Source: UNIVERSITY OF NEW HAMPSHIRE submitted to NRP
ELUCIDATION AND CHARACTERIZATION OF FRANKIA SIGNAL MOLECULES IN THE ACTINORHIZAL SYMBIOSIS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1005242
Grant No.
2015-67014-22849
Cumulative Award Amt.
$399,723.00
Proposal No.
2014-03765
Multistate No.
(N/A)
Project Start Date
Jan 1, 2015
Project End Date
Dec 31, 2019
Grant Year
2015
Program Code
[A1151]- Plant Health and Production and Plant Products: Plant Photosynthetic Efficiency and Nutrient Utilization
Recipient Organization
UNIVERSITY OF NEW HAMPSHIRE
51 COLLEGE RD SERVICE BLDG 107
DURHAM,NH 03824
Performing Department
MCBS
Non Technical Summary
Nitrogen fixation by actinorhizal plants is an important part of the nitrogen budget of the planet. The plants involved are also of economic significance with respect to land reclamation, reforestation, soil stabilization, landscaping, fuel, and as a food source for ruminant animals. Actinorhizal plants provide an excellent mechanism to restore disrupted environmental sites. The ability of Frankia to bind and sequester several toxic heavy metals suggests potential for bioremediation and phytoremediation applications especially on heavy-metal-contaminated-land. The establishment of this beneficial bacterial-plant relationship requires sound communication between the partners. The purpose of this study is to understand the process of this chemical communication between Frankia with its host plants. Compounds secreted by the plants into the environment induces production of an extracellular signal generated by the Frankia which is recognized by the host plant as a friend, and not a foe. Our previously USDA-funded project showed that in response to host plant root exudates, Frankia produced an extracellular factor involved in early pre-infection signaling events. This project is focused on the identification and characterization of the chemical nature of this novel microbial signal molecule and its effects of the host plant involved in its recognition. Our strategy focuses on the use of genomic tools to identify genes that are differentially regulated by potential signaling molecules. We are interested in identifying the gene products involved in plant recognition, infection, nodulation, and nodule maintenance. The identification of signaling molecules for the development of beneficial symbiosis has broad impact on agricultural system and could be exploited for other crops.
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
20640101040100%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
The overall goal of this proposal is the identification and characterization of the Frankia signaling molecule involved in the early signaling events in the actinorhizal symbiosis between actinorhizal plants and the bacterial partner. Analysis of sequenced Frankia genomes has provided a myriad of information on these bacteria and several surprises including the absence of obvious nodulation genes similar to those found in Rhizobia genomes suggesting that the actinorhizal symbiosis uses novel signal compounds during the infection process. Our previously USDA-funded project showed that in response to host plant root exudates, Frankia produced an extracellular factor (termed NINA) involved in early pre-infection signaling events. The goals of this proposal are: (1) Identification and characterization of the Frankia signal molecule and the genetic basis for their biosynthesis and (2) Determination of how the Frankia signal molecule(s) is perceived by the host plant and the effects of its perception on plant physiology. Our strategy focuses on the use of genomics tools to identify genes that are differentially regulated by potential signaling molecules. To elucidate the bacterial signal molecule, we will fractionation our active samples identified by bioassays with transgenic plants with promoter::GUS fusions in genes that specifically expressed at the early stages of the symbiosis. The active fractions will be analyzed by Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS). The effect of the Frankia signaling molecules on actinorhizal plant physiology and gene expression will be determined by the use of interference RNA (RNAi) transgenic plants containing symbiosis development markers, histological and biochemical techniques, and RNASeq. The identification of signaling molecules for the development of beneficial symbiosis has broad impact on agricultural system and could be exploited for other crops.
Project Methods
Building on the tools and plant bioassays developed in our previous project, we will focus on identifying the chemical composition of the Frankia CcI3 signal molecule. A functional genomics approach will be used to study genes involved in signaling and early plant infection events. The project is centered on the identification and characterization of the Frankia signaling molecule(s) in early actinorhizal symbiosis development and determining its downstream effects on Casuarina physiology and gene expression.Objective 1. Identify and characterize the Frankia signal molecule and the genetic basis for their biosynthesis. Through the use of C. glauca transgenic plants, we established a very sensitive and specific biological assay to detect plant responses to Frankia signal molecules. During our previous USDA-funded project, we found that Frankia produces an extracellular signal molecule which has different physical-chemical properties from hydrophobic rhizobial nod factors. The active molecule NINA (termed for NIN-activating factors) was relatively small (1-3 KDa) hydrophilic and active at low concentrations. To identify the NINA factor, we will perform a series of extraction and fractionation steps in conjunction with the sensitive bioassays. Samples of the fractions will be sent to the Montpellier Lab for the bioassays and remaining portions of the samples will be analyzed by the UNH group. A variety of biochemical approaches will be used to identify the chemical composition and structure of NINA. Besides the use of comparative genomics, transcriptomics, proteomics, gain of function mutants and molecular genetic approaches will be used to identify the NINA biosynthetic pathwaysObjective 2. Determine how the Frankia signal molecule(s) is perceived by the host plant and the effects of its perception (i.e. sense and response) on plant physiology. Several common aspects are found among other plant-microbe interactions including a signaling pathway shared between fungal and bacterial root endosymbioses in the actinorhizal plant Casuarina and in legumes. Physiological effects of the potential signaling molecules will be explored. Transcriptome studies are proposed to identify unique genes and gene products that are associated with the development of the actinorhizal symbiosis. The effects of novel Frankia natural products on actinorhizal host plant will be investigated by the use of molecular reporters for each discrete stage of infection process. A genetic approach utilizing RNAi plants will be used to delineate the role of potential receptors predicted from the common symbiosis (SYM) signaling pathway being involved in NINA perception. Furthermore, the effect of NINA perception will be studied at several levels. The effects of NINA stimulation on infection and nodulation processes will be investigated by the use of transgenic C. glauca plants with markers of Frankia infection and through key physiological steps including reactive oxygen species (ROS) production using histological and biochemical techniques. An RNA Sequencing approach will be used to identify early plant transcriptome changes in response to NINA exposure. These different approaches will help identify the key physiological and molecular events for the early plant responses to the bacterium.

Progress 01/01/15 to 12/31/19

Outputs
Target Audience:The actinorhizal symbiosis represents an important ecological and economic role in agriculture and the environment. The diversity of outcomes and impacts suggest that groups working on plant-microbe interactions (beneficial and pathogenic), agricultural and biotechnology industries, land restoration groups, environmental restoration and protection groups, farmers are projected target groups. The educational components of the project target the training of new investigators to agricultural and environmental sciences Changes/Problems:I appoligize for being late. Covid19 caused a problem and my University is shut down. What opportunities for training and professional development has the project provided?For this period of the project, this grant has helped support the work of 4 graduate students (Megan Worth, Taoqing Wang,Noémie de San Nicholas and Erik Swanson), 1 postdoctoral fellow (Celine Pesce) 4 undergraduate students (Kelsey Mercurio, Allison Lafleur, Lilly Friedman, and Jessica Hodgkin). Ms Worth worked on the NINA purification. Ms. Wang worked analytic identification of NINA. Mr. Swanson was involved in the Frankia genome sequencing and comparative analysis. Dr.Pesce aided in the NINA purification and transgenic plant growth and maintenance. Ms de San Nicholas performed complementary NINA experiments in France. The undergraduate students assisted the graduate students on the above research projects. The laboratory portion of my Molecular Microbiology course (GEN 717) was also involved with this project How have the results been disseminated to communities of interest?The preliminary results of this study have been presented at national and international professional meetings and invited seminars. For this period: Tisa, L.S. 2019. Frankia Genomics and Genome-guided approaches toward understanding the actinorhizal symbiosis and signaling. The 3rd International Congress of Biochemistry and Microbiology Applied Technologies (BMAT) October 31 to November 3rd, 2019 in Hammamet, Tunisia (Invited Plenary Talk) Pesce, C., R. Oshone, S.G. Hurst IV, V. Kliener and L.S. Tisa. 2019. Genetic tools in Frankia: Yes we can, from complementation to site-specific mutagenesis. The 18th Congress of the International Society for Molecular Plant- Microbe Interactions (IS-MPMI XVIII). July 14-20, 2019 (Poster). The results of the study have also been written up and submitted to peer-reviewed journals of professional societies. