Progress 01/01/24 to 12/31/24
Outputs
Target Audience:Academic and industry researchers in microbiome science Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Dr. Kleiner provided individual training and mentoring for several team members (including graduate students Anna Garrell, Molly DuBois, Ayesha Awan, Jessie Maier and Olivia Mathieson) on the operation of a high-resolution mass spectrophotometer and its use for metaproteomics including downstream data analysis. Dr. Joel Swift, Postdoc in the Wagner Lab Paid from other sources but contributed to this project Conferences attended (while paid under the grant) Gave an invited talk at the Gordon Research Conference - Multiscale Plant Vascular Biology, University of Southern Maine, Portland, ME. Gave an invited talk at the Botanical Society of America 2024 conference, Grand Rapids, MI. Natalie Ford, technician in the Wagner lab (partially paid from other sources, but contributing 0.25 FTE to this project) Mentored by Dr. Wagner on bioinformatics, project management Anna Garrell, Graduate Student in the Kleiner Lab (paid from other sources, but contributing to this project) Gave a talk at the Genetics of Maize-Microbe Interactions Workshop, Raleigh, NC (Feb. 2024) Presented a poster at the Plant Microbiomes Symposium, Amsterdam, The Netherlands (June, 2024) During the last reporting period four undergraduate students in the Wagner lab were paid from this grant, trained by Dr. Swift, and conducted research on the drought-protective effects of maize-associated bacteria (Lauren Higgins, Kai Sarwinski, Shahzaib Asif, Braxton Grindstaff) Gaurav Pal, Postdoc in the Kleiner Lab Delivered a talk in the Genetics of Maize-Microbes Interactions Workshops, Raleigh, NC (Feb. 2024) Presented a poster in the International Phytobiomes Conference, St. Louis, MO (Nov. 2024) Attended the 8th Partnerships in Biocontrol, Biostimulants and Microbiome Congress, Raleigh, NC (Oct. 2024) Mentored an REU intern (Frank Schaeffer) and conducted research on the effects of seed surface sterilization protocols on seed microbiomes Mentored an undergraduate (Rayna Gracia) and conducted research on seed endophytes (isolation, identification, and functional characterization) How have the results been disseminated to communities of interest?Results have been presented at multiple conferences including conferences focusing on industry stakeholders Poster presentations: Pal, G., Vintila, S., Balint-Kurti, P., Wagner, M. R., Kleiner, M. (2024) "Impact of functional interactions between microbes on microbiota assembly on corn roots. Poster Presentation." International Phytobiomes Conference, St. Louis, MO, USA. Garrell, A.G., Mahmood, A., Cheadle, J., Van Schaik, J., Vintila, S., Crook, N., Beck, A.E., Kleiner, M. (2024) "Understanding key microbial functions related to maize-microbe interactions using metaproteomics". Plant Microbiomes Symposium, Amsterdam, The Netherlands Conference and seminar presentations: ??Wagner MR, NA Ginnan, V Custódio, G Castrillo (Mar. 2024) Adaptation of plants and microbial neighbors to shared abiotic stress. Department of Biology, Colorado State University - Fort Collins, CO, USA Pal, G., Kleiner, M. (Feb. 2024) "Investigating the impact of keystone species on community structure and function of Maize SynCom. Abstract talk". Genetics of Maize-Microbe Interactions Workshop, Raleigh, NC, USA Swift, J. F. (2024) "Too much or too little: The Impact of Water Stress on Roots and the Microbiome." Gordon Research Conference - Multiscale Plant Vascular Biology. University of Southern Maine, Portland, ME, USA. Garrell, A.G., Mahmood, A., Vintila, S.V., Beck, A.E., Kleiner, M. (2024) "Identifying key microbial functions related to maize-microbe interactions using metaproteomics". Genetics of Maize-Microbe Interactions Workshop. Raleigh, NC, USA What do you plan to do during the next reporting period to accomplish the goals? Objective 2c: 6 additional isolates have been sent for Nanopore sequencing, which will be performed in the next reporting period. These genomes will be used for downstream comparative genomics and metaproteomics approaches. Objective 2b: Colonization efficiency will be assessed using gnotobiotic monoinoculation screening that currently is underway. Objective 2d: Complete gnotobiotic monoinoculation experiments. Objective 3a: We currently have a publication in preparation and plan to submit it to a journal in the upcoming reporting period. Additional follow-up experiments will be performed to to investigate the role of the Type VI Secretion System in H. robiniae (HRO) in order to determine which proteins might be secreted and what role the secretion system plays in mediating interactions with other microbes in planta. Objective 3b: Set up follow up experiment for testing the effect of keystone species (Enterobacter ludwigii and Pseudomonas putida) across different maize genotypes. Also perform co-culture experiments with ELU and PPU to identify direct interactions. Objective 3c: Perform drought experiments using single inoculants and the expanded SynCom and harvest roots for metaproteomics analysis to analyze the effect of drought on microbial and plant protein expression in roots.
