FUNCTIONAL GENOMIC APPROACHES TOWARD UNDERSTANDING THE FRANKIA-ACTINORHIZAL PLANT ASSOCIATION AND THEIR RESPONSES TO HARSH ENVIRONMENTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1019869
• Project Start Date: Oct 1, 2019
• Project End Date: Sep 30, 2022
• Program Code: [(N/A)]- (N/A)

Project Director
Tisa, LO, S..

Recipient Organization
UNIVERSITY OF NEW HAMPSHIRE
51 COLLEGE RD SERVICE BLDG 107
DURHAM,NH 03824

Performing Department
Molecular, Cellular and Biomedical Sciences

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 the potential for bioremediation and phytoremediation applications, especially on heavy-metal-contaminated-land. Furthermore, the metabolic versatility of Frankia also suggests bioremediation capabilities against polyaromatic hydrocarbons. A major hindrance in the application of this system is the lack of genetic tools for Frankia, the bacterial partner of the symbiosis. The purpose of this study is the development of tools that will allow the genetic analysis of Frankia physiology and the interactions of these bacteria with their host plants. The use and development of this beneficial symbiosis has a broad impact on the 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
206	4010	1040	60%
206	4010	1100	40%

Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
4010 - Bacteria;

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

Keywords

actinorhizal plants

frankia

bioremediation

stress environments

genetics

crispr-cas9

Goals / Objectives
This NHAES project was developed as a research program that encompasses several different aspects of the Frankia-actinorhizal plant symbiosis. The overall goal of the project is to understand the physiology of Frankia, the bacterial partner of the actinorhizal symbiosis, and its ability to help these plants colonize harsh environments. Analysis of the 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. Another surprise was the presence of many natural product biosynthetic clusters suggest that these bacteria were a rich new source of bioactive molecules. We have initiated studies on non-Frankia actinobacteria associated with actinorhizal plants to elucidate plant-microbe aspects of the symbiosis and have begun comparative bioinformatics studies on Frankia genomes from different host ranges to identify potential host recognition genes. The microbiome of Casuarina nodules under different environmental conditions was determined and several other bacteria were identified as a members of the nodule microbiome. This proposal seeks to continue and expand those studies to actinorhizal plants found in New England region or other parts of the United States. The goals of this proposal are to: [1] establishment of standard protocols for genetic analysis of these bacteria and initiating functional genomic studies, [2] elucidation of the mechanisms involved Frankia stress tolerance and their potential for bioremediation and reclamation, [3] expanding our study of the actinorhizal symbiosis toward understanding the role of non-Frankia actinobacteria endophytes (like Nocardia sp.) and other members of the actinorhizal microbiome, and [4] elucidation of the steps involved in plant-microbe interactions during the development of the symbiosis.Our strategy focuses on the use of genomics tools to identify genes that are differentially regulated by harsh enviromental conditions, involved in Frankia physiology or host plant interactions and to establish genetic tools for Frankia. We are also interested in investigating the role of members of the actinorhizal microbiome and their effect on the ability of actinorhizal plants to survive harsh environmental conditions. This study clearly is focused on a research priority for the USDA NIFA Program and on the long term goals of the program including improved methods of manipulating plant-associated microorganisms.Objectives are:1. Developing new genetic tools for functional analysis of Frankia.2. Elucidating the effects of harsh environmental stress on Frankia and the actinorhizal symbiosis3. Utilize new genetic and genomic tools to study the development of the plant-microbe interaction between Frankia and actinorhizal plants4. Elucidate the role of non-Frankia actinobacteria endophytes (Nocardia sp.) in the actinorhizal symbiosis

