PLANT-MICROBE COMMUNICATION IN THE MEDICAGO TRUNCATULA RHIZOSPHERE: FUNCTIONAL METAGENOMICS, BIOCHEMISTRY, AND COMMUNITY ANALYSIS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0224544
• Grant No.: 2011-67020-30195
• Cumulative Award Amt.: $452,000.00
• Proposal No.: 2010-04981
• Project Start Date: Jun 1, 2011
• Project End Date: May 31, 2016
• Grant Year: 2011
• Program Code: [A1401]- Foundational Program: Soil Health

Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001

Performing Department
Plant and Soil Sciences

Non Technical Summary
Legumes (Fabaceae) comprise one of the largest and most diverse families of vascular plant on the planet, with an estimated 18,000 species spread across all continents except for Antarctica. The Papilionoidaea legume subfamily includes the majority of legumes of agronomic value, such as alfalfa, soybean, chickpea, garden pea, and lentil. Estimates of the dietary intake of legumes show that this family is responsible for ~1/3 of the total protein intake in the human diet. In addition to their human dietary importance, certain legumes (e.g. alfalfa) are major forage crops important in sustaining livestock agriculture worldwide. Most legume species have the ability to form symbioses with nitrogen-fixing bacteria. This is an unusual yet important feature, shared among few plants outside the legume family, enabling them to thrive in environments with minimal nitrogen input. Due to their nitrogen-fixing ability, they are also used as a rotation crop to enhance available soil nitrogen. Barrel medic (Medicago truncatula) has been studied extensively as a model legume since the 1990s, and work on this plant and its associated microbiota has resulted in significant advances in understanding of the basic biology of the legume, mechanisms of plant-microbe communication, and the biology of symbiotic nitrogen fixation. It is now recognized that bacteria and fungi accumulate in large, diverse populations at the plant root-soil interface (the rhizosphere). This microbial community accumulates to take advantage of the rich collection of nutrients secreted by the plant roots (plant root exudate). Some plant root exudate chemicals are demonstrated to act as signals to specific microbes. These signals can result in alterations to the rhizosphere microbial community that enhance plant nutrient uptake, aid in suppression of plant pathogens, and enable a symbiotic relationship between plant and microbe resulting in nitrogen fixation by certain bacteria in legume plant root nodules. We aim to identify genetic features of the plant that enable optimal microbial community assembly in the Medicago truncatula rhizosphere. While our work will be done with a legume, we strongly believe that the research will be relevant to rhizosphere microbial community assembly and function in all agriculture crops. This work will result in an increased knowledge of signaling pathways between plant and microbe that enhance plant health, plant productivity and sustainability of the agricultural system. Ultimately, crop breeders and plant genetic engineers will use knowledge generated from this research to select for crop varieties that will optimize their rhizosphere microbial community to enhance these features.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
102	0110	1100	50%
102	1419	1040	25%
102	4010	1040	25%

Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships;

Subject Of Investigation
1419 - Leguminous vegetables, general/other; 0110 - Soil; 4010 - Bacteria;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology;

Keywords

metagenomics

plant microbe communication

medicago truncatula

rhizosphere

microbial community

Goals / Objectives
The long-term goals of the project are to advance understanding of plant-microbe communication and to understand how genetic diversity within a plant species leads to changes in the taxonomic and functional diversity of its associated rhizosphere microbial community. The objectives described in the proposal focus on several different aspects of these goals. We will identify changes in the Medicago truncatula rhizosphere microbial community as a function of M. truncatula genotype. We have obtained a number of M. truncatula genotypes that display phenotypic variance suggestive of altered microbial signaling capacity. We will characterize features of these plants that may result in changes to the rhizosphere microbial community, such as root architecture and root exudate profile. We will analyze the microbial content of the rhizosphere for each genotype and identify shifts in community structure. We will develop an operon-trap functional metagenomic screen to isolate genes and operons from the M. truncatula rhizosphere metagenome that are up-regulated in the presence of prominent M. truncatula root exudate compounds. DNA sequences from those recombinant clones identified from the screen will be sequenced. The resulting genes and operons will be analyzed to determine the genes, and consequently biological functions, encoded within the DNA sequences and to identify the mechanisms by which these genes are regulated. These expressed biological functions can therefore be attributed to the presence of certain root exudate compounds, and will be expressed in the rhizosphere of those M. truncatula genotypes that include the compound as part of the plant root exudate. The diversity of biological functions expressed in the rhizosphere (which we define as the functional diversity) will be analyzed with respect to those compounds that induce their expression. Thus, the M. truncatula genetic diversity will be linked with both the taxonomic diversity of the rhizosphere as well as the functional diversity.