Impacts
What was accomplished under these goals?
IMPACT: The impacts of this project so far are in the area of creating a richer research infrastructure for plant-microbiome research, as well as obtaining new insights on what microbial genes are involved in corn-root colonization. First, we have optimized and published an evaluation of protein extraction methods for metaproteomics of plant roots. Second, we have isolated more than 1000 bacterial strains associated with maize roots grown in 7 different prairie and agricultural soils. These isolates represent a large proportion of the known diversity of microbes associated with corn roots. We have submitted 88 of these isolates to a public strain repository and sequenced their complete genomes. These isolates and all associated genome information will be available to all researchers and will represent an invaluable resource to researchers studying maize-microbiome interactions to improve crop resilience. Third, we have carried out a large experiment of corn under drought and well-watered conditions in 6 soils collected from a rainfall gradient across Kansas, which allowed us to identify 17 microbial groups impacting plant growth. These microbial groups, along with others isolated previously, are currently being tested in gnotobiotic monoinoculation experiments to assess their ability to recolonize the maize root microbiome and promote plant growth. Fourth, we have carried out experiments to determine gene expression differences between microbes grown in vitro versus in planta and processed the resulting samples for metaproteomics. We have analyzed data and found so that hundreds to thousands of genes in each analyzed microorganism respond to colonization of corn roots providing many targets. Based on these results, we have carried out follow-up experiments to investigate hemicellulose degradation by two bacterial species and the role of the Type VI Secretion System in plant colonization. Objective 1 - Complete Objective 1a: Completed Objective 1b/c/d: Completed, the factorial experiment was completed and a manuscript describing the work has been published. Objective 1e: Mostly complete, to date we have isolated and stored for long-term preservation over 1000 strains originating from surface-sterilized maize roots inoculated with soil microbiomes collected in objective 1a. We have also established a collaboration with Dr. Gabriel Castrillo (University of Nottingham) and Dr. Klaus Schlaeppi (University of Basel), who possess collections of maize-associated bacterial strains with associated genomic sequencing resources. These collections have supplemented our current culture collection by expanding our representation of several bacterial groups. Objective 2a - Complete. We have identified >1000 of the bacterial isolates via Sanger sequencing. Of the identified isolates, we have selected 88 for inclusion in our curated SynCom and isolate collection. The selection process involved: (1) creation of a phylogenetic tree based on full-length 16S rRNA gene sequences, and selection of representatives from all major clades in the tree; (2) matching isolates' 16S sequences to amplicon sequence variants (ASVs) from the greenhouse experiment in Objective 1, and selecting families or genera that were highly prevalent (detected in more samples than expected based on their overall abundance) or associated with better performance under drought conditions; and (3) selecting additional isolates from taxa that have been reported to improve drought tolerance especially in grain crops . Objective 2b: Gnotobiotic in planta screening is underway to assess recolonization of each isolate. 