ENHANCING THE ESTABLISHMENT OF BENEFICIAL ROOT/RHIZOSPHERE INTERACTIONS IN AGRICULTURALLY RELEVANT PLANTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1012482
• Grant No.: 2017-67013-26523
• Cumulative Award Amt.: $496,345.00
• Proposal No.: 2017-02568
• Project Start Date: Apr 15, 2017
• Project End Date: Jan 21, 2020
• Grant Year: 2017
• Program Code: [A1171]- Plant Biotic Interactions

Recipient Organization
INDIANA UNIVERSITY
JORDAN HALL 142
BLOOMINGTON,IN 47401

Performing Department
Molecular & Cellular Biochem

Non Technical Summary
Much scientific attention has deservedly been focused on the rhizobial genera of bacteria that form nitrogen fixing root nodules with legume plants. This well studied beneficial microbial-plant interaction promotes significantly enhanced crop yields under conditions that require reduced or no addition of nitrogen based fertilizer. Less well understood are members of the Azospirillum clade that constitute a secondary class of nitrogen fixing plant-growth promoting bacteria that also associates with plant roots. These latter microorganisms are very interesting as they form beneficial interactions with a much wider number of crop plants then do rhizobial species. Specifically, Azospirillum are known to productively interact with >64 plants including all major crop plants such as corn, rice, wheat, oats, barley, and many vegetables. This group of microorganisms have received less commercial interest owing to the fact that obtaining productive Azospirillum plant colonization in the field is difficult leading to variability in crop yield enhancement.This proposal is centered on understanding how Azospirillum naturally forms a beneficial interaction with crop roots. This understanding will then be used to develop methodologies that enhance this interaction. We have also observed that Azospirillum is capable of stimulating Rhozobium species to form productive nitrogen fixing root nodules. This process will also be studied with a long term goal of determining conditions for reproducible stimulation of nodulation in a broad number of crop plants.

Animal Health Component

0%

Research Effort Categories

Basic

90%

Applied

0%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	1410	1100	50%
206	1411	1000	50%

Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1410 - Beans (dry); 1411 - Beans (fresh, fresh-processed);

Field Of Science
1100 - Bacteriology; 1000 - Biochemistry and biophysics;

Keywords

azospirillum brasilense

phaseolus vulgaris

bio-fertilizer

chemotaxis

rhizosphere

seed microbiome

Goals / Objectives
The three main goals of this proposal remain unaltered from the grant submitted in July 2016.The first goal is to study underlying mechanisms that allow A. brasilense to effectively colonize crop plants under natural (native) conditions. This goal is based on our observation that A. brasilense not only colonizes roots but also actively and aggressively migrates into areal above ground portions of plants. One tissue that has enhanced A. brasilense colonization is ovary reproductive tissue in the developing flower/seed. Our first goal is to determine if there is a compound synthesized by plant reproductive tissue that specifically attracts A. brasilense.The second goal is to address optimization of root colonization by A. brasilense. We have observed that surface sterilized seeds poorly transmit A. brasilense to the roots when they are surface inoculated with A. brasilense after sterilization. In contrast, unsterilized seeds that natively have A. brasilense in the seed coat are effective root colonizers. A second observation is that A. brasilense naturally present in the seed coat appears to be in a dormant encystment state. In this goal we plan to directly address whether A. brasilense in the seed coat is in a replicative or dormant state by undertaking RNA-seq transcriptome profiling of A. brasilense cells in the seed coat. The results of this analysis will drive future studies to optimize seed inoculation using surface applied vegetative versus dormant A. brasilense cells.The third goal is centered on our discovery that a cGMP deficient mutant of A. brasilense is capable of dramatically stimulating the formation of productive nitrogen fixing rhizoidal nodules on common bean plants. We have recently determined that the strain of A. brasilense that we use for studies with common bean does not stimulate increased rhizoidal nodules in other plant species. Consequently, one of the first goals to address will be to isolate Azospirillum and rhizobial species from soybeans and to subsequently determine if these results can be replicated with these strains in this important crop plant. We also plan to probe the molecular mechanism of enhanced root nodulation by A. brasilense. We specifically plan to determine if Azospirillum affects the cytokinin signaling pathway by inhibiting expression of PIN1 and PIN2 polar auxin transporters or perhaps whether Azospirillum stimulates rhizobial nod gene expression.The objectives for goals 1 and 2 can be summarized as:Determine if we can biochemically isolate a compound from seed coats that attracts A. brasilenseDetermine whether A. brasilense in seed coats are in a vegetative or a dormant cyst stateDetermine if there is a chemical signal in maturing seed coats that induces A. brasilense to undergo cyst dormancyAddress whether A. brasilense cysts are effective root colonizers of various cropsThe objectives for goal 3 can be summarized as:Develop methodology to observe A. brasilense stimulation of rhizoidal nodulation under defined conditionsOnce defined nodulation is obtained then we will address the molecular mechanism of enhanced root nodulation by A. brasilense using qRT-PCR to analyze gene expression in the nodulation pathway.Isolate strains of Azospirillum and Rhizobia that are present on soybean roots and address whether these strains also enhance nodulation in this important crop