Project Methods
Agricultural soil from the University of Kentucky horticulture research farm will be collected and used for growth of Medicago truncatula and five M. truncatula mutants in individual rhizobox apparatuses. Plant root exudates and root features will be analyzed among multiple replicates of each genotype and consistent differences in root biochemical features will be noted. Root exudate compounds that differ between genotypes will be structurally characterized. The exudate will be chemically characterized using multiple analytical techniques, including HPLC, HPLC-MS, GC-MS, ESI-MS and NMR where appropriate. The taxonomic diversity of the rhizosphere metagenome from multiple replicates of each M. truncatula genotype will be compared using T-RFLP of 16S rRNA amplicons. Metagenomic DNA libraries will be constructed from the M. truncatula rhizosphere in an "operon-trap" vector. This vector will be designed to identify genes and operons that are upregulated in the presence of individual root exudate compounds. The vector construct will encode two reporter genes (on either side of the metagenomic DNA cloning site) to enable functional screening for readthrough expression in both directions. Depending on which positive report a metagenomic DNA clone is found to produce, it will be apparent in which direction readthrough expression is occurring. Following library construction, the resultant M. truncatula metagenomic DNA library will be screened using fluorescence activated cell sorting in the presence of certain M. truncatula root exudate compounds. Positive recombinant clones will be identified as those that exhibit fluorescence in the presence of the compound but not in its absence. The DNA inserts from positive clones will sequenced using a 454 pyrosequencing run. Once the metagenomic DNA inserts have been sequenced and their inserts assembled, bioinformatic analysis will give information on the identity of the regulatory gene, the biological process mediated by the upregulated gene/operon, and the likely origin of the metagenomic DNA insert. Each gene/operon uncovered will correspond to a different element of functional diversity in the M. truncatula rhizosphere. By compiling these elements of functional diversity, and attributing their expression to particular root exudate compounds, we will be able to infer how plants can alter the functional diversity of their rhizosphere by changing their root exudate profile.

Progress 06/01/11 to 05/31/16

Outputs
Target Audience:The target audience is scientists with an interest in root-microbe interactions. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Students had the opportunity to attend the Argonne Soil Metagenomics conference and the American Society for Microbiology Conference. The PI attended the American Society for Microbiology Conference, the International Symposium on Phyllosphere Microbiology, and the International Symposium on Microbial Ecology. How have the results been disseminated to communities of interest?See publication list. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In the last review period, we had a number of publications come to fruition. The most prominent work related to plant-microbe interactions at the root surface relates to the Szoboszlay, et al. 2016 publication. We developed a "fake root" system and used this to probe how flavonoids commonly found in Medicago root exudate will impact a soil microbial community. We identified differences using 16S rRNA amplicon sequencing and using synthetic flavonoids 7,4'-Dihydroxyflavone and Naringenin.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Szoboszlay M, White-Monsant A, Moe LA. (2016) The Effect of Root Exudate 7,4'-Dihydroxyflavone and Naringenin on Soil Bacterial Community Structure PLoS One. 11(1):e0146555. doi:10.1371/journal.pone.0146555.
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Radkov AD, McNeill K, Uda K, Moe LA. (2016) d-Amino Acid Catabolism is Common Among Soil-Dwelling Bacteria Microbes Environ. 31(2):165-8. doi:10.1264/jsme2.ME15126.
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Yang Q, Wang R, Ren S, Szoboszlay M, Moe LA. (2016) Practical Survey on Antibiotic-Resistant Bacterial Communities in Livestock Manure and Manure-Amended Soil J Environ Sci Health B. 51(1):14-23. doi:10.1080/03601234.2015.1080481.
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: An R, Moe LA. (2016) Regulation of Pyrroloquinoline Quinone-Dependent Glucose Dehydrogenase Activity in the Model Rhizosphere-Dwelling Bacterium Pseudomonas putida KT2440 Appl Environ Microbiol. 82(16):4955-64. doi:10.1128/AEM.00813-16.