20 out of 88 isolates have been tested so far. Objective 2c: In progress. Via Illumina and NanoPore sequencing, we have complete genome sequences for 82 isolates. The remaining 6 isolates have been sent for NanoPore sequencing to generate high-quality, closed genomes. Objective 2d: In progress. Developed single use inoculants for all the bacterial isolates in the maize culture collection. Gnotobiotic in planta screening is underway to assess recolonization of each isolate and plant growth performance under normal growing conditions.Optimized in vitro screenings (auxin production, PEG screening, ACC deaminase activity, phosphate solubilization, and potassium solubilization) for selecting isolates with the ability to tolerate drought stress. Majority of the isolates tested positive for auxin production. Some of the best auxin producers were Enterobacter ludwigii, Chryseobacterium, Microbacterium caowuchnii, Rahnella sp., Stenotrophomonas rhizophilia, etc. Similarly, isolates belonging to Burkholderia, Achromobacter, and Variovorax demonstrated high tolerance against osmotic stress as measured by the PEG screenings. Strong ACC deaminase activity was shown by Burkholderia spp., Pseudomonas spp., Paraburkholderia spp., Variovorax sp., Massilia sp., Rahnella sp., etc. Majority of the isolates demonstrated phosphate solubilization activity while only one of the isolates (Pseudomonas serboccidentalis) showed potassium solubilization activity so far. Objective 3a: The first part of this objective is completed (i.e. in vitro vs in vivo experiments of the species from the Niu-Kolter SynCom). We have developed a bacterial growth medium based on the plant growth MS medium to allow us to run comparisons of in vitro and in vivo growth individual microbes from the 7 member SynCom without confounding factors from widely different media composition. We have grown all 7 species individually in vitro and in planta and have generated the metaproteomic data from these experiments. For each of the microbial species we see hundreds of differentially expressed genes between the two conditions providing a large number of genes associated with growth in planta. Based on this data, we have performed follow-up experiments to further investigate some of the functions identified. We found that several species increased expression of hemicellulases when grown in planta, indicating that they may be degrading the plant cell wall. Additionally, the Type VI Secretion System (T6SS) was found in increased abundance in Herbaspirillum robiniae (HRO) during in planta growth. We generated three knockout mutants of the T6SS and found that without the T6SS, HRO's colonization of the maize root is significantly reduced, indicating that the T6SS plays an important role in mediating interactions between HRO and maize roots. A publication is currently in preparation. We have additionally observed that thousands of plant genes respond differentially to inoculation with different microbial species indicating a specific response of the plant to inoculation with different microbial species. Objective 3b: In progress. We have performed a first inoculation experiments with Enterobacter ludwigii (ELU) and Pseudomonas putida (PPU) as potential keystone species to identify key genes and proteins with strong impact on maize SynCom assembly. In terms of root and shoot weight, PPU and Full SynCom inoculated maize seedlings showed better growth relative to the control. LC-MS/MS was performed and metaproteomic data was generated from these experimental groups. Currently, the data is being analyzed and subsequent experiments are being planned. Proteomics data analysis identified more than thousand of ELU and PPU proteins in single inoculations vs Full SynCom. Specifically, more than 1400 ELU proteins were identified in single inoculations of ELU vs Full SynCom, of which 272 proteins were found to be significant. Similarly, more than 1100 PPU proteins were identified in single inoculations of PPU vs Full SynCom, of which 301 proteins were found to be significant. Currently we plan to expand the keystone experiment to different genotypes (sugar bun, B73 and Mo17) to investigate the roles of different genotypes on the structure of maize SynCom.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Swift, J.F., Kolp, M.R., Carmichael, A., Ford, N.E., Hansen, P.M., Sikes, B.A., Kleiner, M. and Wagner, M.R. Drought stress homogenizes maize growth responses to diverse natural soil microbiomes. Plant Soil (2024).
