FUNCTIONAL CHARACTERIZATION OF NOVEL NODULE-SPECIFIC GRF TRANSCRIPTION FACTORS REGULATING SYMBIOTIC NITROGEN FIXATION IN MEDICAGO TRUNCATULA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1009357
• Project No.: WVA00686
• Project Start Date: Apr 1, 2016
• Project End Date: Mar 31, 2021

Project Director
Benedito, VA, AU.

Recipient Organization
WEST VIRGINIA UNIVERSITY
886 CHESTNUT RIDGE RD RM 202
MORGANTOWN,WV 26505-2742

Performing Department
Plant & Soil Sciences

Non Technical Summary
Nodule development and symbiotic nitrogen fixation require a complete reprogramming of transcriptional networks by transcription factors. Although much progress has been made to understand how legume genes orchestrate nodule development, the roles of key families of transcription factors have not been elucidated. This research addresses a central question in legume biology to understand the molecular mechanisms related to the establishment of symbiotic nitrogen fixation. In particular, we aim at understanding the function of genes essential in this process. Moreover, most symbiotic genes characterized in legumes are involved in the early steps of nodulation, such as establishment of the symbiosis and very early nodule development (the so-called nod- genes, since the mutant roots are not able to develop nodules). Very few nod+/fix- genes (those which knockout allow normal nodule development but no nitrogen fixation) have been functionally characterized. These genes are key to understand how nitrogen fixation is established in the nodule. This research will focus on GRF transcription factors regulatory networks and their connections with symbiotic genes in nodules of the model legume, Medicago truncatula.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1644	1080	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1644 - Winter annual legumes;

Field Of Science
1080 - Genetics;

Keywords

symbiotic nitrogen fixation

nodule

legume

sustainable agriculture

transcription factors

Goals / Objectives
Although several late nodulins have been identified, just a few were molecularly and functionally characterized. Regarding MtN20 and GRF2, our main questions are: 1) what are the molecular functions of GRF gene products during nodule development and establishment of legume N2 fixation? 2) Which genes have their expression coordinated by these GRF gene products? We hypothesize that each of these two nodule-specific GRF proteins are transcription factors coordinating essential, and distinct, symbiotic gene networks involved in nodule development.Therefore, the specific aims of this project are to: #1: characterize the lack-of-function phenotypes of each mutant, and fine-tune their temporal-spatial gene expression profiles; #2: define the subcellular localization of the gene products; #3: characterize their DNA-binding properties and identify their target genes; and #4: define the regulatory gene networks connected with each gene under study. This work will be seminal to shed light on the function of a large eukaryotic subfamily of zinc finger proteins that have been largely ignored so far. Furthermore, this work will clarify the symbiotic function of MtN20, a nodulin identified a long time ago, but which no function has been assigned so far.

