Plant Biologists (ASPB), Honolulu, HI, USA (June 24-28, 2017) and presented the data collected from this project as a poster titled "Role of Mevalonate Pathway in Symbiotic Signaling in Cereal Crops". These professional meetings provided the Co-PD opportunities to learn recent developments in the field of molecular plant-microbe interactions and plant genomics, to meet our collaborators and for networking with eminent plant biologists. How have the results been disseminated to communities of interest?The results obtained from this research project have been disseminated as publications, poster and oral presentations at conferences and symposia. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will continue to make progress towards our goal of understanding how mevalonate-derived metabolites and calcium regulate symbiotic cation channels by examining relationships between channel activity, channel-dependent oscillations of the nuclear calcium concentration (calcium spiking), and calcium spiking-dependent gene expression. We are planning to circumvent issues of metabolite diffusion with injections. We also want to finish the mutant rescue experiments with different mutants of DMI1 in Medicago truncatula and CASTOR in Lotus japonicus. We intend to submit a manuscript on our collaboration with Dr. Youxing Jiang in spring 2018. We have also initiated similar rescue assays in rice mutants but, given the time required to produce transgenic rice plants, this will be included in a subsequent publication. We also intend to continue the work on the role of DMI1 activity in controlling preference for specific rhizobial strains. So far, we have mostly performed competition assays with three strains as described in Grillo et al. (2016). We intend to test competition experiments with two rhizobial strains to simplify the assay and increase the throughput. We also want to examine the effect of this SNP and different rhizobia on the shape and frequency of calcium spiking. Our goal is to correlate channel activity, calcium signatures, gene expression and preference of the plant for some symbionts. We intend to submit a manuscript on this project in fall 2018 before Mr. Shane Bernard's graduation.
Impacts
What was accomplished under these goals?
Objective 1: Characterize the regulation of symbiotic cation channels by candidate second messengers. We estimate that about 80% of this objective was accomplished. To dissect the molecular mechanisms that regulate nuclear ion channels involved in symbiotic signaling, we have 1) followed up our pharmacological screen to identify novel regulators of symbiotic cation channels, 2) continued to build on our collaboration with structural biologists to examine ionic secondary messenger binding sites in symbiotic cation channels, and 3) begun a new collaboration to characterize natural alleles of DMI1 that regulate partner (rhizobia) choice during nodulation. 1. Pharmacological screen of mevalonate-derived metabolites as activators of calcium spiking As we followed up our pharmacological screen in planta, we found this approach to be limited by inconsistencies between our assays (calcium spiking vs. gene expression). Defining the relevant concentrations was also challenging. These issues may be due to the weak diffusion of some metabolites through cell walls and membranes. We are thinking about using microinjection techniques to resolve some of these discrepancies. 2. Regulation of the DMI1, CASTOR and POLLUX potassium channels by calcium. We have continued to develop our understanding of the role of calcium as a second messenger regulating the activity of DMI1 in Medicago truncatula, and POLLUX and CASTOR in Lotus japonicus. X-ray crystallography data from our collaborator, Dr. Youxing Jiang at the University of Texas Southwestern at Dallas suggest that calcium binds to 3 distinct sites in the RCK (regulator of K conductance) domain of L. japonicus CASTOR. Similar data have been recently obtained for the RCK domain of L. japonicus POLLUX. Our laboratory has initiated a site-directed-mutagenesis and mutant rescue approach on these two proteins to test the role of these sites on the activity of these channels in symbiotic signaling. Using rescue assays of M. truncatula mutants could show that these sites control the ability of DMI1 to mediate nodulation with rhizobia. We are currently performing similar experiments with the L. japonicus castor mutants, and mutants of the pollux ortholog M. truncatula DMI1. We are also testing the effect of these mutations on calcium spiking and expression of early nodulin genes in response to purified Nod factors. 3. Relationships between partner (rhizobia) choice and DMI1 regulation in M. truncatula. Dr. Katy Heath at the University of Illinois at Urbana-Champaign recently published a population ecology study in which they found single nucleotide polymorphisms (SNPs) that correlate with the preference of M. truncatula accession for various rhizobia strains (Grillo et al. 2016). Interestingly, one of these SNPs falls into the DMI1 gene suggesting that mutations in this nuclear ion channel could control the preference of M. truncatula for specific strains of rhizobia. Their initial study was purely correlative, but we initiated a collaboration between our laboratories to test this hypothesis using a site-directed-mutagenesis and mutant rescue approach similar the one we follow for calcium binding sites. Strikingly, our initial data confirm that the mutations identified by genome-wide approaches, do regulate the preference for rhizobial strains even when we make single mutations in the DMI1 ion channel. We hypothesize that these mutations affect the channel activity and we are currently testing the effect of these mutations on calcium spiking and expression of early nodulin genes in response to purified Nod factors. We are very excited to pursue and publish data from projects (2) and (3) soon because they both provided exciting information on how the regulation of nuclear ion channels controls not only the onset of symbiotic associations but also, at a subtler level, which symbionts can colonize the host plant. There is also a lot of synergy between the two projects. These projects could potentially lead to improving the ability of legume crops to associate preferentially with high-performing rhizobial inoculants rather than native and often parasitic rhizobial populations. 