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
J. Jacob Parnell, Gaurav Pal, Ayesha Awan, Simina Vintila, Gabriella Houdinet, Christine V. Hawkes, Peter J. Balint-Kurti, Maggie R. Wagner, and Manuel Kleiner (2024) Effective Seed Sterilization Methods Require Optimization Across Maize Genotypes.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Parnell, J. J., S. Vintila, C. Tang, M. R. Wagner, M. Kleiner (2024). Evaluation of ready-to-use freezer stocks of a synthetic microbial community for maize root colonization. Microbiology Spectrum 12: e02401-23
|
Progress 01/01/23 to 12/31/23
Outputs
Target Audience:Academic and industry researchers in microbiome science Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Dr. Kleiner provided individual training and mentoring for several team members (including graduate student Anna Garrell, Clara Tang, Jessie Maier and Olivia Mathieson) on the operation of a high-resolution mass spectrophotometer and its use for metaproteomics including downstream data analysis. Dr. Joel Swift, Postdoc in the Wagner Lab Conferences/Workshops attended (while paid under the grant) Botany 2023, Boise ID AgBioTech Summit (8th Microbiome Movement), Raleigh, NC. Led two workshops on microbiome metadata standards and best practices Swift, J. F. and Finks, S. (Co-led workshop) Standardizing Metadata and Bioinformatics Workflows in Microbiome Research. Pennslyvannia State University, One Health Microbiome Center, State College, PA. Swift, J. F. (Workshop) Microbiome Metadata Best Practices. New Roots for Restoration-Biodiversity Integration Institute Natalie Ford, technician in the Wagner lab (paid from other sources, but contributing to this project) Mentored by Dr. Wagner on bioinformatics, project management Carmen Rodriguez, undergraduate student researcher in the Wagner lab (paid from other sources, but contributing to this project through July 2023) Mentored by Dr. Wagner and Dr. Nichole Ginnan (former Wagner lab postdoc) on experimental design, data analysis, and high-throughput microbial growth assays Anna Garrell, Graduate Student in the Kleiner Lab (paid from other sources, but contributing to this project) Gave a talk at the International Society for Molecular Plant-Microbe Interactions Conference, Providence, RI Presented a poster at the North Carolina Microbiomes Symposium, Research Triangle Park, NC Presented a poster at the Agricultural Microbiomes Symposium, Raleigh, NC Two undergraduate students in the Kleiner Lab have been paid on this grant and involved in the relevant research on plant-microbe interactions (Aisha Mahmood and Nathan Ring) Gaurav Pal, Postdoc in the Kleiner Lab Delivered a talk in PMB Seminar Series in the Department of Plant and Microbial Biology, NC State University (January, 2023) Attended North Carolina Microbiome Symposium, Raleigh, NC (May, 2023) Attended Agricultural Microbiomes Symposium, NC State University, Raleigh, NC (October, 2023) Completed a professional development course in Leadership organized by Leadership Learning Institute, NC State University (May-August, 2023) How have the results been disseminated to communities of interest?Results have been presented at multiple conferences including conferences focusing on industry stakeholders Poster presentations: Swift, J. F., Kolp, M. R., Carmichael, A., Ford, N. E., Hansen, P. M., Sikes, B. A., Kleiner, M., Wagner, M. R. (2023) "Utilizing native Kansas soil microbiomes from across the US Great Plains precipitation gradient for improved drought tolerance in maize. Poster Presentation". AgBioTech Summit (8th Microbiome Movement), Raleigh, NC. Garrell, A.G., Vintila, S.V., Beck, A.E., Kleiner, M. (2023) "Identifying key microbial functions