Project Methods
Characterization of symbiotic function of nodule-specific GRF proteins through knockout/knockdown phenotypes: Medicago truncatula Tnt1 insertional (knockout) mutants will be used for phenotyping anatomy of mature nodules (optical microscopy), cell infection and bacteroid morphology (confocal microscopy), subcellular ultrastructure (transmission electron microscopy), quantification of symbiotic N2 fixation (we have a established protocol for 15N isotope analysis to quantify symbiotically-fixed nitrogen), and fine-tuning of gene expression profile for GRF2 (qPCR and proGRF2::GUS hairy root transformation).Subcellular localization of GRF gene products: a heterologous transient expression system of YFP fused to the proteins of interest using Nicotiana benthamiana leaf Agrobacterium tumefaciens co-infiltration will be performed with the viral silencing inhibitor protein P19. This system has been used successfully to assess nuclear localization of nodule-specific transcription factor in legumes (Laloum et al., 2014). This analysis is currently underway for MtN20.Characterization of protein-DNA binding properties: to further understand the specific function of transcription factors, it is necessary to identify their direct and indirect targets. However, all available techniques are prone to false positives, therefore at least two (but ideally, three) techniques must be used to demonstrate canonical protein-DNA interactions. Additionally, a new technique called TARGET (Transient Transformation System for Genome-Wide Transcription Factor Target Discovery; Bargmann et al., 2013) has recently been developed in Arabidopsis. By using this technique, transcription factors direct targets can be identified very accurately. This technique is not standardized in Medicago and our collaborator Dr. Sinharoy has been taken efforts towards implementing it in our legume model.i) Chromatin immunoprecipitation assay (ChIP-Seq): For both GRF genes, mature nodules of Medicago truncatula (which are indeterminate and retain all developmental zones: meristem, rhizobial infection and bacteroid differentiation, interzone, nitrogen fixation, and, as the nodule grows, senescence) expressing the full cassette (2-kb promoter plus the entire coding region, including introns) fused with GFP will be excised from transgenic roots generated with Agrobacterium rhizogenes strain 'Arqua1' and selected by DsRed fluorescence (root transformation is well established in our lab) for ChIP-Seq using anti-GFP. Immunoprecipitated DNA will be analyzed with Illumina sequencing at the WVU Genomics Core Facility. This approach will define the positions in the legume genome where nodule-specific GRF proteins interact, thus revealing which gene expression they directly coordinate downstream.ii) Yeast one-hybrid (Y1H) assay: The yeast hybrid system has been successfully used for zinc finger transcriptional activators in plants (e.g., Li et al., 2014). The most prominent DNA fragments identified by ChIP will be cloned and used in Y1H assay to further confirm the TF interactions. Cloning will be performed using SMART technology and screening will use the Matchmaker Gold Yeast One-Hybrid System (Clontech).iii) Electrophoretic mobility shift assay (EMSA): Gel shift assay will be employed to define protein binding to cis-elements found positive in the Y1H assay. This technique has been successfully used in Medicago truncatula and other plant species (e.g., Ariel et al., 2010). Competition assays with random and degenerate sequences will be used to evaluate relative binding strength between each GRF protein and the cis-elements under investigation. GRF protein will be expressed in E. coli using the pMAL Protein Fusion and Purification System (New England BioLabs). Biotin end-labeled DNA probes will be synthesized from Y1H-confirmed nucleotide sequences. Visualization will be performed using chemiluminescence with the LightShift EMSA kit (Thermo Scientific).Mapping of regulatory gene networks around MtN20 and GRF2: to define regulatory gene networks centered on GRF transcription factors, we will perform RNA-Seq analysis on wild-type (control) and mutant nodules as well as GRF-overexpressing (non-nodulating) hairy roots. Weighted gene correlation network analysis (WGCNA; Langfelder & Horvath, 2008) will be applied to RNA-Seq data in order to identify co-expressed gene clusters (Wang et al., 2013). The analyses will be performed using Legume Gene Regulatory Network Prediction Server (Wang et al., 2013). Visualization of the network centered on the GRFs under study will be generated using Cytoscape (Saito et al., 2012). According to the spatio-temporal gene regulatory network prediction 32 genes are predicted to be target of Medtr7g086040.1, where lipid metabolism and protein folding, shorting and degradation are two major classes. These analyses will be complementary to protein-DNA binding experiments (aim #3) to provide a clear understanding of the regulatory networks during the establishment of symbiotic nitrogen fixation in legume nodules that are coordinated by the GRFs of this study.

Progress 10/01/20 to 03/31/21

Outputs
Target Audience:The immediate target audience of this research is plant biology scientists involved with research questions related to symbiotic nitrogen fixation in legumes. Ultimately, we aim to understand how nodulation occurs and how nitrogen fixation is maintained in legumes to potentially enhance nitrogen metabolism in legumes and transfer this trait to non-legume crops. Therefore, the ultimate target audiences for this research are the farmers. Changes/Problems:A problem that occurred was that the Ph.D. student involved in this project was unable to gather useful data from biochemical interactions between the MtN20 protein and potential DNA targets. Due to his funding situation, he had to defend his dissertation and move on with his career. We intended to continue the analysis, but the WVU campus closed when the pandemic started, so we could notcontinue the experiment. Fortunately, we gathered enough data to write a manuscript and submit it to a very good journal (PCE). We havea second manuscript on the function of GRF2 in preparation. It willbe finalized and submitted to another peer-reviewed journal in the coming weeks. What opportunities for training and professional development has the project provided?All in all, a Ph.D. student (now, a post-doc at the University of Wisconsin - Madison) was trained and defended a thesis as a result of this research. Additionally, an international visiting scientist on sabbatical leave (Anathi Magdalela from the University of Kwazulu-Natal, South Africa) was able to cooperate with us on parts of this project. Unfortunately, due to the COVID pandemic, the last year of research was conducted solely by the PD. How have the results been disseminated to communities of interest?One paper has been submitted to a top peer-reviewed journal in the field, and another manuscript on a second nodule-specific GRF gene (GRF2) is in preparation. What do you plan to do during the next reporting period to accomplish the goals?This is a final report. In general, the research was successful in establishing two novel nodule-specific GRF genes in symbiotic nitrogen fixation in Medicago truncatula. This project opened new avenues to explore the genetic regulation involved in the making and function of nodules in legumes.