4. Role of mevalonate in symbiotic signaling pathway in cereal crops In the previous year, we have demonstrated a new role for mevalonate as a signaling molecule in legume-rhizobia symbiosis. However, the role of the mevalonate pathway in arbuscular mycorrhizal symbiosis, another essential symbiosis is currently unknown. Utilizing both pharmacological and genetic approaches, we investigated the role of the mevalonate pathway in symbiotic signaling in cereal crops and its effect on growth and development, using rice as a model cereal crop. Rice plants (wild-type and mutant lines) were grown on half-strength Hoagland medium for ten days, and the roots were treated with various signaling molecules such as mevalonate, germinating spore exudates (GSE) of AM fungi and chitin oligomers (COs, that mimic the AM symbiotic signals) or water (negative control). AM-specific gene expression was monitored in wild-type and mutant plants upon treatment with signaling molecules. We also tested the effects of MVA, GSE and COs on plant growth and development, such as the growth of shoots and roots, and lateral root formation. MVA induced the expression of OsAM1, OsAM3 and OsAM11 genes inwild-typerice similar to GSE and COs. However, the MVA-induced OsAM3 and OsAM11 gene expressions were absent in rice pollux mutant suggesting that the expressionof these genes are dependent on symbiotic signaling pathway. MVA induced the shoot growth and lateral root development in wild-type, but not in rice dmi3 mutant suggesting the role of oursignaling pathway in the MVA-induced growth and development. Objective 2: Identify new regulators of these symbiotic cation channels using a genetic suppressor screen.We estimate that about 90% of this objective was accomplished. We screened over 8,500 additional plants mutagenized (M2) in the presence of a knockout of cation channels required for the activation of the symbiotic pathway. We identified six new putative genetic suppressors by their ability to restore the expression of symbiotic genes in our colorimetric assays. One of these lines formed functional nodules. The suppressor's lines identified in the M2 generation were confirmed at the M3 generation for suppressor phenotypes. Currently, these confirmed suppressor lines from the M3 progeny are being crossed to the parental mutant to remove other unwanted mutations. However, our previous attempts to develop introgression lines for the mapping of our suppressor mutations were not successful due to genetic instability. Overall, the seed setting and germination rate was reduced (around 30%) for the F1 hybrid. Of which, only 15-20 % seeds showed introgression of the alleles requisite for our colorimetric assays of symbiotic pathway activation. However, the introgression of alleles of mutant symbiotic cation channels and suppressor mutations in the necessary genetic background could not be verified as the alleles requisite for our colorimetric assays were lost or not stable in F2 population. The success rate of controlled crosses was also affected by the higher incidence of spider mites and thrips infestation in the greenhouse during Summer 2017 in Co-PD's institution. During Fall 2017, we performed crosses (between the mapping parent, a mutant line of cation channel carrying marker for calorimetric assays, and three of the confirmed suppressor lines) to develop new introgression lines for mapping the suppressor mutations. The pods and seeds need to be collected from these crosses and screened for activity in our colorimetric assays, and those consistent with activation are currently being grown for seed multiplication and subsequent backcrossing.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Venkateshwaran, M., Rubeck, S.*, Ackerman, A.*, Theisen, N.*, Brimeyer, N.*, Welch, K.*, Arida, E.*, Annamalai, R. and An�, J.M. 2017. Role of volatile organic compounds in plant symbiotic and defense signaling pathways. Arabidopsis Research in 2017 and Beyond. 28th International Conference on Arabidopsis Research, Hyatt Regency, St. Louis, MO, USA. June 19-23, 2017. (oral presentation).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Venkateshwaran, M., Ackerman, A.*, Theisen, N.*, Pluemer, B.*, Ely, M.* and An�, J.M. 2017. Role of Mevalonate Pathway in Symbiotic Signaling in Cereal Crops. 2017 Plant Biology Meeting organized by the American Society of Plant Biologists (ASPB), Honolulu, HI, USA. June 24-28, 2017. (Poster presentation).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Brimeyer, N.*, Ackerman, A.*, Ely, M.*, Rubeck, S.*, Annamalai, R. and Venkateshwaran, M. 2017. Investigating the role of volatile organic compounds in plant symbiotic and defense signaling. 14th annual Research in the Rotunda: Showcasing UW Undergraduate Research held at Wisconsin Capitol, Madison, WI. April 12th, 2017. (Poster presentation).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Ackerman, A.*, Theisen, N.*, An�, J.M., Venkateshwaran, M. 2017. Role of mevalonate pathway in the symbiotic signaling in cereal crops. UW System Symposium for Undergraduate Research and Creative Activity, UW-Stevens Point, WI. April 21th, 2017. (Poster presentation).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Ely. M.*, Ackerman, A.*, Theisen, N.*, An�, J.M. and Venkateshwaran, M. 2017. Genetic suppressor screening to identify new regulators of symbiotic signaling in the model legume Medicago truncatula. UW System Symposium for Undergraduate Research and Creative Activity, UW-Stevens Point, WI. April 21th, 2017. (Poster presentation).