related to maize-microbe interactions using metaproteomics". North Carolina Microbiomes Symposium, Research Triangle Park, NC Garrell, A.G., Vintila, S.V., Beck, A.E., Kleiner, M. (2023) "Identifying key microbial functions related to maize-microbe interactions using metaproteomics". Agricultural Microbiomes Symposium, Raleigh, NC Conference and seminar presentations: Swift, J. F., Kolp, M. R., Carmichael, A., Ford, N. E., Hansen, P. M., Sikes, B. A., Kleiner, M., Wagner, M. R. (2023, conference talk) "Legacy effects of precipitation and land use impact maize growth and microbiome assembly under drought stress. Oral Presentation". Botany 2023, Boise, ID. Garrell, A.G., Vintila, S.V., Beck, A.E., Kleiner, M. (2023, conference talk) "Identifying key microbial functions related to maize-microbe interactions using metaproteomics". IS-MPMI Conference, Providence, RI M. Kleiner (2023, Invited Virtual Seminar Talk) "Understanding key microbial functions related to maize-microbe interactions using synthetic microbial communities and metaproteomics". Dutch Microbiome Initiative (MiCRop) Seminar Series, Netherlands Wagner MR, NA Ginnan, M Kleiner, A Garrell, F Tso, V Custódio, G Castrillo (Sep. 2023) Adaptation of plants and microbial neighbors to shared abiotic stress. Microbial Systems Initiative Symposium, University of Illinois - Urbana, IL, USA Wagner MR, NA Ginnan, M Kleiner, A Garrell, F Tso, V Custódio, G Castrillo (Mar. 2023) Rapid evolution of microbiome effects on plant phenotype. Hanson-Wade Microbiome Movement AgBiotech Summit - Raleigh, NC, USA Wagner MR, F Salvato, KM Clouse, A Bartlett, S Sermons, A Garrell, F Tso, M Hoffman, JB Holland, PJ Balint-Kurti, M Kleiner (Mar. 2023) Hybridization, heterosis, and the maize microbiome. School of Integrative Biology, University of Illinois - Urbana, IL, USA Wagner MR, NA Ginnan, M Kleiner, A Garrell, F Tso, V Custódio, G Castrillo. Adaptation of plants and microbial neighbors to shared abiotic stress (Mar. 2023) Ecology and Evolutionary Biology, University of Toronto - Toronto, ON, Canada What do you plan to do during the next reporting period to accomplish the goals? Objective 2c: 21 additional isolates have been sent for Illumina sequencing, which will be performed in the next reporting period. Additional NanoPore sequencing will be performed for 30 of these isolates in order to generate high-quality, closed genomes. These genomes will be used for downstream metaproteomics approaches. Objective 3a: Follow-up experiments will be performed to validate functions identified using our comparative proteomics approach. We are working with collaborators to generate knockout mutants of the Type VI Secretion System in H. robiniae (HRO) in order to determine which proteins might be secreted, what role the secretion system plays in maize root colonization, and what role the secretion system plays in mediating interactions with other microbes. We additionally plan to investigate fatty acid degradation in HRO, as it seems to play a role in its growth on maize roots. Furthermore, we will be using fluorescence microscopy to determine the location of HRO in maize roots and to determine whether it is an endophyte. Objective 3b: Complete data analysis and identify key plant and microbial proteins involved in the functional interactions that shape the community structure of the maize SynCom. Include more potential keystone species and investigate their effect on community structure. Objective 3c: Perform drought experiments with the expanded SynCom and harvest roots for metaproteomics analysis to analyze the effect of drought on microbial and plant protein expression in roots.
Impacts
What was accomplished under these goals?