Impacts
What was accomplished under these goals? Specific aim #1: We demonstrated that lack of GRF1 function leads to inefficient nitrogen fixation and characterizedthe exact expression domain of GRF1 (now called MtN20) occurs during the early stages of bacterioid differentiation in the nodule. Specific aim#2: Our analyzes point out the nucleus to be the destination of MtN20, which corroborates its function as a transcription factor. Specific aim #3: This goal was replaced along the way by a more detailed analysis of MtN20 expression, given the timeline of the Ph.D. student involved in this project. Specific aim #4: We constructed an elaborate regulatory gene network centered on MtN20. Various canonical symbiotic genes were captured in this analysis, indicating a central function of MtN20 in establishing nitrogen symbiosis in the nodule.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Maia LGS, Lima D, Benedito VA. MtN20 is an essential GRF-type zinc finger nodulin involved in the first stage of bacteroid differentiation in root nodules of Medicago truncatula. Plant, Cell and Environment (submitted)

Progress 04/01/16 to 03/31/21

Outputs
Target Audience:The immediate target audience of this research is plant biology scientists involved with research questions related to symbiotic nitrogen fixation in legumes. Ultimately, we aim to understand how nodulation occurs and how nitrogen fixation is maintained in legumes to potentially enhance nitrogen metabolism in legumes and transfer this trait to non-legume crops. Therefore, the ultimate target audiences for this research are the farmers. Changes/Problems:A problem that occurred was that the Ph.D. student involved in this project was unable to gather useful data from biochemical interactions between the MtN20 protein and potential DNA targets. Due to his funding situation, he had to defend his dissertation and move on with his career. We intended to continue the analysis, but the WVU campus closed when the pandemic started, so we could not continue the experiment. Fortunately, we gathered enough data to write a manuscript and submit it to a very good journal (PCE). We have a second manuscript on the function of GRF2 in preparation. It will be finalized and submitted to another peer-reviewed journal in the coming weeks. What opportunities for training and professional development has the project provided?All in all, a Ph.D. student (now, a post-doc at the University of Wisconsin - Madison) was trained and defended a thesis as a result of this research. Additionally, an international visiting scientist on sabbatical leave (Anathi Magdalela from the University of Kwazulu-Natal, South Africa) was able to cooperate with us on parts of this project. Unfortunately, due to the COVID pandemic, the last year of research was conducted solely by the PD. How have the results been disseminated to communities of interest?One paper has been submitted to a top peer-reviewed journal in the field, and another manuscript on a second nodule-specific GRF gene (GRF2) is in preparation. What do you plan to do during the next reporting period to accomplish the goals?This is a final report. In general, the research was successful in establishing two novel nodule-specific GRF genes in symbiotic nitrogen fixation in Medicago truncatula. This project opened new avenues to explore the genetic regulation involved in the making and function of nodules in legumes.

Impacts
What was accomplished under these goals? Specific aim #1: We demonstrated that lack of GRF1 function leads to inefficient nitrogen fixation and characterized the exact expression domain of GRF1 (now called MtN20) occurs during the early stages of bacterioid differentiation in the nodule. Specific aim #2: Our analyzes point out the nucleus to be the destination of MtN20, which corroborates its function as a transcription factor. Specific aim #3: This goal was replaced along the way by a more detailed analysis of MtN20 expression, given the timeline of the Ph.D. student involved in this project. Specific aim #4: We constructed an elaborate regulatory gene network centered on MtN20. Various canonical symbioticgenes were captured in this analysis, indicating a central function of MtN20 in establishing nitrogen symbiosis in the nodule.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Maia LGS, Lima D, Benedito VA. MtN20 is an essential GRF-type zinc finger nodulin involved in the first stage of bacteroid differentiation in root nodules of Medicago truncatula. Plant, Cell and Environment (submitted)