Project Methods
All of our initial studies will involve plants grown in pots in a greenhouse which will allow us to optimize light, hydration and temperature for plants as well as the genotype of the Azospirillum inoculation.The identification of compounds in plant flowers that induces attraction of Azospirillum will involve biochemical fractionation of compounds in developing flowers using HPLC chromatography. These separated compounds will be assayed for their ability to stimulate Azospirillum chemotaxis. A similar set of experiments will be undertaken to access if maturing seed coats have a plant compound that stimulates encystment. In this set of experiments we will separate seed coat leachate by HPLC chromatography and microscopically access fractionated samples for their ability to induce encystment.For analysis of Azospirillum stimulation of rhizobial nodulation, we first need to develop a more defined plant growth methodology such as growth on defined medium and or growth hydroponically. Growth under such defined conditions will allow us to control the amount of the inoculant as well as the genotype of Azospirillum and rhizobial strains that are interacting with developing plant roots. Once that is established then we can begin to address which pathway's in the nodulation development cascade are affected by Azospirillum using qRT-PCR amplification of transcripts.As each research goal becomes accomplished, we will be addressing Effort by giving talks at conferences and in publishing our work in high visibility journals. Papers in high quality refereed journals will ensure that the research is undergone rigorous Evaluation for quality and statistical reproducibility. The impact will be measured by the number of citations that our published work received (the PI currently has >6,650 citations of published work from his laboratory).