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Nitrogen fixation by actinorhizal plants is an important part of the nitrogen budget of the planet. The plants involved are also of economic significance with respect to land reclamation, reforestation, soil stabilization, landscaping, fuel, and as a food source for ruminant animals. Actinorhizal plants provide an excellent mechanism to restore disrupted environmental sites. The purpose of this study was the identification of potential molecular signals and genes involved in the interactions of Frankia with its host plants. Compounds secreted by the plants into the environment were tested to see if they cause a change in the microbe that helps them to be recognized by the plant as a friend, and not a foe. The identification of signaling molecules for the development of beneficial symbiosis has broad impact on agricultural system and could be exploited for other crops. The overall impact of this study is a greater understanding of plant-microbe beneficial interactions. The use of these actinorhizal plants in bioremediation, soil stabilization, nurse cropping, biomass production, and land reclamation applications could potentially impact the 320 million people in the United States. Objective 1. Identification and characterization of the Frankia signal molecule and the genetic basis for their biosynthesis (Year 5)- 90% Complete We have continued to focus on the identification and characterization of the potential Frankia signaling molecule(s). These molecules will signal to the host plant that these bacteria are friend and not foes. We have developed a series of bioassays to aid in the purification process and to identify the target molecule. Transgenic Casuarina glauca lines containing the promoter of the CgNIN and CgCCAMK genes fused to reporter genes GUS and GFP were used to visualize the expression of the CgNIN and CCaMK genes. The bioassays with the transgenic plants containing GUS or GFP fusion were used to identify the activity of the Frankia signal molecule (termed NIN activating factor = NINA). NINA was secreted by Frankia strain CcI3 that was induced host plant (Casuarina) root exudates. We have characterized NINA and found that it is small hydrophilic (aqueous) molecule between 500-3,000 Daltons. NINA is protease-sensitive, but insensitive to heat, alkaline conditions, and chitinase treatment. Freezing and acid treatment inactives the molecule, but sonication will restore partial activity. NINA activity was enriched by ethyl acetate extraction. We are able to bind NINA to a C18 column and elution of the column in response to a methanol gradient has results the generation of a single peak containing NINA activity. All of the other fractions including the column washout had no NINA activity. NINA activity of the fraction was stable in solvents used in mass spectral analysis and has allowed us to proceed on analysis. The active C18 fraction was run on an analytical UltraPerformnce-Liquid Chromatography (UPLC) and identified a strong peak and minor peaks associated with the large peak that retain NINA activity. The large peak turns out to probably to be riboflavin, not NINA, which co-migrates with NINA. Riboflavin does not induce activity in the bioassay. IPCLC-PDA-MS analysis identified the riboflavin, but another peak that we were unable to determine its mass. Recently, the C18 fraction and correspondent control C-18 fraction were run on nano-LC-MS system or UPC capillary chromatography coupled with MS MS analysis. We got multiple peaks including the ribroflavin peak but many others with mass under 700-800. We are analyzing the predicted compounds from the nano-LC-MS run and there are some unusual compounds. Transcription experiments of were performed to correlate Frankia expression data to these compounds., More replicates of RNASeq data were obtained Frankia exposed to Casuarina exudates or the controls plant medium or another actinorhizal plant for 1, 3, and 5 days exposure. The majority of the genes specifically up-regulated were hypothetical proteins. and we needed more replicates for better statistical analysis. Although the exact chemical structure of the NINA molecule has not yet been identified, we have made significant progress toward that goal Objective 2. Determination of how the Frankia signal molecule(s) is perceived by the host plant and the effects of its perception on plant physiology (Year 2)- 60% Complete The actinorhizal plants use signaling components of a common symbiosis pathway also found in rhizobia-legumes and arbuscular mycorrhizal interactions. These common features support the hypothesis that these plants (Fabids) have a predisposition to evolve to being nodulation or establishing a symbiosis. Besides expression of NIN and CCAMK genes, root hair curling and Ca2+ spiking described above, we are investigating the effects of NINA on other plant physiological events that are involved in nodulation. To determine whether LysM receptor kinases (LysM-RLKs, the receptors of the rhizobial Nod factors in legumes) are involved in NINA perception, we have generated transgenic C. glauca lines that down-regulate the expression of CgNFP (a member of LysM-RLKs). We have initiated an RNASeq experiment and are analyzing the data.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Nouioui, I., C. Cort�s-albayay, L. Carro, J. F. Castro, M. Gtari, F. Ghodhbane-Gtari, H.-P. Klenk, L. S. Tisa, V. Sangal, and M. Goodfellow 2019. Genome insights into the plant growth promoting potentialities of the genus Frankia. Frontiers in Microbiology 10:1457 (doi:10.3389/micb.2019-01457)
  • Type: Book Chapters Status: Published Year Published: 2020 Citation: Diagne, N., P. I. Djighaly, M. Ngom, C. Pesce, A. Champion, S. Svistoonoff, V. Hocher, and L. S. Tisa. 2019. Advances in Frankia genome studies and molecular aspects of tolerance to environmental stresses In: Salwan, R., V. Sharma, W. Yang, L. Khalil and T. Al-Ani (eds) Molecular Aspects of Plant Beneficial Microbes in Agriculture. Elsevier, Cambridge, MA 02139, USA pp.381-390 (https://doi.org/10.1016/B978-0-12-818469-1.00031-6)
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Belaid, K. E. SWANSON, A. Carr�-Mlouka, V. Hocher, S. Svistoonoff, S. Simpson, K. Morris, W. K. Thomas, S. Amrani, L. S. Tisa, and H. Gherbi. 2020. Draft Genome Sequence of the Symbiotic Frankia sp. strain B2 isolated from root nodules of Casuarina cunninghamiana found in Algeria. J. Genomics 8:00-00 doi 10.7150/jgen.38461
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Pesce, C., R. OSHONE, V. Kleiner, S.G. HURST IV, and L.S. Tisa. 2019. Stable transformation of the actinobacteria Frankia. Applied Environ Microbiol. 85:e00957-19 (doi:10.1128/AEM.00957-19)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Tisa, L.S. 2019. Frankia Genomics and Genome-guided approaches toward understanding the actinorhizal symbiosis and signaling. The 3rd International Congress of Biochemistry and Microbiology Applied Technologies (BMAT) October 31 to November 3rd, 2019 in Hammamet, Tunisia (Invited Plenary Talk)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Pesce, C., R. Oshone, S.G. Hurst IV, V. Kliener and L.S. Tisa. 2019. Genetic tools in Frankia: Yes we can, from complementation to site-specific mutagenesis. The 18th Congress of the International Society for Molecular Plant- Microbe Interactions (IS-MPMI XVIII). July 14-20, 2019 (Poster).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Cissoko M, Gully D, Boisseaux M, Pesce, C, Pignoly S, Tisseyre P, Carre-Mlouka A, Gherbi H, Pujic P, Sy,MO, Hocher V, Tisa L, Svistoonoff S (2019) Signaling molecules involved in actinorhizal symbioses Microbioccitanie fev 18-20, Montpellier, France AF
  • Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: No�mie de SAN NICOLAS Master II IMHE, U. Montpellier Etude des signaux microbiens dans la symbiose fixatrice dazote Frankia/Casuarina glauca. 2019
  • Type: Book Chapters Status: Published Year Published: 2019 Citation: Gherbi, H., V. Hocher, M. Ngom, N. Diagne, J. Fournier, A. Carre-Mlouka, C. Pesce, L. G. Wall, L. S. Tisa and S. Svistoonoff. 2019. Molecular methods for research on actinorhiza. In: Reinhardt, D. and Sharna, A.K. (ed) Methods in Rhizosphere Biology Research. Springer Press, Singapore pp.35-59 (https://doi.org/10.1007/978-981-13-5767-1_4)


Progress 01/01/18 to 12/31/18

Outputs
Target Audience:The actinorhizal symbiosis represents an important ecological and economic role in agriculture and the environment. The diversity of outcomes and impacts suggest that groups working on plant-microbe interactions (beneficial and pathogenic), agricultural and biotechnology industries, land restoration groups, environmental restoration and protection groups, farmers are projected target groups. The educational components of the project target the training of new investigators to agricultural and environmental sciences Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?For this period of the project, this grant has helped support the work of 5 graduate students (Megan Worth, Nate Ennis, Maimouna Cissoko, Marion Boisseaux, and Erik Swanson), 1 postdoctoral fellow (Celine Pesce) 4 undergraduate students (Kelsey Mercurio, Allison Lafleur, Lilly Friedman, and Victoria Kleiner). Ms Worth and Ms Boisseaux worked on the NINA purification. Mr. Swanson was involved in the Frankia genome sequencing and comparative analysis. Dr.Pesce aided in the NINA purification and transgenic plant growth and maintenance. Ms Cissoko's PhD thesis work has centered on developing an assay for the quicker detection of the NINA factor through root hair curling and RT-qPCR expression of plant gene NIN. The undergraduate students assisted the graduate students on the above research projects. The laboratory portion of my Molecular Microbiology course (GEN 717) was also involved with this project. How have the results been disseminated to communities of interest?The preliminary results of this study have been presented at national and international professional meetings and invited seminars. For this period: Signaling Molecules Involved In Actinorhizal Symbioses. Cissoko M, Gully D, Pignoly S, Carre-Mlouka A, Hocher V, Svistoonoff S. The 19th International Conférence on Frankia and Actinorhizal Plants, march 17-19, Hammamet, Tunisia (poster) Signaling pathway in the actinorhizal root nodule symbiosis between Casuarina glauca and Frankia casuarinae. Cissoko M, Svistoonoff S, Gully D, Carré-Mlouka A, Tisseyre P, Tisa, l, Pujic P, Gtari M, Oureye Sy M, Gherbi H, Hocher V (2018) 13th European Nitrogen Fixation Conference, august 18-21, Stockholm, Sweden (oral presentation) Tisa, L.S., S. Svistoonoff, C. Pesce, M. Cissoko, Sheldon Hurst IV, E. Swanson, R. Oshone, V. Hocher, H. Gherbi, and M. O. Sy. 2018. Frankia Genomics and Genome-guided approaches toward understanding the actinorhizal symbiosis and signaling. The 9th International Symbiosis Society Congress held July 15-20, 2018 in Corvallis, OR USA (Invited Talk) Tisa, L.S., E. Swanson, C. Pesce, A. Sen, F. Ghoddhbane-Gtari and M Gtari. 