|
Progress 01/01/16 to 12/31/16
Outputs
Target Audience:State legislators, state leaders and general public Three undergraduate students working with Co-PD presented their research findings from this project to Wisconsin state legislators, state leaders and general public at the Posters in the Rotunda event held at the Wisconsin State Capitol on April 13, 2016. Posters in the Rotunda is an opportunity for students and their faculty advisors across the University of Wisconsin system to present undergraduate research on important topics to state legislators, state leaders, UW alumni and the public. Growers: The significance of plant nutrient acquisition to sustainable agriculture makes our research relevant not only to the scientific community but others interested in plant health. This target audience includes farmers, gardeners, and all voters concerned with the sustainability of our agriculture and its implications for ecosystem stability at the local and global level. We have worked to bridge the gap between our laboratory work and farmers by attending many outreach activities. We continue to make ongoing progress in our connections with the local community of gardeners and voters by participating in multiple community events year-round, including Family Horticulture Day, a springtime family event focused on bringing families together through gardening; Darwin Day, a fall event for graduate students, high school students and general public focused on instilling the participants with the joy and wonder of the discovery of biology; Agronomy/Soils Field Day and Grant County Family Fun Night, which highlight the research on emerging technologies and relevant crop production issues to farmers and those who are interested in sustainable agriculture. Our ongoing efforts to connect with local communities are essential not only for an alignment of our goals with the fulfillment of a societal need but a sustained public understanding of the importance of our research to societal wellbeing. Scientific community: The most relevant target audience is scientists in the field of plant-microbe interactions, as our work investigates one of the key questions regarding the regulation of symbiotic cation channels in the common symbiotic pathway, which are required for plant symbioses by both rhizobia and arbuscular mycorrhizal fungi. These target audiences have been served by our contributions to our field during the most recent funding period through our work with collaborators and our presentation of our work at multiple community-specific symposia/conferences (2016 American Phytopathological Society Annual Meeting, 2016 IS-MPMI XVII Congress, 2016 European Nitrogen Fixation Conference, 2016 Posters in the Rotunda at Wisconsin state capitol, School of Agriculture Internship Night at UW-Platteville). Through these contributions, we have continued to participate in an ongoing effort within our field to understand the mechanisms of plant-microbe symbioses to improve plant nutrient acquisition for the benefit of society. Students (undergraduate, high- and middle-school students): The project has been served as a base for the development of curriculum to teach principles and techniques about Plant Breeding, Plant Biotechnology, and Plant Physiology. Twenty-five students from Plant Breeding class (SCSCI 4240) taught by Co-PD during Fall 2016, learned the crossing techniques and the availability of genetic mapping tools in M. truncatula which are currently pursued through this project. Similarly, students of Plant Physiology class (SCSCI 4340; 51 students from Spring and Fall 2016) and Plant Development and Biotechnology class (SCSCI 3220; 30 students in Spring 2016) learned about gene expression analyses, promoter-GUS assays and Nod factor-induced ENOD11-GUS assays as plant-microbe symbiotic signaling events in demonstration conducted as a part this project. Co-PD held a half-day long outreach program at UW-Platteville on plant-microbe interactions (legume nodulation and biological nitrogen fixation) to 20 underrepresented middle school students through a Full STEAM program which is designed to bring underrepresented middle school student populations to campus to engage in STEM and Agriculture. This outreach activity was held on June 20, 2016. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?At the PD's laboratory, a graduate student (Mr. Shane Bernard) is working on the project to accomplish the tasks towards achieving our goals particularly towards specific objective 1. Mr. Bernard gained experience mentoring undergraduate researchers at the UW-Madison community and worked with the university's technology transfer office to help enhance networks within the UW-Madison community. During 2016, Mr. Bernard was assisted in this research by two graduate students at the University of Wisconsin-Madison, Brent Larsen, and Erin Sweeney. They have participated in cloning genetic constructs, transforming plant roots and assaying nodulation in Medicago truncatula. During the summer of 2016, he also mentored an undergraduate, Miguel Rosas, from the University of California-San Marcos, as part of a summer research experience for undergraduates. Miguel was trained in hairy root transformations and fluorescent microscopy and has expressed interest in attending graduate school to study plant biology. During Mr. Bernard's studies, he has begun to leverage his developing scientific expertise by interning with UW-Madison's technology transfer office to promote networking within the UW community. Specifically, he works to connect researchers across research departments to promote interdisciplinary collaborations and discovery within the scientific community. He also works to connect the university's researchers with technology transfer experts to help return the financial gains of scientific research to the UW-Madison research community. This participation in the UW community was made possible by the research experiences garnered during Mr. Bernard's scientific training. At the Co-PD's laboratory, eight undergraduate students (Mr. Kendell Welch, Mr. Arlyn Ackerman, Ms. Emily Hefty, Ms. Leanna Oltz, Ms. Larissa Ruchotzke, Mr. Tyler Nitz, Ms. Laura Declercq and Ms. Sara Rubeck) and one high-school student (Mr. Adam Compton) have been trained in this project since January 2016. These students have been trained in the fields of Plant Genetics and Breeding, Plant Molecular Biology, Microbiology, and in vitro culture techniques. Mr. Arlyn Ackerman pursued his Summer Research Internship which is a required course for his major in Soil and Crop Science, through working on this project and currently he is working as Research Specialist coordinating all the research activities on Objective 2. Similarly, Ms. Laura Declercq and Mr. Tyler Nitz obtained their research training through SCSCI 3390: Special Problems in Crop Science and ENVHORT: 3370 Undergraduate Research in Horticulture, respectively during Fall 2016. These students learned to work as a team, coordinate various project activities to accommodate research in their class schedules and in mentoring new students in their area of expertise. During the Summer 2016, the Co-PD traveled to the PD's Laboratory at UW-Madison to train an undergraduate student from UW-Platteville (Mr. Arlyn Ackerman) and a high school student (Mr. Adam Compton), on the use of confocal microscopy for calcium imaging and sub-cellular localization of candidate proteins in M. truncatula root hairs. An undergraduate student (Mr. Kendell Welch) who gained undergraduate research experience through working on this project since January 2015, graduated in May 2016 with a major in Environmental Horticulture from the University of Wisconsin-Platteville, who is currently working as Research Associate at Calyxt Inc., New Brighton, MN. The Co-PD attended 2016 American Phytopathological Society (APS) annual meeting held in Tampa, FL on July 30-August 3, 2016 and presented the data collected from this project as a poster titled "A band of misfits: the role of unexpected proteins in the plant symbiotic signaling pathway". The Co-PD attended 2016 USDA-NIFA/DOE Project Director Meeting held at San Diego, CA, on January 8, 2016, and presented the data collected from this project as a poster titled "Regulation of nuclear cation channels controlling the establishment of plant-microbe symbioses". The Co-PD also attended the 2016 XXIV International Plant and Animal Genome Conference held in San Diego, CA, on January 9-13, 2016. These professional meetings provided the Co-PD opportunities to learn recent developments in the field of molecular plant-microbe interactions and plant genomics, to meet our collaborators and for networking with eminent plant biologists. How have the results been disseminated to communities of interest?The results obtained from this research project have been disseminated as publications, posters and oral presentations at the conferences/symposium. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will continue to make progress towards our goal of understanding symbiotic cation channel regulation by identifying compounds responsible for the activation of the symbiotic pathway. Using in planta assays, we will screen compounds that induce calcium oscillations in root cells in Medicago truncatula. Previous analyses of putative compounds that trigger calcium spiking in M. truncatula root hair cells identified that isopentenyl pyrophosphate, one of the compounds in the isoprenoid pathway which is synthesized downstream of mevalonate, did not trigger either the nuclear calcium spiking in the root hair cells nor activation of our pathway in colorimetric assays. However, the absence of signaling upon exogenous application of isopentenyl pyrophosphate could be due to the lack of penetration of this compound into the root epidermal cells as a result of the compound's specific chemistry. To preclude the possibility of false negatives in our screen due to a lack of tissue penetration by candidates, we will expand our suite of candidate compounds within given pathways to include those with varying chemical characteristics. By combining our genetic and biochemical analyses with robust, relatively high-throughput analyses of candidate compounds, guided by a chemically rational narrowing of the putative candidates, these studies will allow us to screen candidate compounds while also verifying their biological significance rapidly. Following identification of secondary regulators of symbiotic cation channels, we will determine the evolutionary specificity or conservation of these messengers by competence to induce calcium oscillations in rice. These studies will describe the evolutionary origin and conserved functions of these messengers across legumes and cereals as part of our larger efforts to engineer nitrogen-fixing symbioses from legumes into cereal crops. Experiments are also in progress to decipher the transcriptomic changes in Medicago truncatula in the presence of mevalonate to determine the relative contribution of mevalonate to early gene expression in the symbiotic signaling pathway. These studies will describe the evolutionary origin and conserved functions of these messengers across legumes and cereals as part of our larger efforts to engineer nitrogen-fixing symbioses from legumes into cereal crops. Objective 2: Identify new regulators of these symbiotic cation channels using a genetic suppressor screen. Following our timeline for the development of a suppressor screen to identify new regulators of symbiotic cation channels, we will continue to screen for suppressors in a cation channel mutant background using the high-throughput procedure that we developed and optimized in the first year. An additional 50,000 lines of mutagenized M2 plants in this background will be analyzed for activity in colorimetric assays, and for nodulation phenotypes. To eliminate the false positives from our screen this year, the putative suppressors will be analyzed for the stable inheritance of suppressor phenotypes at the next generation (M3). Once putative suppressors are isolated, they will be grown to maturity and will be self-fertilized to produce an M3 progeny. First, plants from the M3 progeny will be reevaluated in colorimetric assays and nodulation. Like this year, the additional confirmed suppressor lines from the M3 progeny will be crossed to the parental mutant to remove unwanted additional mutations. To determine the pattern of inheritance of a suppressor mutation, the resulting F1 and F2 progeny from the above crosses will be phenotypically analyzed in colorimetric nodulation assays. For genetic mapping and subsequent cloning of suppressor genes, we will continue to screen the seeds obtained from crossing the mapping parent. These introgressed lines will be confirmed for the presence of alleles of the cation channel mutations and alleles required for colorimetric assays by PCR.
Impacts
What was accomplished under these goals?
Objective 1: Characterize the regulation of symbiotic cation channels by candidate second messengers: We estimate that about 60% of this objective was accomplished. During the last year, we developed tools for the identification of new compounds that participate in activation of the common symbiotic pathway and identified candidate messengers via inter-laboratory collaboration. To identify new compounds that participate in the activation of the common symbiotic pathway, we developed both genetic and biochemical assays to verify candidates. In our genetic validation studies, we used colorimetric assays that produce a visible compound within plant tissues to indicate activation of the symbiotic pathway. We optimized this protocol to increase the signal to noise ratio in these assays, generating a more throughput approach. In our biochemical validation of candidates, we took multiple approaches to the development of a high throughput assay: we assayed the activation of calcium oscillations in human embryonic kidney cells transformed with cation channels from the symbiotic pathway, and activation of the symbiotic pathway in planta. We determined that in planta assays are the most throughput and direct approach for the identification of candidate compounds, and are developing tools to test the compounds that produce symbiotic calcium oscillations in vivo. These tools are being used in our ongoing efforts to identify key regulators of symbiotic cation channels. In addition to our ongoing screen of cation channel regulators, we continued to engage in collaboration with a team of protein structural biologists and, using assays of Medicago truncatula symbioses with symbiotic partners, identified residues in symbiotic cation channels that are responsible for binding to ionic secondary messengers, demonstrating that specific amino acids regulate the activity of cation channels in the symbiotic pathway to control plant-microbe symbioses. These studies fit into our larger goal of identifying the precise secondary messengers responsible for symbiotic cation channel regulation. Objective 2: Identify new regulators of these symbiotic cation channels using a genetic suppressor screen: We estimate that about 70% of this objective was accomplished. During this period, we continued our genetic suppressor screening in two different experimental set up to enhance the sensitivity of the assay as detailed in our previous report. We screened over 12,000 additional plants mutagenized (M2) in the presence of a knockout of cation channels required for the activation of the symbiotic pathway. On an average, we observed a 35% germination for these M2 seeds (two new seed batches), which was comparatively a lower germination rate than the previous batches of seeds used last year. We identified 14 new putative genetic suppressors by their ability to restore the expression of symbiotic genes in our colorimetric assays. The suppressor's lines identified in the M2 generation that demonstrated both an activation of the symbiotic pathway in colorimetric assays and nodulation phenotypes were confirmed at the M3 generation for 6 of the newly identified suppressor lines. Currently, these confirmed suppressor lines from the M3 progeny are being crossed to the parental mutant to remove other unwanted mutations. To develop introgression lines for the mapping of our suppressor mutations, we screened seeds from our previous crossings for introgression of the alleles requisite for our colorimetric assays of symbiotic pathway activation, and alleles of mutant symbiotic cation channels, in the requisite genetic background. Seeds were collected from these crosses and screened for activity in our colorimetric assays, and those consistent with activation are currently being grown for seed multiplication and subsequent backcrossing.