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The immediate target audience of this research is plant biology scientists involved with research questions related to symbiotic nitrogen fixation in legumes. Ultimately, we aim to understand how nodulation occurs and how nitrogen fixation is maintained in legumes to potentially enhance nitrogen metabolism in legumes and transfer this trait to non-legume crops. Therefore, the ultimate target audiences for this research are the farmers. Changes/Problems:In general, it is undeniable that the current pandemic surely created hindrances for research. However, we were able to work on data analyses and finalize a few experiments to conclude the experimental component of this project. What opportunities for training and professional development has the project provided?This project provided training to one work-study undergraduate student. Unfortunately, with the campus shutdown and limited access to facilitates since March 2020, remote training has been provided but challenges limited the full experience of student-mentor training. How have the results been disseminated to communities of interest?Due to the ongoing COVID-19 pandemic, our plans to present our work at conferences were postponed to 2021. Through a collaboration with Chinese investigators, we have published one publication in a top peer-reviewed journal on soybean nodulation that aligns with the goals of this project. We are currently investigating how GRF transcription factors and their gene expression networks evolved in the legume family. In this period, I have also accepted the invitation to co-edit a book on nodulation and symbiotic nitrogen fixation in legumes and accepted writing two chapters for this book, one of which on the orchestration of transcription factors in Medicago truncatula, which will be submitted in the first semester of 2021. What do you plan to do during the next reporting period to accomplish the goals? We will finalize all data analyses, create figures, and write a manuscript for publication. We will also write a more general overview on transcription factor orchestration occurring duringlegume nodulation to be published as a book chapter.

Impacts
What was accomplished under these goals? We have finalized the study of regulatory gene networks centered on GRF transcription factors in Medicago truncatula. A manuscript is currently being written for submission in a peer-reviewed journal in 2021 as a conclusion to this project.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ahmad MZ, Zhang Y, Zeng X, Li P, Wang X, Benedito VA, Zhao J. (2020) Isoflavone malonyl-CoA acyltransferase GmMaT2 is involved in nodulation of soybean (Glycine max) by modifying synthesis and secretion of isoflavones. Journal of Experimental Botany. eraa511, https://doi.org/10.1093/jxb/eraa511

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Plant biology scientists involved with research questions related to symbiotic nitrogen fixation in legumes. Ultimately, we want to understand how nodulation occurs and nitrogen fixation is maintained in legumes to potentially enhance nitrogen metabolism in legumes and transfer this trait to non-legume crops. Therefore, the ultimate target audiences for this research are farmers. Changes/Problems:The major challenge of this period was the availability of workforce in the lab. I had to split the approaches to several people that stay only for a short period of time, thus hindering the seamless continuation of the project. This slows down the progress, but otherwise, we have made good advances. What opportunities for training and professional development has the project provided?In this period, my lab has trained a visiting scholar on sabbatical leave from Kwazulu-Natal University in South Africa. Anathi Magdlela was involved in setting up and harvesting material from the first set of homocitrate experiment in growth chambers. He was also trained in R programming to gain basic skills. I also trained an undergraduate studentfrom Biology (Korina de Jesus, paid from the work-student funding program). She conducted and harvested the second set of homocitrate experiment in greenhouse conditions. I participated in a 3-day bioinformatics workshop at Marshall University to learn advanced features of the software Cytoscape, which will be used to make visualizations of gene networks. I am also very glad to say that a former PhD student who worked on my hatch project (Lina Yang) has been employed by a large pharmaceutical company in the U.S. to lead their gene expression laboratory. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?goal #1: finalize the phenotyping of knockout nodules goal #2: transform roots with vectors for subcellular localization of gene products (our hypothesis is that they are localized to the nucleus of the nodule cells) and visualize using confocal microscopy goal #3: start cloning work todefine whichcis-elements our TFs of interest interact to. goal #4: process RNA-Seq data and create RGNs centered on both GRF TFs of interest to this project.