Project Methods
Building on the tools that were developed in our previous project, we will focus on several different aspects of the Frankia-actinorhizal plant symbiosis.1. Developing new genetic tools for functional analysis of Frankia.The absence of a genetic system for Frankia has been a major obstacle for probing Frankia-actinorhizal symbiosis. A cloning vector was successfully introduced into Frankia that allows the expression of cloned genes. The plasmid has been stably maintained for several years. This is the first step in developing genetic tools, this technique opens up new avenues for research in the field. The experimental strategy involves the use of CRISPR technology to create deletions within Frankia target genes. This gene-editing technique has been used with prokaryotes to generate these mutations. We have constructed a modified CRISPR-Cas9 plasmid with a synthesized Frankia 16S promoter was cloned in front of the Cas9 gene to allow expression of Cas9 in Frankia. The construct also contained the guide RNA (gRNA) from the original plasmid pCRISPRomyces-2 and the ori, rep and mob genes from pBBR plasmids, which allow mobilization and stability in Frankia. This construct will be used to generate "knock-out" mutants for selected Frankia genes. After molecular confirmation of the mutants, they will be studied for effects on plant-microbe interactions and physiological effects.2. Elucidating the effects of harsh environmental stress on Frankia and the actinorhizal symbiosisFrankia and actinorhizal plants are found in many harsh environments but little known about the mechanism and ability of these bacteria to adapt to these extreme environments. We use our 24-well semi-high throughput growth assay to survey the ability to tolerate harsh conditions. By the use of a genome-guided approach, we will extend this screen to investigate the minimum inhibitory concentration (MIC) and maximum tolerable concentration (MTC) values for toxic aromatic hydrocarbons, heavy metals, and stress conditions including salt, osmotic, nitric oxide (NOS) and reactive oxygen (ROS). A bioinformatics genome-guided approach will be used to identify putative degradation operons or resistance pathways and test these compounds on the Frankia strains predicted to have those activities gene expression levels of identified natural product clusters will be measured under several different environmental conditions including the presence of plant exudates. Primers will be designed for key genes in these clusters and gene expression will be measured by qRT PCR from extracted mRNA. Proteomics and transcriptomics methods will also be applied to identify global gene expression. Genetic tools established in objective 1 can also be used to confirm the role of genes in these processes.3. Utilize new genetic and genomic tools to study the development of the plant-microbe interaction between Frankia and actinorhizal plants Besides testing the ability of the mutants generated in Objective 1, we are interested in following the path of the infection process. For this purpose, we will use tagged Frankia strains to monitor the infection process by confocal microscopy. We have generated a pBBR1mcs construct that contains the mCherry gene with the Frankia 16S promoter in front of that gene. The use of this promoter will allow expression with Frankia to fluoresce bright red to allow the tagged cells to be followed throughout the infection process and nodule development. The tagged strain will be used to follow all stages of the infection process up to the development of a functioning root nodule.4. Elucidate the role of non-Frankia actinobacteria endophytes (Nocardia sp.) in the actinorhizal symbiosisThe availability of genome data for 45 Frankia genomes and three nonFrankia actinobacteria endophytes of actinorhizal plants provide a unique opportunity to expand this system to other Frankia strains and endophytes to investigate common themes in the infection process and plant-microbe interaction. By the use of comparative genomics approaches, we will look at common gene homologs that may provide clues on the steps involved in the plant infection and nodule development. Many of the experiments that we have performed on the Frankia CcI3-Casuarina system will be performed on other actinorhizal plant symbioses and the nonFrankia actinobacteria endophytes.Culture-independent and -dependent approaches will be used to investigate the microbial diversity and microbiome from local nodules of Alnus, Myrica, Comptonia, Ceanothus and Elaeagnus plants. The genomic DNA (gDNA) will be extracted from surface sterilized root nodules and rhizosphere and the microbial community will be determined by Next-generation sequencing of 16S rRNA. The metagenomes of these samples will be determined in order to understand the genetic potential of these populations. In tandem with these genomic studies, a culture-dependent approach will be performed that is focused on isolating microbes from local nodules of actinorhizal plants. We will use the appropriate enrichment and culture techniques for unique microbes identified from the molecular work. The genomes of these isolates will be sequenced to provide a larger database.