Progress 04/15/17 to 04/14/20

Outputs
Target Audience: The main target audience are scientists that undertake research in the general area of biological crop enhancement (or biofertilizer). These were reached via submission of several publications as well as by presentation of our work at a scientific conference titled4thPartnerships in Biocontrol, Biostimulants, & Microbiome Congress:Raleigh-Durham, NC, USA. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two undergraduate students, two graduate students and one postdoctoral fellow have/are working on various aspects of this research project. Research from these efforts led to the graduation of one graduate student who quickly obtained a full-time research position at Dupont-Pioneer specifically to research the commercial use of Azospirillum as a biofertilizer. A second graduate student involved in this project obtained a highly competitive summer internship at Bayer (formerly Monsanto) .This student will be defending her thesis in four weeks and has been highly encouraged by Bayer to apply to their Biofertilizer unit for a full-time position. A postdoctoral fellow in my group working on this project has also recently obtained a full-time position in industry working on plant production of plant hormones (auxin etc). Consequently, students and postdoctoral fellows working on this project are highly sought after by corporations that are exploring the use of microbes for crop improvements. The PI has also recently received invitations to high visibility conferences that have sessions dedicated to productive plant-microbe interactions. Graduate students from my laboratory will also be attending several of these conferences and presenting their research results. How have the results been disseminated to communities of interest?Two manuscripts highlighting results from this study have recently (2018) been published and a third is currently is under review at PLOS Genetics. The Symbiosis manuscript has really caught the attention of researchers in this field as it has been highlighted by an editorial commentary regarding its significance at another journal. We also plan to present this work at a conferences this summer. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? IMPACT: Our published results showing that Azospirillum is a member of the seed microbiome (published in the journal Symbiosis) is highly significant. This study demonstrated that developing flowers attract Azospirillum to migrate from the root to the developing seed. This allows seeds to be populated by this beneficial microorganism and to be passed to subsequent plant generations. Our work also established that Azospirillum which is natively present in the seed coat is more effectively transmitted to developing roots then is Azospirillum that is added artificially to the seed coat. One impact of this study is that techniques can be developed to maximally populate sees with Azospirillum in a way that maximizes crop yield enhancement by this beneficial bacterium.These results were recently presented at the "4thPartnerships in Biocontrol, Biostimulants, & Microbiome Congresswhere it stimulated robust discussion amount the participants.We have also undertaken the first genetic dissection of regulatory genes involved in allowing dormant cysts to sense when environmental conditions are favorable for growth for the purpose of initiating germination.This is the first such analysis on how cells emerge from dormancy in any Gram-positive bacterial species. OUTCOME ACHIEVED AND RESULTS ACCOMPLISHED: In the past funding period we continued to study several aspects regarding how Azospirillum interacts with plants. Our results are as follows: 1) We have constructed Azospirillum mutations in the Che3 signal transduction cascade and have obtained mutations that either overproduce or underproduce cysts. We have finished characterizing these mutants and have determined that cells that overproduce cysts actually colonize roots more effectively than do cells that underproduce cysts.A manuscript describing these results should be submitted this summer 2) We are continuing to analyze the role of cGMP in root development and in enhancing nodulation.We have observed that the Azospirillum cGMP mutant affects tap root development leading to the formation of seedlings containing multiple tap roots. We have also observed that beans inoculated with a cGMP defective strain of Azospirillum actually produce nodules that have altered morphology (they are larger than nodules made by plants inoculated with wild type Azospirillum).Furthermore, using GFP expressing strains of Azospirillum, we observe that nodules not only contain rhizobia but also harbor the cGMP defective strain of Azospirillum.We will continue to analyze the role of cGMP in nodulation until its ready for publication 3) We are continuing our analysis of plant signaling compounds present in seeds that signal Azospirillum to form cysts.Our preliminary analysis suggests that the signal may bealkylresorcinol lipids that are present in seed coats.This is very interesting as alkylresorcinol's are made late in seed development seeds to protect seeds from desiccation.The use ofplant synthesized alkylresorcinol to signal Azospirillum to enter dormancy suggests that there is interkingdom signaling from plant to bacteria to ensure that this bacterium becomes dormant as seeds also become dormant.This avenue of research will be continued until its ready for publication. 4). We just finished a detailed analysis of genome wide gene expression changes that occur as cyst cells germinate to form replicating vegetative cells.This study was undertaken to obtain an understanding of signals that lead cysts to undergo the germination process.This is the first study of its kind on the germination of dormant cysts.This work has been completed and submitted to PLOS Genetics.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Ashok, N., & C. E. Bauer (2020) Evidence of defined temporal expression patterns that lead a gram-negative cell out of dormancy. Under revision for PLOS Genetics
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Malinich, E. & C. E. Bauer (2018) Transcriptome Analysis Of Azospirillum brasilense Vegetative And Cyst States Reveals Large Scale Alterations In Metabolic And Replicative Gene Expression. Microbial Genomics 4: doi: 10.1099/mgen.0.000200
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Malinich, E. & C. E. Bauer (2018) The plant growth promoting bacterium Azospirillum brasilense is vertically transmitted in Phaseolus vulgaris (common bean). Symbiosis 76(2), 97-108. https://doi.org/10.1007/s13199-018-0539-2. A commentary of the significance of manuscript was published by: Zawoznik M & M Daniela-Groppa (2019) Viewpoint: Azospirillum in the cloudy boundaries of bacterial endophytes. Applied Soil Ecology 135:194-195
• Type: Theses/Dissertations Status: Submitted Year Published: 2020 Citation: Ashok, N. 2020. Genetic and transcriptomic analysts of cyst germination. Indiana University