Progress 06/01/13 to 05/31/14

Outputs
Target Audience: The target audience during the reporting period is scientists involved in research related to rhizosphere science, plant-microbe interactions, soil microbiology, and metagenomics. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Within the past year, the PI has had the opportunity to attend the following conferences: Argonne soil metagenomics conference, University of Kentucky Natural Products Consortium conference, Kentucky Innovation and Entrepeneurship Conference, Institute of Biological Engineering conference, and the American Society for Microbiology general meeting. The PI engaged in outreach opportunities with the Kentucky Governor's scholars high school summer program, and served in various administrative and mentoring capacities within the Agricultural Biotechnology undergraduate program at the University of Kentucky. The graduate student funded by the grant has had the opportunity to give lectures in a graduate level microbiology course on campus, and is currently mentoring an undergraduate student who is participating in this project. The graduate student also applied to, was accepted to, and attended the 6-week summer course on microbial diversity offered by the Marine Biological Laboratory. How have the results been disseminated to communities of interest? Results from this work have been disseminated through publication in appropriate scholarly journals. What do you plan to do during the next reporting period to accomplish the goals? During the next reporting period, we will continue with data analysis on the flavonoid content. We will conduct additional experiments using the synthetic root system, and will continue with analysis of data on the current experiments. We will analyze the rhizosphere microbial communities from the Medicago truncatula and mutant experiments.

Impacts
What was accomplished under these goals? 1) We have developed a sterile, hydroponic plant growth system that can be used for obtaining plant root exudate under sterile growth conditions. We have processed the exudates from wild type Medicago truncatula and mutants Ifs-1, Ifs-2, and DMI01. Lyophilized exudate material has been analyzed at the West Coast Metabolomics Center by HPLC-MS/MS to identify and quantitate flavonoids present in the exudates. Analysis of flavonoid profiles for each plant is currently underway. Preliminary analysis reveals a handful of flavonoids that are highly abundant, including 7,4'-dihydroxyflavone and naringenin. 2) We have developed a system for studying the impact of individual compounds on soil microbial communities using a synthetic root system. This grew out of a desire to assess how individual flavonoids, which can differ from plant to plant, can impact the structure and function of microbial communities. Using the artificial root system, we have run experiments with addition of a simple mock exudate comprised of organic acids, sugars, and amino acids. We have also run experiments using the mock exudate supplemented with increasing concentrations of either 7,4-dihydroxyflavone or naringenin. From the "rhizosphere" of the synthetic roots, we analyzed microbial community structure using 16S rRNA gene amplicon sequencing on the Illumina MiSeq platform. We also assessed total microbial biomass using soil ATP assays, and are in the process of running enzyme assays to characterize soil nutrient cycling. We are optimizing an operon-trapping methodology to identify genes and operons responsive to the compounds of interest from the "rhizosphere" metagenomes. Soil ATP assays reveal that the total biomass in the "rhizosphere" samples is not significantly different between treatments. Statistical analysis of the 7,4'-dihydroxyflavone 16S amplicon data compared to the mock control reveals significant differences in a number of taxa. Differences in community structure are also noted from samples with varying amounts of 7,4'-dihydroxyflavone added. We are currently running a MiSeq on the naringenin samples for comparison with the mock exudate and the 7,4'-dihydroxyflavone. 3) We have conducted rhizosphere experiments on wild type Medicago truncatula, DMI-1, Sunn, IFS-1, and IFS-2 in field soil as above. Seedlings were transplanted to tubes with a soil/sand mixture and grown in a growth chamber under controlled conditions. Plant biomass, root, and shoot characteristics were noted upon harvest. Rhizosphere soil was collected from each plant, and will be analyzed as above.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: M�rton Szoboszlaya, Julie Lambers, Janet Chappell, Joseph V. Kupper, Luke A. Moe, David H. McNear Jr."Comparing root system architecture and rhizosphere microbial communities of Balsas teosinte and domesticated corn cultivars" accepted at Soil Biology & Biochemistry
• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Atanas D. Radkov and Luke A. Moe "Bacterial synthesis of D-amino acids" Applied Microbiology and Biotechnology 98, 5363-5374