2018. Genes, genomes and genetic of Frankia and non-Frankia bacteria involved in the actinorhizal symbiosis. The 19th Symposium on Frankia and Actinorhizal Plants. March 17-19, 2018 Hammamet, Tunisia (Invited Talk). Gueddou, A., A. Ktari, F. Ghodhbane-Gtari, J. Armengaud, L.S. Tisa and M. Gtari. Deciphering the role of Frankia nod-like genes in actinorhizal symbiosis: A proteogenomic approach The 19th Symposium on Frankia and Actinorhizal Plants. March 17-19, 2018 Hammamet, Tunisia (Talk) The results of the study have also been written up and submitted to peer-reviewed journals of professional societies. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Nitrogen fixation by actinorhizal plants is an important part of the nitrogen budget of the planet. The plants involved are also of economic significance with respect to land reclamation, reforestation, soil stabilization, landscaping, fuel, and as a food source for ruminant animals. Actinorhizal plants provide an excellent mechanism to restore disrupted environmental sites. The purpose of this study was the identification of potential molecular signals and genes involved in the interactions of Frankia with its host plants. Compounds secreted by the plants into the environment were tested to see if they cause a change in the microbe that helps them to be recognized by the plant as a friend, and not a foe. The identification of signaling molecules for the development of beneficial symbiosis has broad impact on agricultural system and could be exploited for other crops. The overall impact of this study is a greater understanding of plant-microbe beneficial interactions. The use of these actinorhizal plants in bioremediation, soil stabilization, nurse cropping, biomass production, and land reclamation applications could potentially impact the 320 million people in the United States. Objective 1. Identification and characterization of the Frankia signal molecule and the genetic basis for their biosynthesis (Year 4)- 75% Complete We have continued to focus on the identification and characterization of the potential Frankia signaling molecule(s). These molecules will signal to the host plant that these bacteria are friend and not foes. We have developed a series of bioassays to aid in the purification process and to identify the target molecule. Transgenic Casuarina glauca lines containing the promoter of the CgNIN and CgCCAMK genes fused to reporter genes GUS and GFP were used to visualize the expression of the CgNIN and CCaMK genes. The bioassays with the transgenic plants containing GUS or GFP fusion were used to identify the activity of the Frankia signal molecule (termed NIN activating factor = NINA). NINA was secreted by Frankia strain CcI3 that was induced host plant (Casuarina) root exudates. We have characterized NINA and found that it is small hydrophilic (aqueous) molecule between 500-3,000 Daltons. NINA is protease-sensitive, but insensitive to heat, alkaline conditions, and chitinase treatment. Freezing and acid treatment inactives the molecule, but sonication will restore partial activity. NINA activity was enriched by ethyl acetate extraction. We are able to bind NINA to a C18 column and elution of the column in response to a methanol gradient has results the generation of a single peak containing NINA activity. All of the other fractions including the column washout had no NINA activity. NINA activity of the fraction was stable in solvents used in mass spectral analysis and has allowed us to proceed on analysis. We are investigating the chemical composition of the active NINA fraction by several analytical methods including IR spectrometry, Mass Spectrometry, NMR and elemental analysis. We have identified several peaks in MS MS analysis that respond to different concentration of the C18 NINA fraction. Our bioassays are quantified by dilution and are responsive to 1/10,000 fold dilution. This dilution was too low for NMR analysis. We have concentrated this fraction by 500-fold and have moved to a more sensitive NMR machine to determine structure. Both H-NMR and C13-NMR identified two compounds in the C18 NINA fraction, which was biologically active. Compound A was in low levels and consisted of about 8-9 amino acids, while compound B was found a higher concentration and is not peptide but consisted of 9 C. By inactiving the C18 NINA preparation, another round of NMR showed loss of compound A. This compound (A) is probably the NINA molecule. Another concentrated C18 NINA fraction showed similar NMR results. A further chromatographic step will be required to separate these two compounds and test biological activity. Objective 2. Determination of how the Frankia signal molecule(s) is perceived by the host plant and the effects of its perception on plant physiology (Year 4)- 40% Complete The actinorhizal plants use signaling components of a common symbiosis pathway also found in rhizobia-legumes and arbuscular mycorrhizal interactions. These common features support the hypothesis that these plants (Fabids) have a predisposition to evolve to being nodulation or establishing a symbiosis. Besides expression of NIN and CCAMK genes, root hair curling and Ca2+ spiking described above, we are investigating the effects of NINA on other plant physiological events that are involved in nodulation. To determine whether LysM receptor kinases (LysM-RLKs, the receptors of the rhizobial Nod factors in legumes) are involved in NINA perception, we have generated transgenic C. glauca lines that down-regulate the expression of CgNFP (a member of LysM-RLKs). The expression of the symbiotic signaling pathway (SYM) genes upon NINA treatment will be investigated by qRT-PCR. Most of this work was being held up until a semi-purified NINA preparation was developed. However, we anticipate this work to be performed in the upcoming year and the NINA-C18-fraction will be used in these experiments.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Gueddou A., E. Swanson, K. Hezbri, I. Nouioui, A. Ktari, S. Simpson, K. Morris, W. K. Thomas, F. Ghodhbane-Gtari, M. Gtari, and L. S. Tisa. 2019. Draft Genome Sequence of the Symbiotic Frankia sp. strain BMG5.30 isolated from root nodules of Coriaria myritifolia in Tunisia. Antonie van Leeuwenhoek. https://doi.org/10.1007/s10482-018-1138-1
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Signaling Molecules Involved In Actinorhizal Symbioses. Cissoko M, Gully D, Pignoly S, Carre-Mlouka A, Hocher V, Svistoonoff S. The 19th International Conf�rence on Frankia and Actinorhizal Plants, March 17-19, Hammamet, Tunisia (poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Signaling pathway in the actinorhizal root nodule symbiosis between Casuarina glauca and Frankia casuarinae. Cissoko M, Svistoonoff S, Gully D, Carr�-Mlouka A, Tisseyre P, Tisa, L, Pujic P, Gtari M, Oureye Sy M, Gherbi H, Hocher V (2018) 13th European Nitrogen Fixation Conference, august 18-21, Stockholm, Sweden (oral presentation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Tisa, L.S., E. Swanson, C. Pesce, A. Sen, F. Ghoddhbane-Gtari and M Gtari. 2018. Genes, genomes and genetic of Frankia and non-Frankia bacteria involved in the actinorhizal symbiosis. The 19th Symposium on Frankia and Actinorhizal Plants. March 17-19, 2018 Hammamet, Tunisia (Invited Talk).
  • Type: Book Chapters Status: Accepted Year Published: 2018 Citation: Gherbi, H., V. Hocher, M. Ngom, N. Diagne, J. Fournier, A. Carre-Mlouka, C. Pesce, L. G. Wall, L. S. Tisa and S. Svistoonoff. 2018. Molecular methods for research on actinorhiza In: Reinhardt, D. and Sharna, A.K.(eds) Methods in Rhizosphere Biology Research. Springer Press
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Cissoko, M., V. Hocher, H. Gherbi, D. Gully, A. Carr�-Mlouka, S. Sane, S. Pignoly, A. Champion, M. Ngom, P. Pujic, P. Fournier, M. Gtari, E. Swanson, C. Pesce, L. S. Tisa, M. O. Sy, and S. Svistoonoff. 2018. Actinorhizal signaling molecules: Frankia root hair deforming factor shares properties with NIN inducing factor. Frontiers in Plant Science 9:1494 https://doi.org/10.3389/fpls.2018.01494
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Griesmann, M., Chang, Y., Liu, X., Song, Y., Haberer, G., Crook, M. B., Billault-Penneteau, B., Lauressergues, D., Keller, J., Imanishi, L., Roswanjaya, Y. P., Kohlen, W., Pujic, P., Battenberg, K., Alloisio, N., Liang, Y., Hilhorst, H., Salgado, M. G., Hocher, V., Gherbi, H., Svistoonoff, S., Doyle, J. J., He, S., Xu, Y., Xu, S., Qu, J., Gao, Q., Fang, X., Fu, Y., Normand, P., Berry, A. M., Wall, L. G., An�, J.-M., Pawlowski, K., Xu, X., Yang, H., Spannagl, M., Mayer, K. F. X., Wong, G. K.-S., Parniske, M., Delaux, P.-M., and Cheng, S. 2018. Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis. . Science Vol. 361, Issue 6398, eaat1743; DOI: 10.1126/science.aat1743 http://science.sciencemag.org/content/361/6398/eaat1743
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Hocher, V., Ngom, M., Carr�-Mlouka, A., Tisseyre, P., Gherbi, H., and Svistoonoff, S. (2018). Signalling in actinorhizal root nodule symbioses. Antonie Van Leeuwenhoek, https://doi.org/10.1007/s10482-018-1182-x
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Gueddou, A., A. Ktari, F. Ghodhbane-Gtari, J. Armengaud, L.S. Tisa and M. Gtari. Deciphering the role of Frankia nod-like genes in actinorhizal symbiosis: A proteogenomic approach The 19th Symposium on Frankia and Actinorhizal Plants. March 17-19, 2018 Hammamet, Tunisia (Talk).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Tisa, L.S., S. Svistoonoff, C. Pesce, M. Cissoko, Sheldon Hurst IV, E. Swanson, R. Oshone, V. Hocher, H. Gherbi, and M. O. Sy. 2018. Frankia Genomics and Genome-guided approaches toward understanding the actinorhizal symbiosis and signaling. The 9th International Symbiosis Society Congress held July 15-20, 2018 in Corvallis, OR USA (Invited Talk)
  • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: BOISSEAUX Marion. "Molecular dialogue in the nitrogen-fixing root symbiosis Casuarina/Frankia: characterization of Frankias symbiotic signal molecules". MsC (Engineer) in Horticulture and Landscaping ; AgroParisTech, 27 September 2018


Progress 01/01/17 to 12/31/17

Outputs
Target Audience:The actinorhizal symbiosis represents an important ecological and economic role in agriculture and the environment. The diversity of outcomes and impacts suggest that groups working on plant-microbe interactions (beneficial and pathogenic), agricultural and biotechnology industries, land restoration groups, environmental restoration and protection groups, farmers are projected target groups. The educational components of the project target the training of new investigators to agricultural and environmental sciences Changes/Problems:The PI (Tisa) has had medical issues this year that have also caused a delay in progress. What opportunities for training and professional development has the project provided?For this period of the project, this grant has helped support the work of 3 graduate students (Rediet Oshone, Maimouna Cissoko, and Erik Swanson), 1 postdoctoral fellow (Celine Pesce) 4 undergraduate students (Celeste Souza, Sean Vigeant, Ashely Harrington, and Victoria Kliener). As part of Mr. Oshone's PhD thesis research, he worked on the NINA purification and performed the RNASeq experiments. Mr. Swanson was involved in the Frankia genome sequencing and comparative analysis. Dr.Pesce aided in the NINA purification and transgenic plant growth and maintenance. Ms Cissoko's PhD thesis work has centered on developing an assay for the quicker detection of the NINA factor through root hair curling and RT-qPCR expression of plant gene NIN. The undergraduate students assisted the graduate students on the above research projects. The laboratory portion of my Molecular Microbiology course (GEN 717) was also involved with this project. How have the results been disseminated to communities of interest?The preliminary results of this study have been presented at national and international professional meetings and invited seminars. For this period: Tisa, L.S. 2017. Everybody is talking: Communication during actinorhizal symbiosis. Seminar presented to the University of Tunis. Tunisia March 17.2017. Tisa, L.S. 2017. Everybody is talking: Communication during actinorhizal symbiosis. Seminar presented to Institut Supérieur de Biotechnologie, Université de La Manouba, Sidi Thabit, Tunisia March 21, 2017 Tisa, L.S. 2017. Everybody is talking: Communication during actinorhizal symbiosis. Seminar presented to Center de Biotechnologie Borj Cedria, Tunisia March 23. 2017 Pesce, C. R. Oshone and LS Tisa. 2017. Development and application of CRISPR/cas9 genome editing tool on the symbiotic actinobacteria Frankia. The 118th General Meeting of the American Society for Microbiology June 1-4, 2017 in New Orleans, LA. Harrington, K., C. Pesce, F. Chu and L.S. Tisa. 2017. Identification of signal molecules from Frankia that induce the early step of nodule development in Casuarina. The 26th Annual COLSA Undergraduate Research Conference April 22nd, 2017 (poster). Cissoko M, Gully D, Mlouka Carre A, Pignoly S, Gherbi H, Hocher V, Tisa L, Chu F, Svistoonoff S., Sy MO. Identification et caractérisation des molécules signal impliquées dans la symbiose actinorhizienne Casuarina-Frankia. Sustainable Intensification Conference "Biodiversity and ecological engineering for sustainable intensification of agriculture" 2017 April 24-26, Dakar, Senegal (Poster) Cissoko M, Gully D, Mlouka Carre A, Pignoly S, Gherbi H, Hocher V, Tisa L, Chu F, Svistoonoff S., Sy MO. Identification et caractérisation des molécules signal impliquées dans la symbiose actinorhizienne Casuarina-Frankia. 5th Meeting on Nitrogen-Fixing Root Endosymbioses" October 12-13 octobre 2017, Toulouse, France (Presentation) Cissoko M, Gully D, Mlouka Carre A, Pignoly S, Hocher V, Tisa L, Chu F, Svistoonoff S., Sy MO. Identification et caractérisation des molécules signal impliquées dans la symbiose actinorhizienne Casuarina-Frankia. 2nd edition des journees scientifiques du campus de Bel Air. Oct 4th, Dakar, Senegal (Presentation) The results of the study have also been written up and submitted to peer-reviewed journals of professional societies. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Nitrogen fixation by actinorhizal plants is an important part of the nitrogen budget of the planet. The plants involved are also of economic significance with respect to land reclamation, reforestation, soil stabilization, landscaping, fuel, and as a food source for ruminant animals. Actinorhizal plants provide an excellent mechanism to restore disrupted environmental sites. The purpose of this study was the identification of potential molecular signals and genes involved in the interactions of Frankia with its host plants. Compounds secreted by the plants into the environment were tested to see if they cause a change in the microbe that helps them to be recognized by the plant as a friend, and not a foe. The identification of signaling molecules for the development of beneficial symbiosis has broad impact on agricultural system and could be exploited for other crops. The overall impact of this study is a greater understanding of plant-microbe beneficial interactions. The use of these actinorhizal plants in bioremediation, soil stabilization, nurse cropping, biomass production, and land reclamation applications could potentially impact the 320 million people in the United States. Objective 1. Identification and characterization of the Frankia signal molecule and the genetic basis for their biosynthesis (Year 2)- 50% Complete During this reporting period, we have continue to focus on the identification and characterization of the potential Frankia signaling molecule(s). These molecules will signal to the host plant that these bacteria are friend and not foes. We have developed a series of bioassays to aid in the purification process and to identify the target molecule. Transgenic Casuarina glauca lines containing the promoter of the CgNIN and CgCCAMK genes fused to reporter genes GUS and GFP were used to visualize the expression of the CgNIN and CCaMK genes. These plant genes signal the development of the plant-microbe association at the very early stages preceding infection. The reporter constructs provide a quick system to screen for the Frankia signal molecule. These transgenic plants were transported from France to the US partner to help streamline the purification, characterization, and analysis process. The bioassays with the transgenic plants containing GUS or GFP fusion were used to identify the activity of the Frankia signal molecule (termed NIN activating factor = NINA). Previously, we reported that NINA was secreted by Frankia strain CcI3 that was induced host plant (Casuarina) root exudates. Other Casuarina compatible Frankia strains (Thr and other) also showed this signaling effect, which was restricted to this Frankia lineage. Other Frankia lineages or other actinobacteria (Streptomyces) did not produce a signal. Thus, the signal was specific to the host plant symbionts. Besides repeating work performed in France, we have successfully maintained these transgenic lines and continued in our characterization of the Frankia signal molecule. Frankia infection of the host plant causes root hair curling. We have developed a bioassay for this effect and this root hair curling has been correlated with NIN and CCAMK expression in root hairs. Both transgenic bioassays were used to characterize the properties of the NINA factor. Our preliminary physical and chemical properties show that the NINA factor is unique and distinctly different from the microbial signaling molecules of the legume-Rhizobium and mycorrhizal-plant symbioses, which are amphiphilic and chitin-based. The size of the NINA factor was estimated to be a small hydrophilic (aqueous) molecule between 0.5-5 KDa, which was active even at 1:10.000 dilution of the culture supernatant fluid. Thus, this represents a novel signaling molecule and a potentially new paradigm for plant-microbe signaling. Early chromatography studies showed that NINA was very hydrophilic passing quickly through any column, but we have found that NINA will bind to a C18. NINA activity eluted from the column 6-10% methanol. We have test the stability of NINA in solvents used in mass spectral analysis and determined that NINA is stable. The analysis of the fractions work will be accomplished during the upcoming year. We have also found that there is another factor that can induce root hair curling and elutes from the column after NINA. This factor only induces root hair curling and has no effect on our bioassay. A manuscript on this difference is in parathion. To determine the genetic basis for the biosynthesis of the Frankia signal molecule, we have taken multiple approaches. Several Frankia genomes including more Casuarina compatible isolates were generated and comparative genomics approaches are being performed to identify unique genes for each host compatibility group. The Frankia global gene expression profiles in response to host (Casuarina) and nonhost (Hippophae) plant root exudates and to the control BD plant medium. RNA from Frankia cultures that were exposed to these extracts for 1, 2, 3 and 5 days was collected and RNA-seq has been performed. About 79 genes were up-regulated with exposure to host plant root exudates compared to the two controls. The cultures were exposed to these conditions. Currently, these data are in analysis. We hope to identify genes in Frankia that involved on NINA biosynthesis by this approach. Objective 2. Determination of how the Frankia signal molecule(s) is perceived by the host plant and the effects of its perception on plant physiology (Year 2)- 40% Complete The actinorhizal plants use signaling components of a common symbiosis pathway also found in rhizobia-legumes and arbuscular mycorrhizal interactions. These common features support the hypothesis that these plants (Fabids) have a predisposition to evolve to being nodulation or establishing a symbiosis .Besides expression of NIN and CCAMK genes, root hair curling and Ca2+ spiking described above, we are investigating the effects of NINA on other plant physiological events that are involved in nodulation. To determine whether LysM receptor kinases (LysM-RLKs, the receptors of the rhizobial Nod factors in legumes) are involved in NINA perception, we have generated transgenic C. glauca lines that down-regulate the expression of CgNFP (a member of LysM-RLKs). The expression of the symbiotic signaling pathway (SYM) genes upon NINA treatment will be investigated by qRT-PCR. Most of this work is being held up until a semi-purified NINA preparation is developed, but we anticipate this work to be performed in the upcoming year.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Nouioui, I., F. Ghodhbane-Gtari, M. C. Montero-Calasanz, M. Rohde, L. S. Tisa, M. Gtari, and H.-P. Klenk. 2017. Frankia inefficacis sp. nov. an actinobacterial endophyte inducing ineffective, non nitrogen-fixing, root nodules on its actinorhizal host plants. Anton van Leeuw. 110:313-320. DOI 10.1007/s10482-016-0801-7