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Venkateshwaran, M., Wiley-Kalil, A., Jayaraman, D., Banba, M., Binder, A., Bernard, S., Maeda, J., Otegui, M., Imaizumi-Anraku, H., Parniske, M., An�, J.M. 2016. A band of misfits: the role of unexpected proteins in the plant symbiotic signaling pathway. Poster presented at the 2016 Annual Meeting of American Phytopathological Society, Tampa, FL, USA (July 30-August 3, 2016).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Venkateshwaran, M., Wiley-Kalil, A., Jayaraman, D., Banba, M., Binder, A., Bernard, S., Maeda, J., Otegui, M., Imaizumi-Anraku, H., Parniske, M., An�, J.M. 2016. A band of misfits: the role of unexpected proteins in the plant symbiotic signaling pathway. Poster presented at the XVII Congress of International Society of Molecular Plant-Microbe Interactions (IS-MPMI), Portland, Oregon, USA (July 17-21, 2016).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Hefty, E.*, Welch, K.*, Oltz, L., An�, J.M., and Venkateshwaran, M. 2016. Genetic suppressor screening to identify new regulators of symbiotic genes in the model legume Medicago truncatula. Poster presented at the 13th annual Posters in the Rotunda: Showcasing UW Undergraduate Research held at Wisconsin Capitol, Madison, WI. (April 13th, 2016).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Venkateshwaran, M., Wiley-Kalil, A., Bernard, S., Welch, K., Otegui, M. and Ane, J.M. 2016. Regulation of nuclear cation channels controlling the establishment of plant-microbe symbioses. Poster presented at the 2016 USDA-NIFA/DOE Project Director Meeting, San Diego, CA, USA (January 8, 2016).
- Type:
Journal Articles
Status:
Submitted
Year Published:
2016
Citation:
Wiley-Kalil, A., Binder, A., Han, L., Li, W., Venkateshwaran, M., Maeda, J., Bernard, S., Delaux, P.M., Mysore, K., Wen, J., Imaizumi-Anraku, H., Parniske, M., Otegui, M. and An�, J.M. NUP107-160 nuclear pore sub-complex members are required for the proper localization of symbiotic ion channels in Lotus japonicus and Medicago truncatula. Manuscript submitted to New Phytologist.
|
Progress 01/01/15 to 12/31/15
Outputs
Target Audience: Scientific community The most relevant target audience is scientists in the field of plant-microbe interactions, as our work investigates one of the key questions regarding the regulation of DMI1 in the common symbiotic pathway, which is required for plant symbioses by both rhizobia and arbuscular mycorrhizal fungi. These target audiences have been served by our contributions to our field during the most recent funding period through our work with collaborators, our presentation of our work at multiple community-specific symposia/conferences (Plant Biology 2015-ASPB, International Molecular Mycorrhiza Meeting 2015, School of Agriculture Internship Night at UW-Platteville) and through the publication of our work as scientific literatures, specifically in reputed articles, such as PNAS and the Plant Cell, which have broad readership. Through these contributions, we have continued to participate in an ongoing effort within our field to understand the mechanisms of plant-microbe symbioses in order to improve plant nutrient acquisition for the benefit of society. Growers and General Public The significance of plant nutrient acquisition to sustainable agriculture makes our research relevant not only to the scientific community, but to others interested in plant health. This target audience includes farmers, gardeners, and all voters concerned with the sustainability of our agriculture and its implications for ecosystem stability at the local and global level. We have worked to bridge the gap between our laboratory work and farmers by attending many outreach activities. We continue to make ongoing progress in our connections with the local community of gardeners and voters by participating in multiple community events year-round, including: Family Horticulture Day, a spring-time family event focused on bringing families together through gardening; Darwin Day, a fall event for graduate students, high school students and general public focused on instilling the participants with the joy and wonder of discovery of biology; Agronomy/Soils Field Day and Grant County Family Fun Night, which highlight the research on emerging technologies and relevant crop production issues to farmers and those who are interested in sustainable agriculture. Our ongoing efforts to connect with local communities are essential not only for an alignment of our goals with the fulfillment of societal need, but for a sustained public understanding of the importance of our research to societal well-being. Undergraduate Students The project has been served as a base for the development of curriculum to teach principles and techniques pertaining to Plant Breeding, Plant Biotechnology and Plant Physiology. Twenty one students from Plant Breeding class (SCSCI 4240) taught by Co-PD during Fall 2015, learned and appreciated the crossing techniques and the availability of genetic mapping tools in M. truncatula which are currently pursued through this project. Similarly, students of Plant Physiology class (SCSCI 4340; 47 students from Spring and Fall 2015) and Plant Development and Biotechnology class (SCSCI 3220; 28 students in Spring 2015) learned about gene expression analyses, promoter-GUS assays and Nod factor-induced ENOD11-GUS assays as plant-microbe symbiotic signaling events in demonstration conducted as a part this project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? At the PD's laboratory, a graduate student (Mr. Shane Bernard) is working on the project to accomplish the tasks towards achieving our goals particularly towards our specific objective 1. This project provided the opportunity for the graduate student working on this project to attend the International Molecular Mycorrhizal Conference (iMMM), where he gathered insight from former lab members and collaborators whose work inspired this project. Attendance at iMMM also allowed him to network with experts in our field, both providing new insights into both our research and promoting his development as a scientist. Our research project also permitted this graduate student to visit a collaborating lab, where he learned newer, faster protocols for a method that is being applied directly to his research, as well as the research occurring throughout our laboratory. This method, called fluorescence microscopy, couples the production of light with DMI1's activation such that, whenever DMI1 translates a signal, light is produced within the cell's nucleus. By observing the conditions required for DMI1 to translate signals and produce light, we can begin to determine how DMI1 translates complicated messages into a single signal for