Progress 04/15/17 to 01/21/20

Outputs
Target Audience: The main target audience are scientists that undertake research in the general area of biological crop enhancement (or biofertilizer). These were reached via submission of several publications as well as by presentation of our work at a scientific conference titled4thPartnerships in Biocontrol, Biostimulants, & Microbiome Congress:Raleigh-Durham, NC, USA. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two undergraduate students, two graduate students and one postdoctoral fellow have/are working on various aspects of this research project. Research from these efforts led to the graduation of one graduate student who quickly obtained a full-time research position at Dupont-Pioneer specifically to research the commercial use of Azospirillum as a biofertilizer. A second graduate student involved in this project obtained a highly competitive summer internship at Bayer (formerly Monsanto) .This student will be defending her thesis in four weeks and has been highly encouraged by Bayer to apply to their Biofertilizer unit for a full-time position. A postdoctoral fellow in my group working on this project has also recently obtained a full-time position in industry working on plant production of plant hormones (auxin etc). Consequently, students and postdoctoral fellows working on this project are highly sought after by corporations that are exploring the use of microbes for crop improvements. The PI has also recently received invitations to high visibility conferences that have sessions dedicated to productive plant-microbe interactions. Graduate students from my laboratory will also be attending several of these conferences and presenting their research results. How have the results been disseminated to communities of interest?Two manuscripts highlighting results from this study have recently (2018) been published and a third is currently is under review at PLOS Genetics. The Symbiosis manuscript has really caught the attention of researchers in this field as it has been highlighted by an editorial commentary regarding its significance at another journal. We also plan to present this work at a conferences this summer. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? IMPACT: Our published results showing that Azospirillum is a member of the seed microbiome (published in the journal Symbiosis) is highly significant. This study demonstrated that developing flowers attract Azospirillum to migrate from the root to the developing seed. This allows seeds to be populated by this beneficial microorganism and to be passed to subsequent plant generations. Our work also established that Azospirillum which is natively present in the seed coat is more effectively transmitted to developing roots then is Azospirillum that is added artificially to the seed coat. One impact of this study is that techniques can be developed to maximally populate sees with Azospirillum in a way that maximizes crop yield enhancement by this beneficial bacterium.These results were recently presented at the "4thPartnerships in Biocontrol, Biostimulants, & Microbiome Congresswhere it stimulated robust discussion amount the participants.We have also undertaken the first genetic dissection of regulatory genes involved in allowing dormant cysts to sense when environmental conditions are favorable for growth for the purpose of initiating germination.This is the first such analysis on how cells emerge from dormancy in any Gram-positive bacterial species. OUTCOME ACHIEVED AND RESULTS ACCOMPLISHED: In the past funding period we continued to study several aspects regarding how Azospirillum interacts with plants. Our results are as follows: 1) We have constructed Azospirillum mutations in the Che3 signal transduction cascade and have obtained mutations that either overproduce or underproduce cysts. We have finished characterizing these mutants and have determined that cells that overproduce cysts actually colonize roots more effectively than do cells that underproduce cysts.A manuscript describing these results should be submitted this summer 2) We are continuing to analyze the role of cGMP in root development and in enhancing nodulation.We have observed that the Azospirillum cGMP mutant affects tap root development leading to the formation of seedlings containing multiple tap roots. We have also observed that beans inoculated with a cGMP defective strain of Azospirillum actually produce nodules that have altered morphology (they are larger than nodules made by plants inoculated with wild type Azospirillum).Furthermore, using GFP expressing strains of Azospirillum, we observe that nodules not only contain rhizobia but also harbor the cGMP defective strain of Azospirillum.We will continue to analyze the role of cGMP in nodulation until its ready for publication 3) We are continuing our analysis of plant signaling compounds present in seeds that signal Azospirillum to form cysts.Our preliminary analysis suggests that the signal may bealkylresorcinol lipids that are present in seed coats.This is very interesting as alkylresorcinol's are made late in seed development seeds to protect seeds from desiccation.The use ofplant synthesized alkylresorcinol to signal Azospirillum to enter dormancy suggests that there is interkingdom signaling from plant to bacteria to ensure that this bacterium becomes dormant as seeds also become dormant.This avenue of research will be continued until its ready for publication. 4). We just finished a detailed analysis of genome wide gene expression changes that occur as cyst cells germinate to form replicating vegetative cells.This study was undertaken to obtain an understanding of signals that lead cysts to undergo the germination process.This is the first study of its kind on the germination of dormant cysts.This work has been completed and submitted to PLOS Genetics.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Ashok, N., & C. E. Bauer (2020) Evidence of defined temporal expression patterns that lead a gram-negative cell out of dormancy. Under revision for PLOS Genetics
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Malinich, E. & C. E. Bauer (2018) Transcriptome Analysis Of Azospirillum brasilense Vegetative And Cyst States Reveals Large Scale Alterations In Metabolic And Replicative Gene Expression. Microbial Genomics 4: doi: 10.1099/mgen.0.000200
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Malinich, E. & C. E. Bauer (2018) The plant growth promoting bacterium Azospirillum brasilense is vertically transmitted in Phaseolus vulgaris (common bean). Symbiosis 76(2), 97-108. https://doi.org/10.1007/s13199-018-0539-2. A commentary of the significance of manuscript was published by: Zawoznik M & M Daniela-Groppa (2019) Viewpoint: Azospirillum in the cloudy boundaries of bacterial endophytes. Applied Soil Ecology 135:194-195
• Type: Theses/Dissertations Status: Submitted Year Published: 2020 Citation: Ashok, N. 2020. Genetic and transcriptomic analysts of cyst germination. Indiana University