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Nouioui, I., M. C. Montero-Calasanz, F. Ghodhbane-Gtari, M Rohde, L. S Tisa, H.-P. Klenk, and M. Gtari. 2017. Description of Frankia discarae sp. nov. an infective and effective microsymbiont isolated from root nodule of Discaria trinervis. Arch. Microbiol. 199:641-647 DOI 10.1007/s00203-017-1337-6
  • Type: Other Status: Published Year Published: 2017 Citation: Gueddou,A., E. Swanson, A. Ktari, I. Nouioui, K. Hezbri, F. Ghodhbane-Gtari, S. Simpson, K. Morris, W.K. Thomas, A. Sen, M. Gtari, and L.S. Tisa. 2017. Permanent Draft Genome Sequences for Three Frankia Strains that are Atypical, Noninfective (Nod-) Ineffective (Fix-) Isolates. GenomeA5:e00174-17. DOI:10.1128/genomeA.00174-17
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Swanson, E., R. Oshone, I. Nouioui, F. Abebe-Akele, S. Simpson, K. Morris, W.K. Thomas, A. Sen, F. Ghodhbane-Gtari, M. Gtari, and L.S. Tisa. 2017. Permanent Draft Genome Sequence for Frankia sp. Strain Cc1.17, a Nitrogen-Fixing Actinobacterium Isolated from the Root Nodules of Colletia cruciata. GenomeA 5:e00530-17 doi: 10.1128/genomeA.00530-17
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Pesce, C. E. Swanson, S. Simpson, K. Morris, W.K. Thomas, L.S. Tisa, and A. Sellstedt. 2017. Draft Genome Sequence for Frankia sp. Strain KB5 Isolated from the Root Nodules of Casuarina equisetifolia. J. Genomics 5: 64-67. doi: 10.7150/jgen.20887
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Ktari, A., I. Nouioui; T. Furnholm, E. Swanson, F. Ghodhbane-Gtari, L. S Tisa,.and M. Gtari. 2017. Permanent draft genome sequence of Frankia sp. NRRL B-16219 reveals the presence of canonical nod genes, which are highly homologous to those detected in Candidatus Frankia Dg1 genome. Standards in Genomic Science 12:e51 doi:10.1186/s40793-017-0261-3
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Mansour,S., E. SWANSON, Z. MCNUTT, C. Pesce, K. HARRINGTON, F. Abebe-Alele, S. Simpson, K. Morris, W. K. Thomas, and L. S. Tisa. 2017. Permanent Draft Genome sequence for Frankia sp. strain CcI49, a Nitrogen-Fixing Bacterium Isolated from Casuarina cunninghamiana that Re-infects Elaegnaceae. J. Genomics 5:115-119 doi: 10.7150/jgen.22138
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Pesce, C. R. Oshone and LS Tisa. 2017. Development and application of CRISPR/cas9 genome editing tool on the symbiotic actinobacteria Frankia. The 118th General Meeting of the American Society for Microbiology June 1-4, 2017 in New Orleans, LA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Harrington, K., C. Pesce, F. Chu and L.S. Tisa. 2017. Identification of signal molecules from Frankia that induce the early step of nodule development in Casuarina. The 26th Annual COLSA Undergraduate Research Conference April 22nd, 2017 (poster)
  • Type: Other Status: Other Year Published: 2017 Citation: Tisa, L.S. 2017. Everybody is talking: Communication during actinorhizal symbiosis. Seminar presented to the University of Tunis. Tunisia March 17.2017.
  • Type: Other Status: Other Year Published: 2017 Citation: Tisa, L.S. 2017. Everybody is talking: Communication during actinorhizal symbiosis. Seminar presented to Institut Sup�rieur de Biotechnologie, Universit� de La Manouba, Sidi Thabit, Tunisia March 21, 2017
  • Type: Other Status: Other Year Published: 2017 Citation: Tisa, L.S. 2017. Everybody is talking: Communication during actinorhizal symbiosis. Seminar presented to Center de Biotechnologie Borj Cedria, Tunisia March 23. 2017
  • Type: Other Status: Other Year Published: 2016 Citation: Tisa, L.S. 2017. Genes, genomes, and genetics of Frankia and the actinorhizal symbiosis. Seminar presented to Institut de Rechrche pour le D�veloppement (IRD) and the University of Cheikh Anta Diop, Dakar, Senegal October 18, 2016.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Nouioui, I., F. Ghodhbane-Gtari, M. C. Montero-Calasanz, M. G�ker, J. P. Meier-Kolthoff, P. Schumann, M. Rohde, M. Goodfellow, M. P. Fernandez, P Normand, L. S. Tisa, H.-P. Klenk, and M. Gtari. 2016. Proposal of a type strain for Frankia alni (Woronin 1866),and recognition Frankia casuarinae sp. nov. and Frankia elaeagni sp. nov. Int J. Syst.. Evol. Microbiol. 66:5201-5210. doi: 10.1099/ijsem.0.001496
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Cissoko M, Gully D, Mlouka Carre A, Pignoly S, Gherbi H, Hocher V, Tisa L, Chu F, Svistoonoff S., Sy MO. Identification et caract�risation des mol�cules signal impliqu�es dans la symbiose actinorhizienne Casuarina-Frankia. Sustainable Intensification Conference Biodiversity and ecological engineering for sustainable intensification of agriculture 2017 April 24-26, Dakar, Senegal (Poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Cissoko M, Gully D, Mlouka Carre A, Pignoly S, Gherbi H, Hocher V, Tisa L, Chu F, Svistoonoff S., Sy MO. Identification et caract�risation des mol�cules signal impliqu�es dans la symbiose actinorhizienne Casuarina-Frankia. 5th Meeting on Nitrogen-Fixing Root Endosymbioses" October 12-13 octobre 2017, Toulouse, France (Presentation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Cissoko M, Gully D, Mlouka Carre A, Pignoly S, Hocher V, Tisa L, Chu F, Svistoonoff S., Sy MO. Identification et caract�risation des mol�cules signal impliqu�es dans la symbiose actinorhizienne Casuarina-Frankia. 2nd edition des journees scientifiques du campus de Bel Air. Oct 4th, Dakar, Senegal (Presentation)


Progress 01/01/16 to 12/31/16

Outputs
Target Audience:The actinorhizal symbiosis represents an important ecological and economic role in agriculture and the environment. The diversity of outcomes and impacts suggest that groups working on plant-microbe interactions (beneficial and pathogenic), agricultural and biotechnology industries, land restoration groups, environmental restoration and protection groups, farmers are projected target groups. The educational components of the project target the training of new investigators to agricultural and environmental sciences Changes/Problems:As reported previously, we obtained APHIS approval {notification No. 15-307-106n (526853)] and this year we received the transgenic plants used in our bioassay. It has taken a while to get these transgenic plants up and growing. This caused a delay in our progress. We were able to repeat the work performed in France. This should help streamline the purification, characterization, and analysis process. Lastly, the PI (Tisa) has had a medical issues this year that have also caused a delay in progress. What opportunities for training and professional development has the project provided?For the duration of this project year, this grant has helped support the work of 4 graduate students (Rediet Oshone, Maimouna Cissoko, Tim D'Angelo, Erik Swanson,), 4 undergraduate students (Zakkary McNutt, Ashely Harrington, and Victoria Kliener). As part of Mr. Oshone's PhD thesis research, he worked on the NINA purification and performed the RNASeq experiments. Mr. Swanson was involved in the Frankia genome sequencing and comparative analysis. Mr. D'Angelo aided in the NINA purification and transgenic plant growth and maintenance. Ms Cissoko's PhD thesis work has centered on developing an assay for the quicker detection of the NINA factor through root hair curling and RT-qPCR expression of plant gene NIN. The undergraduate students assisted the graduate students on the above research projects. The laboratory portion of my Molecular Microbiology course (GEN 717) was also involved with this project. How have the results been disseminated to communities of interest?The preliminary results of this study have been presented at national and international professional meetings. For this period: Tisa, L.S.. R. Oshone, A. Sen, F. Ghodhbane-Gtari, and M Gtari. 2016. Genes, genomes and genetics of Frankia and the actinorhizal symbiosis. The 15th Biological Nitrogen Fixation Non-legume (BNF-NL)-Symposium held on 23rd/24th August 2016 as satellite symposium for The 12th European Nitrogen Fixation Conference in Budapest, Hungary. (Invited Talk) The results of the study have also been written up and submitted to peer-reviewed journals of professional societies. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Nitrogen fixation by actinorhizal plants is an important part of the nitrogen budget of the planet. The plants involved are also of economic significance with respect to land reclamation, reforestation, soil stabilization, landscaping, fuel, and as a food source for ruminant animals. Actinorhizal plants provide an excellent mechanism to restore disrupted environmental sites. The purpose of this study was the identification of potential molecular signals and genes involved in the interactions of Frankia with its host plants. Compounds secreted by the plants into the environment were tested to see if they cause a change in the microbe that helps them to be recognized by the plant as a friend, and not a foe. The identification of signaling molecules for the development of beneficial symbiosis has broad impact on agricultural system and could be exploited for other crops. The overall impact of this study is a greater understanding of plant-microbe beneficial interactions. The use of these actinorhizal plants in bioremediation, soil stabilization, nurse cropping, biomass production, and land reclamation applications could potentially impact the 320 million people in the United States. Objective 1. Identification and characterization of the Frankia signal molecule and the genetic basis for their biosynthesis (Year 2)- 40% Complete During this reporting period, we focused on the identification and characterization of the potential Frankia signaling molecule(s). These molecules will signal to the host plant that these bacteria are friend and not foes. A series of bioassays were developed to help in the purification process and to identify the target molecule. Transgenic Casuarina glauca lines containing the promoter of the CgNIN and CgCCAMK genes fused to reporter genes GUS and GFP were used to visualize the expression of the CgNIN and CCaMK genes. These plant genes signal the development of the plant-microbe association at the very early stages preceding infection. The reporter constructs provide a quick system to screen for the Frankia signal molecule. These transgenic plants were transported from France to the US partner to help streamline the purification, characterization, and analysis process. We were able to repeat the work performed in France. Second, transgenic C. glauca plant lines that express the nuclear chameleon protein (Nup:YC2.1) were generated (New Phytologist 2016 209:86-93) to detect Ca+2 spiking during the infection process and used to confirm the activity of the Frankia signal molecule. Third, Frankia infection of the host plant causes root hair curling. We have developed a bioassay for this effect and this root hair curling has been correlated with NIN and CCAMK expression in root hairs. The bioassays with the transgenic plants containing GUS or GFP fusion were used to identify the activity of the Frankia signal molecule (termed NIN activating factor = NINA). From these bioassay, we found that cultures treated with host plant root exudates produced a signal that was present in culture supernatant fluid from the Frankia CcI3 strain, which is compatible with Casuarina. Other Casuarina compatible Frankia strains (Thr and other) also showed this signaling effect. Root-exudates-treated or untreated Frankia strains from a different plant host groups (BCU110501 and others) or other actinobacteria (Streptomyces) did not produce a signal. Both cells and culture supernatant fluids were tested and no signal was detected for these non-host bacteria. Thus, the signal was specific to the host plant symbionts. Both transgenic bioassays were used to characterize the properties of the NINA factor. The size of the factor was estimated by the use of dialysis tubing. Both ProCgNIN::GFP expression and Ca2+ spiking activities indicate that NINA was a small molecule between 0.5-5 KDa. Ethyl acetate and chloroform extraction of culture supernatant fluid showed that the aqueous phase contained the active NINA indicating the molecule had a hydrophilic nature. Similar results were obtained using a butanol extraction: NINA activity was found only in the hydrophilic (aqueous) phase and not in the lipophilic (hydrophic or water repelling) phase. Treatment of the active aqueous fraction derived from Frankia CcI3 supernatant with chitinase failed to block both ProCgNIN activation and nuclear Ca2+ spiking. These preliminary results indicate that NINA is a small hydrophilic molecule, which was active even at 1:10.000 dilution of the culture supernatant fluid. These preliminary physical and chemical properties demonstrate that the NINA factor is unique and distinctly different from the microbial signaling molecules of the legume-Rhizobium and mycorrhizal-plant symbioses, which are amphiphilic and chitin-based. Thus, this represents a novel signaling molecule and a potentially new paradigm for plant-microbe signaling. Early chromatography studies showed that NINA was very hydrophilic passing quickly through any column. Recently, we have been able to bind NINA to a C18. NINA activity eluted from the column 6-10% methanol. We are optimizing the chromatography step prior to analysis of the fractions. This work will be accomplished during the upcoming year. To determine the genetic basis for the biosynthesis of the Frankia signal molecule, we have taken multiple approaches. Several Frankia genomes including more Casuarina compatible isolates were generated and comparative genomics approaches are being performed to identify unique genes for each host compatibility group. We have started to investigate the Frankia global gene expression profiles in response to host (Casuarina) and nonhost (Hippophae) plant root exudates and to the control BD plant medium. RNA from Frankia cultures exposed to these extracts have been collected and RNA-seq has been performed. The cultures were exposed to these conditions for 1, 2, 3 and 5 days. Currently, these data are in analysis. We hope to identify genes in Frankia that involved on NINA biosynthesis by this approach. Objective 2. Determination of how the Frankia signal molecule(s) is perceived by the host plant and the effects of its perception on plant physiology (Year 2)- 30% Complete The actinorhizal plants use signaling components of a common symbiosis pathway also found in rhizobia-legumes and arbuscular mycorrhizal interactions. These common features support the hypothesis that these plants (Fabids) have a predisposition to evolve to being nodulation or establishing a symbiosis .Besides expression of NIN and CCAMK genes, root hair curling and Ca2+ spiking described above, we are investigating the effects of NINA on other plant physiological events that are involved in nodulation. To determine whether LysM receptor kinases (LysM-RLKs, the receptors of the rhizobial Nod factors in legumes) are involved in NINA perception, we have generated transgenic C. glauca lines that down-regulate the expression of CgNFP (a member of LysM-RLKs). The expression of the symbiotic signaling pathway (SYM) genes upon NINA treatment will be investigated by qRT-PCR. Most of this work is being held up until a semi-purified NINA preparation is developed, but we anticipate this work to be performed in the upcoming year.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Ngom, M., R. Oshone, S. Hurst IV, F. Abebe-Akele, S. Simpson, K. Morris, M. O. Sy, A. Champion, W. K. Thomas, and L. S. Tisa. 2016. Permanent Draft Genome sequence for Frankia sp. strain CeD, a Nitrogen-Fixing Actinobacterium Isolated from the Root Nodules of Casuarina equistifolia Grown in Senegal. Genome Announc. 4(2):e00265-16 (doi: 10.1128/genomeA.00265-16).
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Oshone R., M. Ngom, S. Hurst IV, F. Abebe-Akele, S. Simpson, K. Morris, M. O. Sy, A. Champion, W. K. Thomas, and L. S. Tisa. 2016. Permanent Draft Genome Sequences for Frankia sp. Strain Allo2, a Salt-Tolerant Nitrogen-Fixing Actinobacterium Isolated from the Root Nodules of Allocasuarina. Genome Announc. 4(3) e00388-16 (doi: 10.1128/genomeA.00388-16).
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Franche, C., P. Normand, K. Pawlowski, L.S. Tisa and D. Bogusz. 2016. An update on research on Frankia and actinorhizal plants on the occasion of the 18th meeting of the Frankia-actinorhizal plant symbiosis. Symbiosis 70:1-4 (DOI 10.1007/s13199-016-0431-x)
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Tisa, L.S., R. Oshone, A. Sen, I. Sarkar, M. Gtari and A. Ktari. 2016. Genomic approaches toward understanding the actinorhizal symbiosis: An update on the status of the Frankia genomes. Symbiosis 70:5-16 (doi: 10.1007/s13199-016-0390-2)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Tisa, L.S.. R. Oshone, S. Hurst IV, C. Pesce, A. Sen, F. Ghodhbane-Gtari, and M Gtari. 2016. Genes, genomes and genetics of Frankia and the actinorhizal symbiosis. The 15th Biological Nitrogen Fixation Non-legume (BNF-NL)-Symposium held on August 24th 2016 as satellite symposium for The 12th European Nitrogen Fixation Conference in Budapest, Hungary. (Invited Talk)
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Ghodhbane-Gtari, F., N. Beauchemin, A. Gueddou, K. Hezbri, A. Ktari, M. Louati, I. Nouioui, A. Chen, M. Huntemann, N. Ivanova, N. Kyrpides, V. Markowitz, K. Mavrommatis, I. Pagani, A. Sen, L. Wall, T. Woyke, M. Gtari and L. S. Tisa. 2016 Permanent Draft Genome sequence of Nocardia sp. BMG111209, an actinobacterium isolated from nodules of Casuarina glauca. Genome Announc 4(4):e00770-16 doi:10.1128/genomeA.00770-16.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Ghodhbane-Gtari, F., N. Beauchemin, A. Gueddou, K. Hezbri, A. Ktari, M. Louati, I. Nouioui, A. Chen, M. Huntemann, N. Ivanova, N. Kyrpides, V. Markowitz, K. Mavrommatis, I. Pagani, A. Sen, L. Wall, T. Woyke, M. Gtari and L. S. Tisa. 2016. Permanent improved high quality draft genome sequence of Nocardia casuarinae strain BMG51109 an endophyte of actinorhizal root nodules of Casuarina glauca. Genome Announc 4(4):e00799-16 doi:10.1128/genomeA.00799-16
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: DAngelo, T, R. Oshone, F. Abebe-Akele, S. Simpson, K. Morris, W. K. Thomas, and L. S. Tisa. 2016. Permanent Draft Genome Sequence for Frankia sp. Strain BR, a Nitrogen-Fixing Actinobacterium Isolated from the Root Nodules of Casuarina equisetifolia. Genome Announc 4(5):e01000-16 doi:10.1128/genomeA.01000-16.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Nouioui, I., M. Gtari, M. G�ker, F. Ghodhbanne-Gtari, L.S. Tisa, M. Fernandez, P. Normand, M. Huntemann, A Clum, M. Pillay, N. Varghese, T.B.K. Reddy, N. Ivanova, T. Woyke, N.C. Kyrpides, and H.-P. Klenk. 2016. Draft Genome Sequence of Frankia Strain G2, a NitrogenFixing Actinobacterium Isolated from Casuarina equisetifolia, and Able to Nodulate Actinorhizal Plants of the Rhamnales. Genome Announc. 4(3)e00437-16 (doi:10.1128/genomeA.00437-16)
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: DAngelo, T, R. Oshone, F. Abebe-Akele, S. Simpson, K. Morris, W. K. Thomas, and L. S. Tisa. 2016. Permanent Draft Genome Sequences for Frankia sp. Strain EI5c, a Single Spore Isolate of a Nitrogen-Fixing Actinobacterium Isolated from the Root Nodules of Elaeagnus angustifolia. Accepted Genome Announc 4(4)e00600-16 (doi: 10.1128/genomeA.00600-16).