the nucleus to interpret. Our current method permits observation of a few plant nuclei prepared under laborious preparatory conditions. However, our collaborators have designed a protocol for the rapid preparation and imaging of these samples, resulting in a significant decrease in the time required for sample preparation and imaging. This protocol will allow our researchers to perform many more experiments and save our laboratory dozens of research hours in the coming years. This research is being completed by Mr. Bernard with assistance from his undergraduate mentee (Brent Larsen), who has been trained in the methods of hairy root transformation to assist in these studies. At the Co-PD's laboratory, four undergraduate students (Ms. Katie Martin, Ms. Emily Hefty, Mr. Kendell Welch, Ms. Leanna Oltz) have been trained in this project since spring 2016, and 3 of them (Ms. Emily Hefty, Mr. Kendell Welch, Ms. Leanna Oltz) are currently working on accomplishing the tasks towards achieving our goals, particularly towards our specific objective 2. These students have been trained in the fields of Plant Genetics and Breeding, Plant Molecular Biology, Microbiology, and in vitro culture techniques. Mr. Kendell Welch, pursued his Summer Research Internship which is a required course for his major in Environmental Horticulture, through working on this project and currently he is working as Research Specialist coordinating all the research activities pertaining to Objective 2. Similarly, Ms. Leanna Oltz, Ms. Emily Hefty and Ms. Katie Martin obtained their research training through SCSCI 3390: Special Problems in Plant Biotechnology, BIOLOGY 4920: Special Problems in Biology and ENVHORT: 3370 Undergraduate research in Ornamental Horticulture, respectively during spring 2015. While Ms. Katie Martin has graduated, currently Ms. Emily Hefty, Ms. Leanna Oltz are working as student help to accomplish the project tasks, specifically towards Objective 2. These students learned to work as a team, coordinate various project activities to accommodate research in their class schedules and in mentoring new students in their area of expertise. During spring and summer 2015, the Co-PD traveled to the PD's laboratory at UW-Madison to train the graduate student (Mr. Shane Bernard) and a visiting scholar, Judith Salles from the Valdés-López Lab the University of Mexico, on the use of confocal microscopy for calcium imaging in M. truncatula root hairs. During these training sessions, Mr. Kendell Welch (from UW-Platteville) accompanied Co-PD to learn the techniques of calcium imaging and confocal microscopy. The Co-PD attended 2015 Plant Biology annual meeting organized by American Society of Plant Biologists (ASPB) held at Minneapolis, MN, USA on July 26-30, 2015 and presented the data collected from this project as a poster titled "Role of mevalonate pathway in plant-microbe symbiotic signaling pathway". This also provided the Co-PD an opportunity to learn recent developments in the field of molecular plant-microbe interactions, to meet our collaborators and for networking with eminent Plant Biologists. How have the results been disseminated to communities of interest? The results obtained from this research project have been disseminated as publications, poster and oral presentations at the conferences/symposium. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will continue to make progress towards our goal of understanding DMI1 regulation by identifying compounds responsible for DMI1 activation in legumes. Following identification, we will characterize their role in mediating the formation of plant-microbe symbioses in legumes and examine their possible role in plant-microbe signaling in cereals such as rice. I- Characterize the regulation of symbiotic cation channels by candidate second messengers Previous analyses of putative compounds that trigger calcium spiking in M. truncatula root hair cells identified that isopentenyl pyrophosphate (IPP), one of the compounds in the isoprenoid pathway which is synthesized downstream of mevalonate, did not trigger either the nuclear calcium spiking in the root hair cells or ENOD11 expression which is evident from pENOD11-GUS assays. However, this lack of these signaling events upon exogenous application of IPP could be due to the lack of penetration of this compound into the root epidermal cells due to its specific chemistry. To assay compounds that may provide false-negatives due to a lack of tissue penetration, we will assay candidate second messengers/regulators in the Human Embryonic Kidney 293 cell system that we have established to analyze the potential ligands that trigger calcium spiking. Combining different approaches, such as pENOD11-GUS expression and calcium imaging in HEK293 cells, will allow us to both rapidly screen candidate compounds while also verifying their biological significance. Following the identification of secondary messengers that activate DMI1, we will characterize their role in mediating the formation of plant-microbe symbioses in legumes by examining how these messengers contribute to calcium spiking as triggered by mycorrhizal and rhizobial symbiotic signals to determine the relative contribution of these messengers to symbiotic calcium spiking. To determine the evolutionary specificity or conservation of these messengers, we will examine their role in plant-microbe signaling in rice using rice line stably transformed with a calcium sensitive reporter to assay calcium spiking induced by candidate messengers. These studies will describe the evolutionary origin and conserved functions of these messengers across legumes and cereals as part of our larger efforts to engineer nitrogen fixing symbioses from legumes into cereal crops. II- Identify new regulators of these symbiotic cation channels using a genetic suppressor screen. We will continue to screen for suppressors in dmi1-2 mutant background using the high-throughput procedure that we developed and optimized this year. We also conduced another mutagenesis experiment to generate more seeds for screening purposes. Mutagenesis was carried out on 4,000 homozygous dmi1-2 mutant seeds using 0.15% ethyl methanesulfonate (a chemical mutagen) at the PD laboratory. The pods and seeds will be collected from these mutagenized population will be grown to obtain M2 population. Approximately 100,000 lines of mutagenized M2 plants in dmi1-2 mutant background will be analyzed for ENOD11 expression and nodulation phenotypes. In order to eliminate the false positives from our screen this year, the putative suppressors will be analyzed for the stable inheritance of suppressor phenotypes (ENOD+ Nod- or ENOD- Nod+ or ENOD+ Nod+; here ENOD refers to ENOD11 expression, Nod refers to nodulation) at the next generation (M3). Once putative suppressors are isolated they will be grown to maturity and will be self-fertilized to produce an M3 progeny. First, plants from the M3 progeny will be reevaluated for ENOD11 expression and nodulation. Like this year, the additional confirmed suppressor lines from the M3 progeny will be crossed to the parental mutant, dmi1-2(pENOD11:GUS), to remove unwanted additional mutations. To determine the pattern of inheritance of a suppressor mutation, the resulting F1 and F2 progeny from the above crosses (e.g. suppressor line for dmi1-2 mutant × dmi1-2(pENOD11:GUS) will be phenotypically analyzed for ENOD11 expression and nodulation. For genetic mapping and subsequent cloning of suppressor genes, this year we crossed the mapping parent A20 with A17 (pENOD11-GUS) and dmi1-2 (pENOD11-GUS). We will collect the pods and seeds from these crosses and analyze for the introduction (introgression) of pENOD11-GUS and dmi1-2 mutation in A20 line. This introgressed line will be confirmed for the presence of dmi1-2 mutation and pENOD11:GUS via PCR and sequencing.