IMPACT: The impacts of this project so far are in the area of creating a richer research infrastructure for plant-microbiome research, as well as obtaining new insights on what microbial genes are involved in corn-root colonization. First, we have optimized and published an evaluation of protein extraction methods for metaproteomics of plant roots. Second, we have isolated more than 100 bacterial strains associated with maize roots grown in 7 different prairie and agricultural soils. These isolates represent a large proportion of the known diversity of microbes associated with corn roots. Currently we are submitting these isolates to a public strain repository and finishing up sequencing of their complete genomes. These isolates and all associated genome information will be available to all researchers and will represent an invaluable resource to researchers studying maize-microbiome interactions to improve crop resilience. Third, we have carried out a large experiment of corn under drought and well-watered conditions in 6 soils collected from a rainfall gradient across Kansas, which allowed us to identify 16 microbial groups impacting plant growth. These 16 microbial groups will be further tested. Fourth, we have carried out experiments to determine gene expression differences between microbes grown in vitro versus in planta and processed the resulting samples for metaproteomics. We have started analysis of this data and found so far that hundreds of genes in each analyzed microorganism respond to colonization of corn roots providing many targets for the study of microbial genes involved in plant root colonization. Objective 1 - Complete Objective 1a: Completed, soils were selected and collected from across the Kansas precipitation gradient (n = 6, both agricultural and prairie). Objective 1b/c/d: Completed, the factorial experiment was completed and a manuscript describing the work has been deposited on bioRxiv, with submission to target journal (Plant and Soil) imminent. Briefly, we found that well-watered plants when inoculated with inocula derived from prairie soils showed a negative association between shoot mass accumulation rate and mean annual precipitation of the site. We also identified bacterial and fungal families that were correlated with promotion or repression of plant growth, these represent key groups to evaluate from our strain collection with further in planta testing. Objective 1e: Mostly complete, to date we have isolated and stored for long-term preservation over 1000 strains originating from surface-sterilized maize roots inoculated with soil microbiomes collected in objective 1a. Smaller-scale follow-up efforts were undertaken to increase representation of key bacterial groups missing or under-represented in the original collection. These have resulted in the inclusion of isolates of Massilia sp., Paenarthrobacter sp., Rahnella sp., Telluria sp., and Enterobacter sp. We have also established a collaboration with Dr. Gabriel Castrillo (University of Nottingham) who possesses a collection of maize-associated bacterial strains with associated genomic sequencing resources. We are in the process of importing this collection to the United States to be housed in replicate at the University of Kansas. This collection will supplement our current culture collection by expanding our representation of several bacterial groups; including Bacillus spp., Streptomycetes spp., Arthrobacter spp., and Sphingomonas spp., among others. Objective 2a - Complete. We have identified >1000 of the bacterial isolates via Sanger sequencing and also recorded colony morphology. Of the identified isolates, we have selected ~100 for inclusion in our curated SynCom and isolate collection. The selection process involved: (1) creation of a phylogenetic tree based on full-length 16S rRNA gene sequences, and selection of representatives from all major clades in the tree; (2) matching isolates' 16S sequences to amplicon sequence variants (ASVs) from the greenhouse experiment in Objective 1, and selecting families or genera that were highly prevalent (detected in more samples than expected based on their overall abundance) or associated with better performance under drought conditions; and (3) selecting additional isolates from taxa that have been reported to improve drought tolerance especially in grain crops . Objective 2b: Not started yet. Objective 2c: In progress. Biomass for DNA extraction has been produced for 102 isolates. Illumina sequencing has been performed for 81 isolates thus far. The remaining 21 isolates have been sent for Illumina sequencing. Additional NanoPore sequencing will be performed for 30 of these isolates in order to generate high-quality, closed genomes. Objective 2d: Not started yet Objective 3a: The first part of this objective is completed (i.e. in vitro vs in vivo experiments of the species from the Niu-Kolter SynCom). We have developed a bacterial growth medium based on the plant growth MS medium to allow us to run comparisons of in vitro and in vivo growth individual microbes from the 7 member SynCom without confounding factors from widely different media composition. We have grown all 7 species individually in vitro and in planta