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

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:For objective 4, we would like to expand our plant microbiome investigation to local maple trees beyond that of just actinorhizal trees. The same techniques would be used inclusding 16S amplicon and next generation sequencing but with maple trees. This change will allow us the opportunity to expand the effects of these microbes on another local plant. What opportunities for training and professional development has the project provided?For the duration of the project period, this grant has helped support the work of 1 postdoctoral fellow (Céline Pesce), 2 graduate students (Erik Swanson and Megan Worth), and 4 undergraduate students (Delancey Hirsch, Alison Lafluer, Lilly Friedman, and Kelsey Mecurio). Ms. Worth's PhD research is focused on elucidate the role of non-Frankia actinobacteria endophytes in the actinorhizal symbiosis and the effects of stress conditions on nodulation of actinorhizal plants. Mr. Swanson's PhD research is directed toward exploring the degradation of polyromantic hydrocarbon compounds including polychlorinated biphenyls by Frankia and the potential of actinorhizal plants for phytoremediation. He was also involved in community profiling of the phytobiome of actinorhizal plants. Dr. Pesce work has focused on the development of a genetic system for Frankia and she is also involved in studies on role of microbiome the actinorhizal symbiosis and tolls for study on the symbiosis. 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 Covid pandemic affected professional conferences and several canceled or postponed. Two of these evens (The 20th International Conference on Frankia and Actinorhizal Plants and The preliminary results of this study have been presented at national and international professional meetings and at invited talks. 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. and L.S. Tisa. 2019. Genetic tools inFrankia. New England Workshop on Symbiosis. November 9, 2019 at University of Vermont; Swanson, E. and L. S. Tisa. 2019. The phytomicrobiome ofCoriaria myrtifolia. New England Workshop on Symbiosis. November 9, 2019 at University of Vermont; and Mercurio, K., I. Davis, C. Pesce, E. Swanson and L.S. Tisa. 2020. Frankia and Friends: Roles of various nodule inhabitants in the actinorhizal symbiosis. The 29th Annual COLSA Undergraduate Research Conference, April 24th. 2020). 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? As preface to this section, I would like to point out that the pandemic caused serious delays in the progress of this project. My laboratory was shut down for over 6 months causing many delays especially propagating plants for infection studies. 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. A major hindrance in the application of this system is the lack of genetic tools for Frankia, the bacterial partner of the symbiosis. The purpose of this study is the development of tools that will allow the genetic and genomic analysis of Frankia physiology and the interactions of Frankia with its host plants. 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. Continue development of genetic tools for functional analysis of Frankia. Building off our previous results of our last progress report with the pHTK1 plasmid, we have made significant progress in the development of genetic tools for Frankia. A cloning vector was successfully introduced into Frankia that allows the expression of cloned genes. Furthermore, a plasmid with the green fluorescent protein (GFP) was used and allowed expression of GFP in Frankia. This is the first report on introduction plasmids into Frankia and their expression of GFP and a cloned gene. The cloned gene provided increased salt tolerance in the transformed Frankia. Currently, we have initiated experiments to develop site-specific mutagenesis techniques for Frankia The results of this objective are providing the framework the development of genetic approaches toward the study of the bacterial partner, Frankia, of the actinorhizal symbiosis, a major breakthrough for the field. Objective 2. Continue investigating the effects of harsh environmental stress on Frankia and the actinorhizal symbiosis To understand of the mechanisms that Frankia aids the plants to overcome harsh environmental conditions, we have examined Frankia cultures grown under harsh environmental conditions. Selenite is major contaminate of soil and several Frankia strains are tolerant to selenite. These strains reduce selenite to elemental selenium, a nontoxic toxic form and generated nanosphere particles containing selenium. The shut down on my laboratory delayed efforts at global transcriptome studies on the effect of selenite on Frankia inefficax (strain EuI1c). However, proteome analysis of F. inefficax indicated that several proteins were expressed under selenite-stress. The proposed transcriptome work will provide a complete picture of the physiological response to selenite stress. A comparative genomics approach was used to detect regions in Frankia genomes homologous to a known dioxin-degrading operon (bph) in a closely related species, Rhodococcus RAH1. Only 6 of the 39 available Frankia genomes, including Frankia strain EUN1f and F. inefficax (EuI1c) contain the bph operon with the putative biphenyl and dioxin-like compound degradation genes. Frankia strains EUN1f and EuI1c were able to metabolize dioxin-like compounds as a sole carbon and energy source. Quantitative reverse-transcriptase PCR (qPCR)assays show that expression of the bph operon was induced in Frankia stain EuI1c after exposure to three different dioxin-like compounds (biphenyl, 4-chlorobiphenyl, and dibenzofuran). A dyed based assay was used to quantify cellular respiration in Frankia EUN1f and EuI1c after exposure to dioxin-like compound. These assays confirm that Frankia is able to maintain cellular functions after dioxin-like compound exposure to respire when dioxin-like compounds are the only carbon source available. The metabolic potential of Frankia is being elucidated by these studies and shows an opportunity for use in bioremediation. Global transcriptome (RNASeq) analysis of Frankia EUN1f under biphenyl-stress provides insight on the genes involved in the process and these results were confirmed by qPCR. The results of this objective have increased over understanding of how these beneficial microbes aid the plant in its ability to reclaim degraded land especially land affected by salt or pollutants. The ability of the microbe to tolerant these harsh conditions influences how the plants will survive under these environments. Objective 3. Utilize new genetic and genomic tools to study the development of the plant-microbe interaction between Frankia and actinorhizal plants. We are continuing to develop vectors with different fluorescent proteins and promoters. The Frankia 16S promoter is being introduced in front of different genes for fluorescent proteins to allow continuous expression of the Green Fluorescent Protein (GFP) or mCherry within Frankia. Under UV light, GFP fluoresces green, while mCherry fluoresces red. The GFP-tagged and mCherry-tagged strains will be used to follow the symbiont throughout the infection process and nodule development. We should be able to visualize the infection pathway for the establishment of this beneficial symbiosis. Objective 4. Elucidate the role of non-Frankia actinobacteria endophytes (Nocardia sp.) in the actinorhizal symbiosis. We have begun to elucidate the microbiome of local actinorhizal plants. Culture-dependent and -independent approaches were used to identify the microbiome of nodules of Alnus trees in NH. Several samples are being taken seasonally and the microbiome will determined using next-generation of the 16S rRNA genes. The pandemic shut down affected these studies. However, we had previously initiated culture-dependent approach isolating microbes from samples collected from local nodules of Alder plants. We have isolated over 50 different bacteria. For ten of these isolates, the genomes of these isolates were sequenced to provide a larger database and published results. To study the plant-microbe interaction between actinorhizal plants and nonFrankia actinobacteria, we have continued to study two of these NH isolates, Streptomyces 4R-3d and Rhodococcus 1R11 physiologically. Analysis of these genomes revealed an absence of any known nitrogenase genes, but they contained biosynthetic pathways for several plant-growth-promoting factors (i.e phytohormones). Co-inoculation of Frankia QA3 with Streptomyces 4R-3d did not alter the nodulation onset versus Frankia alone, but promoted greater plant health. Prolonged incubation (over a month) of Alnus plants with Streptomyces 4R-3d promoted plant health but not as extensive as co-inoculation with Frankia. These preliminary results show the potential of these nonFrankia actinobacteria at enhancing beneficial effects on plant health.