Progress 01/01/15 to 12/31/15

Outputs
Target Audience:The actinorhizal symbiosis represents an important ecological and economic role in agriculture and the environment. The diversity of outcomes and impacts suggest that groups working on plant-microbe interactions (beneficial and pathogenic), agricultural and biotechnology industries, land restoration groups, environmental restoration and protection groups, farmers are projected target groups. The educational components of the project target the training of new investigators to agricultural and environmental sciences Changes/Problems:We had a few problems that may have a significant impact on our rate of expenditures. First, there were some complication with the subcontract between the IRD and UNH that caused a prolonged delay (greater than 6 months) in the transfer of funds to the IRD. This has been since corrected. Two, the lab of the Co-PI (Chu) moved to another building at UNH causing a major disruption. Lastly, the PI (Tisa) has obtained permission to use transgenic plants at UNH and was recently approved by APHIS (Notification No. 15-307-106n (526853) to import the transgenic plants used as a bioassay (NIN:GUS; NIN:GFP). This change should help streamline the characterization process. What opportunities for training and professional development has the project provided?For the duration of the project, this grant has helped support the work of 2 graduate students (Rediet Oshone, Maimouna Cissoko), 3 undergraduate students (Zakkary McNutt, Melissa E McLaughlin, and Emily Lundstedt). As part of Mr. Oshone's PhD thesis research, he developed our Frankia CcI3 and Allo2 fosmid library that is expressed in Escherichia coli and Streptomyces and performed the RNASeq experiments. Ms Cissoko's PhD thesis work has centered on developing an assay for the quicker detection of the NINA factor through root hair curling and RT-qPCR expression of plant gene NIN. The undergraduate students assisted the graduate students on the above research projects. The laboratory portion of my Molecular Microbiology course (GEN 717) was also involved with this project. How have the results been disseminated to communities of interest? The preliminary results of this study have been presented at national and international professional meetings. For this period: [TISA, L.S., T. Furnholm, R. Oshone, M. Ngom, E. Baker, M. Rehan, E. Swanson, J. Wishart, N. Diagne, D. Diouf, V. Hocher, M. O. Sy, L Laplaze, and A. Champion. 2015. Genome-guided approaches toward understanding the ability of Frankia to tolerant harsh environmental conditions. Invited speaker to the 3rd Conference of LAPSE (LMI Adaptation des Plantes et microorganisms associates aux Stress Environmentaux. April 1-2, 2015 in Dakar, Senegal (INVITED TALK); Tisa, L.S., S. Svistoonoff, N. Beauchemin, S. Hurst IV, T. Furnholm, R. Oshone, V. Hocher, H. Gherbi, C. Franche, and D. Bogusz. 2015. Frankia genomics and genome-guided approaches toward understanding the actinorhizal symbiosis. The 8th Congress for the international Symbiosis Society July 12-18, 2015 in Lisbon, Portugal (INVITED TALK); Tisa, L.S., T Furnholm, R Oshone, M. Ngom, E Baker, M Rehan, E Swanson, F. Ghodhbane-Gtari, N. Beauchemin, S. Hurst IV, J Wishart, A Sen, and M Gtari. 2015 Genome-guided approaches toward understanding the actinorhizal symbiosis and the ability of Frankia to tolerant harsh environmental conditions. The 18th International Conference on Frankia and Actinorhizal Plants. August 24th-27th 2015 in Montpellier, France (INVITED TALK); Cissoko,M., V. Vaissayre, M. Ngom, F. Chu, V. Hocher, A. Champion, L.S. Tisa, M. Ourèye Sy, and S. Svistoonoff. 2015. Identification and characterization of signalling molecules involved in actinorhizal symbioses. The 18th International Conference on Frankia and Actinorhizal Plants. August 24th-27th 2015 in Montpellier, France (poster); Tisa, L.S., R. Oshone, A. Sen, F. Ghodhbane-Gtari, and M. Gtari. 2015. Genes, genomes and genetics of Frankia and nonFrankia actinobacteria involved in the actinorhizal symbiosis. Invited plenary speaker to the 23rd North American Symbiotic Nitrogen Fixation Conference in Ixtapa, Mexico December 6-10, 2015 (Invited Talk)]. The results of the study have also been written up and submitted to peer-reviewed journals of professional societies. How have the results been disseminated to communities of interest?The preliminary results of this study have been presented at national and international professional meetings. For this period [TISA, L.S., T. Furnholm, R. Oshone, M. Ngom, E. Baker, M. Rehan, E. Swanson, J. Wishart, N. Diagne, D. Diouf, V. Hocher, M. O. Sy, L Laplaze, and A. Champion. 2015. Genome-guided approaches toward understanding the ability of Frankia to tolerant harsh environmental conditions. Invited speaker to the 3rd Conference of LAPSE (LMI Adaptation des Plantes et microorganisms associates aux Stress Environmentaux. April 1-2, 2015 in Dakar, Senegal (INVITED TALK); Tisa, L.S., S. Svistoonoff, N. Beauchemin, S. Hurst IV, T. Furnholm, R. Oshone, V. Hocher, H. Gherbi, C. Franche, and D. Bogusz. 2015. Frankia genomics and genome-guided approaches toward understanding the actinorhizal symbiosis. The 8th Congress for the international Symbiosis Society July 12-18, 2015 in Lisbon, Portugal (INVITED TALK); Tisa, L.S., T Furnholm, R Oshone, M. Ngom, E Baker, M Rehan, E Swanson, F. Ghodhbane-Gtari, N. Beauchemin, S. Hurst IV, J Wishart, A Sen, and M Gtari. 2015 Genome-guided approaches toward understanding the actinorhizal symbiosis and the ability of Frankia to tolerant harsh environmental conditions. The 18th International Conference on Frankia and Actinorhizal Plants. August 24th-27th 2015 in Montpellier, France (INVITED TALK); Cissoko,M., V. Vaissayre, M. Ngom, F. Chu, V. Hocher, A. Champion, L.S. Tisa, M. Ourèye Sy, and S. Svistoonoff. 2015. Identification and characterization of signalling molecules involved in actinorhizal symbioses. The 18th International Conference on Frankia and Actinorhizal Plants. August 24th-27th 2015 in Montpellier, France (poster); Tisa, L.S., R. Oshone, A. Sen, F. Ghodhbane-Gtari, and M. Gtari. 2015. Genes, genomes and genetics of Frankia and nonFrankia actinobacteria involved in the actinorhizal symbiosis. Invited plenary speaker to the 23rd North American Symbiotic Nitrogen Fixation Conference in Ixtapa, Mexico December 6-10, 2015 (Invited Talk)]. The results of the study have written up and submitted to peer-reviewed journals of professional societies. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Nitrogen fixation by actinorhizal plants is an important part of the nitrogen budget of the planet. The plants involved are also of economic significance with respect to land reclamation, reforestation, soil stabilization, landscaping, fuel, and as a food source for ruminant animals. Actinorhizal plants provide an excellent mechanism to restore disrupted environmental sites. The purpose of this study was the identification of potential molecular signals and genes involved in the interactions of Frankia with its host plants. Compounds secreted by the plants into the environment were tested to see if they cause a change in the microbe that helps them to be recognized by the plant as a friend, and not a foe. The identification of signaling molecules for the development of beneficial symbiosis has broad impact on agricultural system and could be exploited for other crops. The overall impact of this study is a greater understanding of plant-microbe beneficial interactions. The use of these actinorhizal plants in bioremediation, soil stabilization, nurse cropping, biomass production, and land reclamation applications could potentially impact the 320 million people in the United States. Objective 1. We are interested in identification and characterization of potential Frankia signaling molecules. These molecules will signal to the host plant that these bacteria are friend and not foes. A series of bioassays have been developed to help in the purification process and to identify the target molecule. We have shown that the CgNIN gene is required for actinorhizal nodulation in Casuarina glauca. Furthermore, CgNIN expression is correlated with infection by Frankia including pre-infection stages in developing root hairs (New Phytologist 2015 208:887-903). Transgenic C. glauca lines containing the promoter of the CgNIN and CgCCAMK genes fused to reporter genes GUS and GFP were generated. These constructs are used to visualize the expression of the CgNIN and CCaMK genes that signal the development of the plant-microbe association at the very early stages preceding infection. The reporter constructs provide a quick system to screen for the Frankia signal molecule. Second, transgenic C. glauca plant lines that express the nuclear chameleon protein (Nup:YC2.1) were generated (New Phytologist 2016 209:86-93) to detect Ca+2 spiking during the infection process and used to confirm the activity of the Frankia signal molecule. Third, Frankia infection of the host plant causes root hair curling. We are developing a bioassay for this effect and trying to correlate this activity with NIN and CCAMK expression in root hairs. The development of an RNA extraction protocol for small roots has been initiated. The bioassays with the transgenic plants containing GUS or GFP fusion were used to identify the activity of the Frankia signal molecule (termed NIN activating factor = NINA). From these bioassay, we found that cultures treated with host plant root exudates produced a signal that was present in culture supernatant fluid from the Frankia CcI3 strain, which is compatible with Casuarina. Root-exudates-treated or untreated Frankia strains from a different plant host group (BCU110501) or other actinobacteria (Streptomyces) did not produce a signal. Both cells and culture supernatant fluids were tested and no signal was detected for these non-host