Progress 04/15/18 to 04/14/19

Outputs
Target Audience:The main target audience are scientists thatundertake research in the general area of biological crop enhancement (or biofertilizer).These were reached via submission of several publications as well as by presentation of our work at scientific conferences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students and one postdoctoral fellow have/are working on various aspects of this research project.Research from these efforts recently led to the graduation of one graduate student who quickly obtained a full-time research position at Dupont-Pioneer specifically to research the commercial use of Azospirillum as a biofertilizer.A second graduate student recently obtained a highly competitive summer internship at Bayer (formerly Monsanto) who will also work on biofertilizers.Consequently, students and postdoctoral fellows working on this projectappear to be highly sought after by corporations that are exploring the use of microbes for crop improvements. The PI has also recently received invitations to three high visibility conferences that have sessions dedicated to productive plant-microbe interactions.Graduate students and a postdocrtoral fellowfrom my laboratory will be attending several of these conferences and presenting their research results. How have the results been disseminated to communities of interest?Two manuscripts highlighting results from this study have recently (2018) been published and a third is currently inpreparation.The Symbiosis manuscript has really caught the attention of researchers in this field as it has been highlighted byan editorial commentary regarding its significance at another journal as well as a recentinvitation to write a review on Azospirillum-plant endosymbiosis (which we have accepted).We also plan to present this work at several conferences this year that have session on plant-microbe interactions. What do you plan to do during the next reporting period to accomplish the goals?Overall the main goals of the originally submitted grant proposal remain intact. We will continue our analysis of the enhancement of root growth that is stimulated by Azospirillum that is naturally present on seeds vs Azospirillum that is artificially added to the seed coat using Azospirillum that is traceable with GFP and mCherry reporters.This analysis should allow us to continue to optimize the delivery of Azospirillum to developing roots during seed germination. We will continue to analyze the production of auxin by Azospirillum as well as the role of Azospirillum produced auxin in enhancing root growth. We will continue to analyze the effect of cGMP production, as well as auxin production, on enhancing nodulation in common bean. We will continue to address techniques allowing us to rapidly and reproducibly monitor motility of Azospirillum to plant flower extracts with the goal of identifying the plant compound that stimulates migration of Azospirillum from roots to developing seeds.