Impacts
What was accomplished under these goals? goal #1: we have re-worked our cloning strategy and finalized binary vectors for knocking out both genes of interest. Medicago truncatula roots have been transformed with A. rhizogenes and are currently developing for inoculation with rhizobium and nodule phenotyping. This approach will give us a clear picture of the function of each gene during nodulation and nitrogen fixation. We expect to have these results in the first quarter of the year. goal #2: we used MoClo to clone the vectors for subcellular localization and plan on transforming roots in the first semester of this year. goal #3: not started yet. goal #4: we have finalized the second repetition of our experiment using engineered rhizobium bacteria expressing homocitrate synthase in order to define the essentiality of homocitrate supply by the legume to the rhizobia. The tissues have been collected and noduleRNA samples will be sent to RNA-Seq to define regulatory networks involved with homocitrate biochemistry and nitrogen fixation. This set of experiments will help us create RGNs centered on GRF transcription factors. In the same period, we have been working on perfecting the modelling software we are going to use for this analysis (e.g., WGCNA parameters).

Publications

• Type: Book Chapters Status: Published Year Published: 2019 Citation: Raul B., Kryvoruchko I., Benedito V.A., Bandyopadhyay K., Sinharoy S. (2019) Root Nodule Development in Model Versus Non-canonical Plants. In: Khurana S., Gaur R. (eds) Plant Biotechnology: Progress in Genomic Era. pp. 397-428. Springer, Singapore. doi: 10.1007/978-981-13-8499-8_18

Progress 10/01/17 to 09/30/18

Outputs
Target Audience: p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 10.0px Arial} Plant biology scientists involved with research questions related to symbiotic nitrogen fixation in legumes. Ultimately, we want to understand how nodulation occurs and nitrogen fixation is maintained in legumes to potentially enhance nitrogen metabolism in legumes and transfer this trait to non-legume crops. Therefore, the ultimate target audiences for this research are farmers. Changes/Problems:Confocal microscopy remains a challenge to observe the subcellular location of gene products. We have moved on a modular cloning strategy to allow us more flexibility on the number of fluorescence channels we can stack up for better visualization. What opportunities for training and professional development has the project provided?I have worked on a successful Fulbright proposal for a visiting faculty scholar from Kwazulu-Natal to work on this project. He is currently conducting an experiment to assess the impact of GRF expression according to homocitrate supply availability. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?1) finalize the CRISPR experiment and assess the impact of GRF knockout on symbiotic nitrogen fixation; 2) confirm nuclearlocalization of GRF TFs with confocal microcopy; 3) assess the impact of homocitrate supply on GRF gene expression; 4) build a regulatory gene network centeredon both GRF genes.

Impacts
What was accomplished under these goals? During the reporting period, we have: 1) cloned gRNAs into transformation vectors for CRISPR editing of GRF genes in transgenic roots. Currently, we are setting up the genetic transformation experiments to assess the impact of gene editing on symbiotic nitrogen fixation (aim #1); 2) Since the lastattempt to visualize the labeledprotein did not work, weset up modular cloning (MoCo) strategy to clone the GRF open reading frame fused with fluorescent labels for subcellular localization on confocal microscope (aim #2)

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Kryvoruchko IS, Routray P, Sinharoy S, Torres-Jerez I, Tejada-Jiménez M, Finney LA, Nakashima J, Pislariu CI, Benedito VA, Gonzalez-Guerrero M, Roberts DM, Udvardi MK (2018) An iron-activated citrate transporter, MtMATE67, is required for symbiotic nitrogen fixation. Plant Physiology 176(3): 2315-2329

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Plant biology scientists involved withresearch questions related to symbiotic nitrogen fixation in legumes. Ultimately, we want to understand how nodulation occurs and nitrogen fixation is maintained in legumes to potentially enhance nitrogen metabolism in legumes and transfer this trait to non-legume crops. Therefore, the ultimate target audiences for this research arefarmers. Changes/Problems:Subcellular localization of our proteins of interest fused with a fluorescent marker has been a challenge, possibly due to problems with the vector used. We have replaced the vector and have finishedthe new cloning at this point. What opportunities for training and professional development has the project provided?A graduate student was trained in molecular biology and computational approaches to tackle the research questions. How have the results been disseminated to communities of interest?by means of peer-reviewed publications. The work with GRF transcription factors is yet to be written, which will occur when all evidence has been collected and analyzed for the story. What do you plan to do during the next reporting period to accomplish the goals?Goal #2: finalize subcellular localization of gene products Goal #3: analyze which cis-elements are overrepresented in genes which expressionissignificantlly associated with our genes of interest. Initiate DNA-binding assays to test these promoterelements. Goals #4: refine our regulatory gene network.