bacteria. Thus, the signal was specific to the host plant symbionts. The kinetics of the response to the Frankia factor NINA revealed gene expression initiated at 14 h in small lateral roots in the zone where root hairs differentiate and activation was maximal at 24 h after inoculation. Residual reporter gene expression was still detectable by 48h after inoculation, but was completely absent at 3 days after inoculation. Besides reporter gene expression results, these Frankia CcI3 extracts also showed nuclear Ca+2 spiking activity in the C. glauca host plants. Cell-free culture supernatants of the compatible Frankia CcI3 elicited sustained high frequency Ca2+ spiking in host root hairs. Fractions from Frankia CcI3 induced Ca+2-spiking, while fractions from Frankia BCU11501 or growth medium did not. Frankia BCU 11501 is not infective for Casuarina plant. Nod factor purified from the broad host-range NGR234 Rhizobium strain failed to elicit a Ca2+ spiking response. These results demonstrate that the compatible symbiont produces an extracellular signal to the host plant. Both transgenic bioassays were used to characterize the properties of the NINA factor. The size of the factor was estimated by the use of dialysis tubing. Both ProCgNIN::GFP expression and Ca2+ spiking activities indicate that NINA was a small molecule between 0.5-5 KDa. We have initiated experiments to determine the chemical nature of NINA. Ethyl acetate and chloroform extraction of culture supernatant fluid showed that the aqueous phase contained the active NINA indicating the molecule had a hydrophilic nature. Similar results were obtained using a butanol extraction: NINA activity was found only in the hydrophilic (aqueous) phase and not in the lipophilic (hydrophic or water repelling) phase. Butanol extraction is routinely used to isolate rhizobial nod factors (NFs) or the chemical signal involved the rhizobia-legume association. However in this case, rhizobial NFs remain in the lipid phase suggesting major differences in terms of hydrophobicity between NINA and rhizobial NFs. The best known microbial endosymbiotic signaling factors (rhizobial and arbuscular mycorrhizal) have chitin-based structures that are hydrophobic. Treatment of the active aqueous fraction derived from Frankia CcI3 supernatant with chitinase failed to block both ProCgNIN activation and nuclear Ca2+ spiking. These preliminary results indicate that NINA is a small hydrophilic molecule, which was active even at 1:10.000 dilution of the culture supernatant fluid. These preliminary physical and chemical properties demonstrate that the NINA factor is unique and distinctly different from the microbial signaling molecules of the legume-Rhizobium and mycorrhizal-plant symbioses, which are amphiphilic and chitin-based. Thus, this represents a novel signaling molecule and a potentially new paradigm for plant-microbe signaling. To determine the genetic basis for the biosynthesis of the Frankia signal molecule, we have taken multiple approaches. We have started to investigate the Frankia global gene expression profiles in response to host (Casuarina) and nonhost (Hippophae) plant root exudates and to the control BD plant medium. RNA from Frankia cultures exposed to these extracts have been collected and RNA-seq has been performed. The cultures were exposed to these conditions for 1, 2, 3 and 5 days. Currently, these data are in analysis. We hope to identify genes in Frankia that involved on NINA biosynthesis by this approach. Objective 2. The actinorhizal plants use signaling components of a common symbiosis pathway also found in rhizobia-legumes and arbuscular mycorrhizal interactions. These common features support the hypothesis that these plants (Fabids) have a predisposition to evolve to being nodulation or establishing a symbiosis .Besides expression of NIN and CCAMK genes, root hair curling and Ca2+ spiking described above, we are investigating the effects of NINA on other plant physiological events that are involved in nodulation. To determine whether LysM receptor kinases (LysM-RLKs, the receptors of the rhizobial Nod factors in legumes) are involved in NINA perception, we have generated transgenic C. glauca lines that down-regulate the expression of CgNFP (a member of LysM-RLKs). The expression of the symbiotic signaling pathway (SYM) genes upon NINA treatment will be investigated by qRT-PCR, a method to detect and qualify the expression.of individual genes.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Clavijo, F., I. Diedhiou, V. Vaissayre, J. Acolatse, D. Moukouanga, A. Crabos, F. Auguy, C. Franche, H. Gherbi, A. Champion, V. Hocher, D. Bogusz, L S. Tisa, and S. Svistoonhoff. 2015. The Casuarina NIN gene is a transcriptionally activated throughout Frankia root infection as well as in response to bacterial diffusible signals. New Phytologist 208:887-903 doi: 10.1111/nph.13506
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Chabaud M., H. Gherbi, E. Pirolles, V. Vaissayre, J. Fournier, D. Moukouanga, C. Franche, D. Bogusz, L.S.Tisa, D.G. Barker and S. Svistoonoff. 2015. Chitinase-resistant hydrophilic symbiotic factors secreted by Frankia activate both Ca2+ spiking and NIN gene expression in the actinorhizal plant Casuarina glauca. New Phytol. 209:8693 doi: 10.1111/nph.13732
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Tisa, L.S., N. Beauchemin, M. N. Cantor, T. Furnholm, F. Ghodhbane-Gtari, L. Goodwin, A. Copeland, M.r Gtari, M. Huntemannd, N. Ivanova, N. Kyrpides, V. Markowitz, K. Mavrommatis, N. Mikhailova, I. Nouioui, R. Oshone, G. Ovchinnikova, I. Pagani, K. Palaniappan, A. Pati, A. Sen, N. Shapiro, E. Szeto, L. Wall, J. Wishart, and T. Woyke . 2015. Permanent Draft Genome sequence of Frankia sp. strain DC12, an atypical, non-infective (Nod-) ineffective (Fix-) isolate from Datisca cannabina. Genome Announc 3(4):e00889-15. doi:10.1128/genomeA.00889-15.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Swanson, E., R. Oshone, K. Morris, F. Abebe-Akele, S. Simpson, W. K. Thomas, and L. S. Tisa. 2015. Draft Genome sequence of Frankia sp. strain ACN1ag, a nitrogen-fixing actinobacterium isolated from the root nodule of Alnus glutinosa,.Genome Announc 3(6):e01483-15. doi:10.1128/genomeA.01483-15.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Chabaud,M., H. Gherbi, E. Pirolles, V. Vaissayre, J. Fournier, D. Moukouanga, C. Franche, D. Bogusz, L.S.Tisa, D.G. Barker, and S. Svistoonoff. 2014. Chitinase-resistant symbiotic factors secreted by Frankia activate both Ca2+ spiking and NIN gene expression in the actinorhizal plant Casuarina glauca. The 11th European Nitrogen Fixation Conference. September 9-14, 2014, Canary Islands, Spain.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Tisa, L.S., S. Svistoonoff, N. Beauchemin, S. Hurst IV, T. Furnholm, R. Oshone, V. Hocher, H. Gherbi, C. Franche, and D. Bogusz. 2015. Frankia genomics and genome-guided approaches toward understanding the actinorhizal symbiosis. The 8th Congress for the international Symbiosis Society July 12-18, 2015 in Lisbon, Portugal.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Tisa, L.S., T Furnholm, R Oshone, M. Ngom, E Baker, M Rehan, E Swanson, F. Ghodhbane-Gtari, N. Beauchemin, S. Hurst IV, J Wishart, A Sen, and M Gtari. 2015 Genome-guided approaches toward understanding the actinorhizal symbiosis and the ability of Frankia to tolerant harsh environmental conditions. The 18th International Conference on Frankia and Actinorhizal Plants. August 24th-27th 2015 in Montpellier, France
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Cissoko,M., V. Vaissayre, M. Ngom, F. Chu, V. Hocher, A. Champion, L.S. Tisa, M. Our�ye Sy, and S. Svistoonoff. 2015. Identification and characterization of signalling molecules involved in actinorhizal symbioses. The 18th International Conference on Frankia and Actinorhizal Plants. August 24th-27th 2015 in Montpellier, France (poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Tisa, L.S., R. Oshone, A. Sen, F. Ghodhbane-Gtari, and M. Gtari. 2015. Genes, genomes and genetics of Frankia and nonFrankia actinobacteria involved in the actinorhizal symbiosis. Invited plenary speaker to the 23rd North American Symbiotic Nitrogen Fixation Conference in Ixtapa, Mexico December 6-10, 2015 (Invited Talk)
  • Type: Book Chapters Status: Published Year Published: 2015 Citation: Beauchemin, N. F. Ghodhbane-Gtari, T. Furnholm, J. Lavenus, S. Svistoonoff, P. Doumas, D. Bogusz, L. Laplaze, and L. S. Tisa. 2015. Actinorhizal Plant Root Exudates Alter the Physiology, Surface Properties, and plant Infectivity of Frankia. Chapter 35 in Biological Nitrogen Fixation Vol. 1. F.J. de Bruijn (Ed.) John Wiley & Sons, Inc. pp. 359-363.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Cissoko M, Vaissayre V, Ngom M, Chu F, Hocher V, Champion A, Tisa L, Sy MO, Svistoonoff S. 2015 Identification and characterization of signalling molecules involved in actinorhizal symbioses. 2�mes Journ�es Scientifiques du CAMES Nov 23-25th, Dakar, Senegal (poster).
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Swanson, E., R. Oshone, S. Simpson, K. Morris, F. Abebe-Akele, S. Simpson, W. K. Thomas, and L. S. Tisa. 2015. Permeant draft genome sequence of Frankia sp. strain AvcI1, a nitrogen-fixing actinobacterium isolated from the root nodule of Alnus viridis ssp. crispa grown in Canada. Genome Announc. 3(6):e01511-15. doi:10.1128/genomeA.01511-15.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: TISA, L.S., T. Furnholm, R. Oshone, M. Ngom, E. Baker, M. Rehan, E. Swanson, J. Wishart, N. Diagne, D. Diouf, V. Hocher, M. O. Sy, L Laplaze, and A. Champion. 2015. Genome-guided approaches toward understanding the ability of Frankia to tolerant harsh environmental conditions. Invited speaker to the 3rd Conference of LAPSE (LMI Adaptation des Plantes et microorganisms associates aux Stress Environmentaux. April 1-2, 2015 in Dakar, Senegal.