Impacts
What was accomplished under these goals? IMPACT: The results that we recently published in the journalSymbiosisare highly significant.This study demonstrated that Azospirillum is a normal part of the seed microflora in many crops.It also demonstrates that developing flowers appearto attract Azospirillum to migrate from the root to the developing seed. This allows seeds to be populated by thisbeneficial microorganism and to bepassed to subsequent plant generations.Our work also established that Azospirillum which is natively present in the seed coat is more effectively transmitted to developing roots then is Azospirillum that is added artificially to the seed coat.One impact of this study is thattechniques can be developed to maximally populate sees with Azospirillum in a way that will maximize crop yield enhancement by this beneficial bacterium. OUTCOME ACHIEVED AND RESULTS ACCOMPLISHED:Last funding period we continued to study several aspects regarding how Azospirillum interacts with plants. Our results are as follows: 1) We have constructed Azospirillum mutants that are defective in auxin production as well as a strain that is defective in NO production.These strains are currently being analyzed for their effect on root growth stimulation.Note also that there are reports of the presence of a receptor of ethylene encoded by the Azospirillum genome.We have identified this gene and are in the process of disrupting it to see if plant derived ethylene (and this receptor) are involved in regulating Azospirillum auxin production. 2) We have constructed Azospirillummutations in the Che3 signal transduction cascade and have obtained mutations that either overproduce or underproduce cysts.These mutants are currently being assayed for their effect onproductive colonization of roots. 3) We continue to observe enhanced nodulation of common bean seedlings with plants grown from seeds that are inoculated with a strain of Azospirillum that is defective in the production of cGMP.Interestingly, we have observed that theAzospirillum cGMP mutant affects tap root development. Specifically,plant seeds inoculatedwith this strain lead to theformation of seadlings containing multiple tap roots.The enhanced nodulation phenotype obtained with this strain only occurs with seeds planted in non-sterile soil that naturally contain rhizobial species. Specifically, we do not observe enhanced nodulation in plants growing in defined sterile medium that is inoculated with both Azospirillum and a defined rhizobium species.This indicates that the enhancement may involve a complex interaction of Azospirillum with multiple bacterial species and/or nutritional complexities that are only observed with soil. It shoul dbe noted that this is actually good news as it indicates that transfeer of this phenomina to the field may very well be doable. 4) We are continuing our studies on the identification of plant signaling compounds that stimulate the migration of Azospirillum from roots to developing seeds.Unfortunately, the standard capillary chemoattractive assay that we have used for measuring motility has proven to be unwieldy and also exhibits high variability which has hampered this analysis.To overcome this problem, we are currently investigating the use of other techniques for measuring motility response to plant extracts.If one of these other procedures works better, then we will once again address this goal.

Publications

• Type: Theses/Dissertations Status: Accepted Year Published: 2018 Citation: A NOVEL PROCESS OF AZOSPIRILLUM BRASILENSE  PLANT INTERACTIONS: ASSOCIATION WITH THE BEAN SEED
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Invited speaker and Session Chairperson, Bacterial Locomotion and Signal Transduction in Microorganisms (BLAST), New Orleans, LA
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Malinich, E. & C. E. Bauer (2018) The plant growth promoting bacterium Azospirillum brasilense is vertically transmitted in Phaseolus vulgaris (common bean). Symbiosis 76(2), 97-108. https://doi.org/10.1007/s13199-018-0539-2
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Malinich, E. & C. E. Bauer (2018) Transcriptome Analysis Of Azospirillum brasilense Vegetative And Cyst States Reveals Large Scale Alterations In Metabolic And Replicative Gene Expression. Microbial Genomics 4: doi: 10.1099/mgen.0.000200

Progress 04/15/17 to 04/14/18

Outputs
Target Audience:The target audience during this funding period were scientists that have interest in the crop yield enhancement potential of Azospirillum. A seminar about our research with Azospirillum was presented in the Agriculture School of Purdue University. Two manuscripts describing our resent results with Azospirillum (supported by NIFA) were submitted to journals. One publication is in press in the journal Symbiosis and the other is under review. Changes/Problems:There are no major changes or problems in approach that have occurred. What opportunities for training and professional development has the project provided?So far we have focused this year on getting good traction on the specific aims of this proposal. Data and manuscript are now being generated so we are now in the position to disseminate our results by attending and presenting our work at conferences. We suspect that the PI, graduate student and postdoctoral fellow that is working on this project will attend and present their work at several conferences during the next two years. How have the results been disseminated to communities of interest?One manuscript is in press in Symbiosis, a second is under review and the third is currently being written. What do you plan to do during the next reporting period to accomplish the goals?-We will continue our analysis of Azospirillum enhancement of nodulation as described above. -We will continue to try to identify plant compounds that induce encystment as described above. -We will construct NO and auxin production mutants of Azospirillum and access the effect of these mutation ion nodulation enhancement as well as on lateral root and root hair development. -We plan to initiate a study to determine whether Azospirillum present in a broad group of crop seeds are more effective in root colonization then is Azospirillum in soil. This study is based on our recently published observation that Azospirillum colonization of green bean roots is most effective Azospirillum cells that are present in seed coats and not with Azospirillum added to the surface of seeds. We want to determine is this also occurs with a wide variety of crop plants.