Progress 06/01/12 to 05/31/13

Outputs
Target Audience: The target audience of this work is scientists interested in the interactions between plants and microbes in the rhizosphere. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training: All members of the project have had the opportunity to mentor undergraduate students in laboratory research. The funded graduate student has been trained in metagenomic analysis at the Michigan State University microbial metagenomics summer course, and has been trained in Illumina next-gen sequencing at the University of Kentucky next-generation sequencing and data analysis workshop. Professional Development: The PI has attended a number of relevant conferences during the reporting period (Botanical Society of America conference; Kentucky Innovation and Entrepreneurship Conference; University of Kentucky Natural Products Consortium; ASA-CSSA-SSSA annual meeting; KY-TN regional branch of the American Society for Microbiology; NIFA AFRI microbial programs awardee meeting). The funded graduate student attended the KY-TN regional branch of the American Society for Microbiology meeting. The PI also had the opportunity to conduct outreach with students in Eastern Kentucky through collaborative work with the Big Sandy Community and Technical College in Prestonsburg, KY. How have the results been disseminated to communities of interest? The results have been disseminated through publication in scholarly journals and through presentations at relevant meetings. What do you plan to do during the next reporting period to accomplish the goals? During the next reporting period, we plan to analyze the data on flavonoid content from each Medicago mutant. We will conduct the plant growth and rhizosphere microbial community analysis on these plants using metagenomic methodologies and the operon trap. We will also develop a fake root system using mini-rhizons as a method for delivering a mock root exudate to soils. We will use this technique to deliver different flavonoids and will analyze the differences in "rhizosphere" soil around the rhizons under these conditions.

Impacts
What was accomplished under these goals? We have developed a hydroponic method for plant growth and plant root exudate collection using float trays and sterile magenta boxes. We have used this method to collect root exudate from wild type Medicago truncatula and mutants that are defective in plant-microbe signaling. The exdudate has been sent for analysis of flavonoid content to a metabolomics center. We have developed, optimized and utilized terminal restriction fragment length polymorphism (TRFLP) methods for analyzing the bacterial and fungal composition of rhizosphere microbial communities in different varieties of corn. We have also used soil enzyme assays for assessing the functional diversity of rhizosphere microbial communities from different corn varieties, and have identified key differences in these processes that may result from plant-based selection for rhizosphere microbial partners. We believe we have an optimal soil for conducting our plant growth experiments and rhizosphere soil microbial community analyses. We should have sufficient seed for this work at this point, and preliminary analysis of the plant growth conditions suggests that plant-microbe communication mechanisms are optimal under these conditions.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2013 Citation: Szoboszlay, M., Lambers, J., Chappell, J., Kupper, J.V., Moe, L.A., and McNear, D.H., Jr. Getting to the root of corn domestication: comparing root system architecture and rhizosphere processes of Balsas teosinte and domesticated corn cultivars submitted to Soil Biology and Biochemistry
• Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Moe, L.A. (2013) Amino acids in the rhizosphere: from plants to microbes American Journal of Botany 100, 1692-1705
• Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Radkov, A.D., and Moe, L.A. (2013) Amino acid racemization in Pseudomonas putida KT2440 Journal of Bacteriology

Progress 06/01/11 to 05/31/12

Outputs
OUTPUTS: Activities to report during this period include work on plant growth conditions to optimize rhizosphere metagenomics studies, and optimization of methodologies for collection and analysis of plant root exudates. The operon trap metagenomic screening vector was developed and optimized, and preliminary analysis of rhizosphere soils was conducted in this vector. T-RFLP methods were optimized using a different set of rhizosphere soils. The PI engaged undergraduate students in two relevant courses during this time period, "experimental techniques in biotechnology" and "molecular genetics", and mentored three graduate students and two undergraduate students in laboratory research. Events to report during this period include the Plant and Animal Genome Conference (San Diego, CA; January 14-18, 2012) and an invited lecture at Miami University (Ohio) on October 12, 2011. Services to report include academic counseling of graduate students in Plant & Soil Sciences, Chemistry, and Geography at UK, and undergraduate students in Agricultural Biotechnology at UK. Products to report include data on T-RFLP analysis of rhizosphere soils, construction and validation of a metagenomic operon trap vector and associated metagenomic screening methods. The Moe laboratory website was constructed (http://moelab.weebly.com) for dissemination. PARTICIPANTS: Luke Moe (PI) David McNear (Co-PI) Seth DeBolt (Co-PI) Marton Szoboszlay (graduate student) TARGET AUDIENCES: Researchers involved in rhizosphere biology, plant-microbe interactions, and metagenomics. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Preliminary data points to a significant effect of plant genotype on the makeup and function of the rhizosphere microbial community. Work underway is aimed at identifying the biochemical and genetic features that contribute to these differences, as well as identifying the genes from rhizosphere-dwelling microbes that impact functional differences within the rhizosphere.

Publications

• No publications reported this period