Impacts
What was accomplished under these goals? Goal #1: transposon insertional mutants forMtN20 and GRF2 gene were phenotyped for nodule development and we identified lines that showed normal development but no nitrogen fixation, indicating both genes are essential for SNF. We also defined the precise domains of expression of each of these genes: MtN20 expresses in the distal portion of zone II, just whenrhizobia are engulfed by the nodule cell whereas GRF2 expressed in the interzone (ZII-III) when bacteroids are finalizing maturation in order to start to fix nitrogen. Goal #2: technical difficulties were faced by the graduate studentwith visualization of the fluorescent marker in transformed nodules. New cloning was carried out and is ready for hairy root transformation. Goal #3: not started yet Goal #4: regulatory gene networks based on RNA-Seq was carried out for both genes of interest and is being now refined with the introduction of a larger dataset.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang J, Hou Q, Yang L, Li P, Chen B, Sun X, Benedito VA, Mysore KS, Wen J, Zhao J (2017) Diverse functions of multidrug and toxin extrusion (MATE) transporters in citric acid efflux and metal homeostasis in Medicago truncatula. Plant Journal 90: 79-95
• Type: Book Chapters Status: Submitted Year Published: 2018 Citation: Kleinert A, Benedito VA, Morcillo RJL, Dames J, Cornejo-Rivas P, Zuniga-Feest A, Delgado M, Munoz G (2018) Morphological and symbiotic root modifications for mineral acquisition from nutrient-poor soils. In: Springer Root Biology Series Volume 83. Giri B, Prasad R and Varma A. Springer-Verlag Heidelberg.

Progress 04/01/16 to 09/30/16

Outputs
Target Audience:- WVU students and faculty: teaching, advising and - plant biology community through publications and other interactions Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?- A graduate student is being trained in molecular genetic techniques forfunctional characterization of genes involved in symbiotic nitrogen fixation in a legume model; - The graduate student was provided the opportunity to go to off-site training in Bioinformatics. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Goal #1: Visualization of nodule phenotype with defective GRF genes and characterization of symbiotic nitrogen fixation under their respective lack-of-function; Goal #2: Definitionof subcellular localization under confocal microscopy; Goal #3: Definition of the techniques to be used to assess DNA-binding properties of GRF transcription factors under study; Goal #4: Medicago truncatula expression data will be used to generate regulatory gene networks.

Impacts
What was accomplished under these goals? Goal #1: A) we have cloned both genes of interest into a CRISPR/Cas9 vector for targeteddeletion. Clones have been introduced into A. rhizogenes for root transformation (currently underway). B) promoter-GUS vectors have been produced for both genes and are in A. rhizogenes for root transformation (currently underway). Goal #2: transcripts have been cloned into RFP fusion vectors, and confirmation of correct clones are underway. Goal #3: Thegraduate student is becoming familiarized withthe techniques to assess this question. Goal #4: The graduate student has been learning how to work with command line and getting familiar with software for data analysis and visualization.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Wang J, Hou Q, Yang L, Li P, Chen B, Sun X, Benedito VA, Mysore KS, Wen J, Zhao J (2017) Diversified functions of multidrug and toxin extrusion (MATE) transporters in citric acid efflux and metal homeostasis in Medicago truncatula. Plant Journal (accepted)
• Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Valentine AJ, Kleinert A, Benedito VA (2017) Adaptive strategies for nitrogen metabolism in phosphate deficient legume nodules. Plant Science 256: 46-52
• Type: Book Chapters Status: Published Year Published: 2016 Citation: Valentine AJ, Kleinert A, Thuynsma R, Chimphang S, Dames J, Benedito VA (2016) Physiology and spatio-temporal relations of nutrient acquisition by roots and root symbionts. In: L�ttge U, C�novas FM, Matyssek R. Progress in Botany 77. Springer Berlin Heidelberg, Springer International, 1-67
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Kryvoruchko IS, Sinharoy S, Torres-Jerez I, Sosso D, Pislariu CI, Guan D, Murray J, Benedito VA, Frommer WB, Udvardi MK (2016) MtSWEET11, a nodule-specific sucrose transporter of Medicago truncatula root nodules. Plant Physiology 171:554-65