Impacts
What was accomplished under these goals?
Our project aims at understanding the molecular mechanism of legume-rhizobia symbiosis in the model legume, Medicago truncatula. Specifically, we aim to understand how a symbiosis-related protein, named Does not Make Infections 1 (DMI1) in M. truncatula is regulated during legume-rhizobia symbiotic interactions. Medicago plants that are mutants for DMI1 cannot associate with either rhizobia or arbuscular mycorrhizal fungi (another group of symbiotic microorganisms necessary for plants' nutrient acquisition, including phosphorous and nitrogen). DMI1 is a cation channel that resides on the nuclear envelope within a plant cell, particularly in the root hair cells where the legume-rhizobia interaction begins. Besides understanding the regulatory mechanism of Medicago DMI1, we are also interested in understanding how its close relatives, such as CASTOR and POLLUX proteins, in rice and other non-leguminous plants are regulated. In order to accomplish our goals, we have utilized pharmacological, biochemical and genetic approaches to make significant progress under the following specific objectives. I- Characterize the regulation of symbiotic cation channels by candidate second messengers During legume-rhizobia interactions, the legume plants recognize the presence of rhizobia (their symbiotic partners) through the perception of symbiotic signaling molecules known as Nod factors. The perception of rhizobial Nod factors triggers a cascade of events (signal transduction) during which one gene activates another gene and so on, until the necessary responses are achieved. This includes accommodation of rhizobia in a specialized organs, known as root nodules. In general, signal transduction involves four main components: signals, receptors, signal transduction machinery and response. Among the signal transduction machinery, second messengers play a crucial role. The second messengers are small molecules produced upon the activation of genes upstream in the cascade right after the perception of symbiotic signals. These second messengers then activate genes which play roles downstream in the signal transduction cascade, such as DMI1. During the 2015 funding period, we identified mevalonate (MVA) as one such candidate second messenger. MVA is the product of biochemical reaction catalyzed by the enzyme HMG CoA Reductase (HMGR). In M. truncatula, HMGR1 has been shown to play a role in legume-rhizobia and arbuscular mycorrhizal symbioses. During this period, we have shown that exogenous application of MVA triggers sustained calcium oscillations (known as calcium spiking) in the nuclear and peri-nuclear region in the root hair cells of legumes (M. truncatula and Lotus japonicus) and non-legumes (rice and carrot). It is interesting to note that MVA is a ubiquitous metabolite in many living organisms. MVA-induced nuclear calcium spiking is analogous to Nod factor-induced nuclear calcium spiking, suggesting that an increase in the cellular MVA level might be responsible for the nuclear calcium spiking. We also showed that this phenomenon is conserved not only in legumes but also in non-leguminous plants. Our data also suggest that MVA likely activates DMI1 directly, resulting in nuclear calcium spiking during legume-rhizobia symbiotic signaling. Our data regarding the role of the mevalonate pathway in the regulation of DMI1 was recently published in PNAS, providing our community with the first connection between plasma membrane recognition of microbial signals and consolidation of these signals in the nucleus (Venkateshwaran et al., 2015). In addition to our ongoing screen for the regulators of DMI1, we recently engaged in a collaboration with a team of protein structural biologists, yielding new data regarding the role ionic secondary messengers in DMI1 regulation. This collaboration, in combination with our ongoing efforts to identify DMI1 regulators, will permit us to achieve our goal of identifying the secondary messengers responsible for DMI1 regulation. II- Identify new regulators of these symbiotic cation channels using a genetic suppressor screen. In parallel to the targeted approaches on identifying specific second messengers, we also pursued a genetic approach, known as suppressor screening, to identify the genes and the gene products that regulate DMI1. As mentioned above, the dmi1 mutants are affected for symbiotic signaling responses, hence do not form root nodules. In the genetic suppressor screening approach, we took a mutant allele of DMI1 (dmi1-2), and introduced additional mutations in this mutant and looked for the reversal of phenotypes (their ability to form root nodules). In addition, we are screening for the expression of a symbiosis-specific marker gene, known as Early Nodulin 11 (ENOD11). The expression of ENOD11 in the root epidermal cells is induced upon the perception of rhizobia or Nod factors, and the expression can be detected through a visual screening using a reporter, GUS enzyme (beta-galactosidase). Briefly, the promoter of the gene ENOD11 is fused to GUS reporter and this cassette was stably transformed in the mutant population for easy visual screening to identify genetic suppressors. Upon the perception of rhizobia or Nod factors, the promoter of ENOD11 will induce the expression of GUS reporter