Publications

• Type: Books 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)
• 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: 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: Journal Articles Status: Published Year Published: 2020 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: 2020 Citation: Davis, I., J. SEVIGNY, V. Kleiner, K. Mercurio, C. Pesce, E. SWANSON, W. K. Thomas and L. S. Tisa. 2020. Draft Genome Sequences of 10 Bacterial Strains Isolated from Root Nodules of Alnus Trees in New Hampshire. Microbiol. Res. Annoc. 9:e01440-19 (DOI:10.1128/MRA.01440-19)
• 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. 2020. Advances in Frankia genome studies and molecular aspects of tolerance to environmental stresses. Chapter 30 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: 2020 Citation: Ghodhbane-Gtari, F. E. SWANSON, A. Gueddou, S. Simpson, K. Morris, W. K. Thomas, M Gtari, and L. S. Tisa. 2020. Draft Genome sequence for Frankia sp. strain BMG5.11, a Nitrogen-Fixing Bacterium Isolated from Elaeagnus angustifolia. Microbiol Res Announc 9:e00824-20. https://doi.org/10.1128/MRA.00824-20.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Mansour, S., E. Swanson, C. Pesce, S. Simpson, K. Morris, W. K. Thomas, and L. S. Tisa. 2020. Draft Genome Sequences for the Frankia sp. Strains CgS1, CcI156 and CgMI4, Nitrogen-Fixing Bacteria Isolated from Root Nodules of Casuarina sp. in Egypt. J. Genomics 8: 84-88. doi: 10.7150/jgen.51181
• 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. and L.S. Tisa. 2019. Genetic tools in Frankia. New England Workshop on Symbiosis. November 9, 2019 at University of Vermont.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Swanson, E. and L. S. Tisa. 2019. The phytomicrobiome of Coriaria myrtifolia. New England Workshop on Symbiosis. November 9, 2019 at University of Vermont
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Mercurio, K., I. Davis, C. Pesce, E. Swanson and L.S. Tisa. 2020. Frankia and Friends: Roles of various nodule inhabitants in the actinorhizal symbiosis. The 29th Annual COLSA Undergraduate Research Conference, April 24th. 2020.