Impacts
What was accomplished under these goals? We have made considerable progress during the first funding year and have no significant changes to the specific aims of the proposal. During this past funding period we made progress in understanding the migratory pattern of Azospirillum through plant tissues during plant development. This work, which cites support from NIFA, has recently been accepted for publication in the journal Symbiosis. Deep sequencing transcriptomic analysis clearly establishes that Azospirillum is a major component of crop plant seeds and that this organism is passed from plant to plant via seed transmission. We further showed that the establishment of a productive Azospirillum- root interaction is highly enhanced when the root tip emerges from seeds that are natively inoculated with Azospirillum in the seed coat. This is in contrast to poor establishment of Azospirillum with roots when these bacteria are added to surface sterilized seeds. These results are a paradigm shift in the understanding of how this bacterial plant relationship is formed. Specifically, this provides a solid basis of understanding of what is needed to form an effective establishment of Azospirillum at the roots where this species has the biggest impact on crop enhancement. It opens up an avenue for maximizing beneficial Azospirillum-crop interaction in the future for example by pre-inoculating seeds that are to be used for growing crops that are subsequently used as seed stocks. We are also continuing to investigate Azospirillum stimulation of nodulation by Rhizobia. For this analysis we have moved from work in poorly defined soil conditions to a chemically defined hydroponic medium with growth occurs in a commercial germination pouch system (Mega International). The benefits of this hydroponic approach are that it allows us to control the nitrogen content and the concentrations of both Azospirillum and Rhizobium sp. It also allows us to easily microscopically study root tip emergence and to harvest root tips for analysis of microbial interaction. Importantly, we have been able to replicate Azospirillum stimulation of common green bean nodulation in the pouch system. Currently we are seeing Azospirillum increase the number of nodules in soybean, alfalfa and common green beans. The best enhancement of nodulation appears to be with common green bean so we are settling with that as an experimental system. Knock out strains of Azospirillum are being constructed that disrupt NO and auxin production to probe their role in the nodulation pathway. This winter we have begun studying in earnest the effect of the seed coat in stimulating Azospirillum encystment. A manuscript was recently submitted (also citing NFIA support) where we tracked genome-wide transcription changes that occur when cells transition from vegetative to cyst states (a manuscript that describes these results is under review). Recently, we completed analysis of the metabolic state (vegetative vs cyst states) that Azospirillum is in when seeds mature. Specifically, we observed that Azospirillum is in the vegetative state early in seed development and are in the cyst state late in cyst development. Finally, we have also recently initiated HPLC analysis to isolate a compound(s) in seed coats that stimulate Azospirillum encystment. To make it easier to screen for these compounds we are constructing GFP reporter plasmids that express GFP only when cells are in the cyst state. Finally, While Arabidopsis studies may not be a major focus of NIFA, this plant species does have a lot of tools and mutants that are useful in addressing the plant side of the Azospirillum-plant interaction. We are pleased to report that we have observed that Azospirillum does generate a strong response in terms of enhanced root growth of Arabidopsis. It initially appeared that Arabidopsis were responding to Azospirillum production of auxin as demonstrated by a decrease in primary root length coupled with a significant increase in lateral roots and root hairs. Interestingly, this may not be the case. Specifically, the Arabidopsis auxin resistant mutants axr2-1 (a dominant mutant that confers resistance to auxin, abscisic acid and ethylene) and axr5-1 (a mutant that is resistant to auxin) produce no root hairsandfew, if anylateral roots.However, when inoculated withAzospirillum, these plantshave dramaticformation in lateral roots androot hairs. This suggests that there are other signals besides auxin that Azospirillum is making to affect Arabidopsis root growth. There is a body of research that indicates that gibberellins and ethylene may be involved in root growth along with auxin but most of the literature points to auxin as the primary hormone involved in the stimulation of root growth caused by Azospirillum. It is also possible that NOis playing a role which will be quicktocheck onceknockoutsinthe NOpathway are made in Azospirillum.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Malinich, E. & C. E. Bauer (2018) The plant growth promoting bacterium Azospirillum brasilense is vertically transmitted in Phaseolus vulgaris (common bean). Symbiosis In press
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Malinich, E. & C. E. Bauer (2018) Transcriptome Analysis Of Azospirillum brasilense Vegetative And Cyst States Reveals Large Scale Alterations In Metabolic And Replicative Gene Expression. Submitted for review