gene, leading to the production of the enzyme, beta-galactosidase. This enzyme will convert a colorless substrate (X-Gluc) to a blue color product, staining the tissues (where ENOD11 expression supposed to happen) blue in a tissue-specific manner. During this period, genetic suppressor screening was performed in two different experimental set up (on trays filled with Turface® and sand, and on plates over Fahraeus medium, a plant growth medium) to enhance the sensitivity of the assay. For screening on the 1:1 mix of Turface® : Sand in a tray, we followed the procedures outlined in our proposal. In the alternative experimental set up (plates), the A17 (pENOD11-GUS) and M2 plants were grown on the Fahraeus medium (plant growth medium) plates for 7 days at the rate of 15 plants per plate, and inoculated with rhizobia. This experimental set up was considered because, A17 (pENOD11:GUS) lines displayed visibly striking blue coloration during the vital-GUS assay, indicating the expression of pENOD11:GUS. Likewise, when incubated for an additional period of 7 days post vital-GUS staining, the A17 (pENOD11-GUS) seedlings formed root nodules enabling nodulation assays on the same seedlings which were screened through vital-GUS assay. During this period, we have screened over 8,000 dmi1-2(pENOD11:GUS) mutagenized (M2) plants. On an average, we observed 55% germination for dmi1-2(pENOD11:GUS) M2 seeds. We identified 10 putative genetic suppressors by their ability to restore ENOD11 expression. In each experiment, we used A17(pENOD11:GUS) plants as positive controls. Genetic suppressors isolated were grown in the growth-chamber and allowed to self-fertilize to produce M3 progeny. The ENOD11 expression and nodulation phenotypes of suppressor lines identified in M2 generation have been confirmed at the M3 generation. Currently, these confirmed suppressor lines from the M3 progeny are being crossed to the parental mutant, dmi1-2(pENOD11:GUS), to remove other unwanted mutations. Now that we have identified few putative genetic suppressors of dmi1-2, our next step is to identify the mutations (or corresponding genetic loci of the suppressor genes) which resulted in the reversal of dmi1-2 phenotype. For genetic mapping and subsequent cloning of suppressor genes, we need to transfer these mutations to A20 ecotype of M. truncatula, which will be used as mapping parent. Hence, we performed crosses between A20 × A17 (pENOD11-GUS), A20 × dmi1-2 (pENOD11-GUS) and A20 × putative suppressor. We will collect the pods and seeds from these crosses and analyze for the introgression of pENOD11-GUS and dmi1-2 mutation in A20 line.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Venkateshwaran, M., Jayaraman, D., Chabaud, M., Genre, A., Balloon, A.J., Maeda, J., Forshey, K., Den Os, D., Kwiecien, N.W., Coon, J.J., Barker, D.G., An�, J.M. 2015. A role for the mevalonate pathway in early plant symbiotic signaling. Proceedings of the National Academy of Sciences USA, 112: 97819786.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Sun, J. Miller, J.B., Granqvist, E., Wiley-Kalil, Gobbato, E., Maillet, F., Cottaz, S., Samain, E., Venkateshwaran, M., Fort, S., Morris, R.J., Ane, J.M., Denarie, J. and Giles, E.D. Activation of symbiosis signalling by arbuscular mycorrhizal fungi in legumes and rice. Submitted to The Plant Cell, 27: 823838.
- Type:
Book Chapters
Status:
Accepted
Year Published:
2015
Citation:
Venkateshwaran, M. 2015. Exploring the feasibility of transferring nitrogen fixation to cereal crops. In: Principles of Plant-Microbe Interactions Microbes for Sustainable Agriculture (Ben Lugtenberg ed.) Springer International Publishing AG, Cham, Switzerland.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2015
Citation:
Wiley-Kalil, A., Binder, A., Han, L., Li, W., Venkateshwaran, M., Maeda, J., Bernard, S., Delaux, P.M., Mysore, K., Wen, J., Imaizumi-Anraku, H., Parniske, M., Otegui, M. and An�, J.M. NUP107-160 nuclear pore sub-complex members are required for the proper localization of symbiotic ion channels in Lotus japonicus and Medicago truncatula. The manuscript is in preparation for submission to The Plant Cell.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Venkateshwaran, M., Jayaraman, D., Chabaud, M., Genre, A., Barker, D.G., An�, J.M. Role of mevalonate pathway in plant-microbe symbiotic signaling pathway. Poster presented at the Plant Biology 2015 organized by American Society of Plant Biologists, Minneapolis, MN, USA (July 26-30, 2015).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Welch, K.R. and Venkateshwaran, M. Genetic suppressor screening to identify novel genes in the legume-rhizobia symbiotic signaling. Poster presented at the School of Agriculture Internship Night held at the University of Wisconsin-Platteville, WI, USA (September 28, 2015).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Bernard, S., Wiley-Kalil A., Binder, A., Banba, M., Venkateshwaran, M., Madea, J., Otegui, M., Imaizumi-Anraku, H., Parniske, M., An�, J.M. Nup107-160 nuclear pore sub-complex members are required for the proper localization of symbiotic ion channels in Lotus japnicus and Medicago truncatula. Poster presented at the International Molecular Mycorrhiza Meeting 2015, Cambridge, UK (September 3-4, 2015).
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