and have generated the metaproteomic data from these experiments. Currently we are analyzing the data. For each of the microbial species we see hundreds of differentially expressed genes between the two conditions providing a large number of genes associated with growth in planta. Based on this data, we have performed follow-up experiments to confirm and further investigate some of the functions identified. We found that several species increased expression of hemicellulases when grown in planta, indicating that they may be degrading the plant cell wall. We therefore used fluorescence imaging to determine whether these species are endophytes and growing within the plant tissue. Additionally, the Type VI Secretion System was found in increased abundance in Herbaspirillum robiniae (HRO) during in planta growth. We have been investigating which environmental factors trigger this expression and are working with collaborators to generate knockout mutants to investigate the role it has in root colonization and interactions with other microbes. We have additionally observed that thousands of plant genes respond differentially to inoculation with different microbial species indicating a specific response of the plant to inoculation with different microbial species. Objective 3b: Partially complete. We have performed a first inoculation experiments with Enterobacter ludwigii (ELU) and Pseudomonas putida (PPU) as potential keystone species to identify key genes and proteins with strong impact on maize SynCom assembly. Six groups (Full SynCom, Full SynCom-ELU, ELU, Full SynCom-PPU, PPU, and Untreated Control) were tested. Roots from all the six experimental groups were harvested and processed for bacterial load estimation as well as proteomics analysis. Root and shoot growth parameters were also recorded. Bacterial load data suggested a strong influence of ELU on community structure of maize SynCom as its removal resulted in the collapse of the SynCom. However, PPU did not show such a significant influence on the community structure. From the absolute abundance load data, we observed that ELU has a positive effect on growth of SMA, BPI, CIN, HRO and PPU and negative effect on CPU. In case of PPU, a positive effect was observed on SMA, CIN, and HRO while a negative effect on BPI, CPU, and ELU. In terms of root and shoot weight, PPU and Full SynCom inoculated maize seedlings showed better growth relative to the control. LC-MS/MS was performed and metaproteomic data was generated from these experimental groups. Currently, the data is being analyzed and subsequent experiments are being planned. Objectives 3c and 3d: Not started yet.
Publications
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Swift, J.F., Kolp, M.R., Carmichael, A., Ford, N.E., Hansen, P.M., Sikes, B.A., Kleiner, M. and Wagner, M.R., 2023. Legacy effects of precipitation and land use impact maize growth and microbiome assembly under drought stress. Preprint, bioRxiv https://doi.org/10.1101/2023.04.11.536405
- Type:
Other
Status:
Published
Year Published:
2022
Citation:
Wilson, N. J., C. M. Smith-Moore, Y. Xu, B. Edwards, C. La Hovary, S. Barampuram, K. Li, D. Aslett, M. Ji, X. Lin, S. Vintila, M. Kleiner, D. Xie, Y. Shachar-Hill, A. Grunden, and H. Sederoff (2022). Introduction of a condensed, reverse tricarboxylic acid cycle for additional CO2 fixation in plants. bioRxiv https://www.biorxiv.org/content/10.1101/2022.03.04.483018v1.full
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Parnell, J. J., S. Vintila, C. Tang, M. R. Wagner, M. Kleiner (2023). Evaluation of ready-to-use freezer stocks of a synthetic microbial community for maize root colonization. bioRxiv https://www.biorxiv.org/content/10.1101/2023.05.10.540175v1
|
Progress 01/01/22 to 12/31/22
Outputs
Target Audience:Academic and industry researchers in microbiome science Changes/Problems:The Wagner lab encountered some technical complications while shipping DNA samples for Sanger sequencing (excessive evaporation due to summer heat wave). This was resolved by increasing the reaction volumes and repeating the work, but it did delay the timeline and increase expenditures on research supplies. Hiring of the postdoc for this project in the Kleiner Lab was delayed due to an initial failed search that required an additional search. A postdoc has now been hired and started work on this project on November 30 2022. What opportunities for training and professional development has the project provided? Dr. Kleiner provided individual training and mentoring for several team members (including graduate student Anna Garrell, Clara Tang, Jessie Maier and Olivia Mathieson) on the operation of a high-resolution mass spectrophotometer and its use for metaproteomics including downstream data analysis. Dr. Joel Swift, Postdoc in the Wagner Lab Conferences/Workshops attended (while paid under the grant) Botany 2022 (Anchorage, AK, July 24-27th). "Unveiling the secret of underground: technologies for visualizing root and rhizosphere" (Hybrid format; Tsukuba, Japan, November 4th, 2022). "Intro to reviews and meta-analysis" (Hybrid format; Hosted by BOTANY360, November 7th, 2022) Root Phenotyping Workshop (Hybrid format; Hosted by Phenome Force, November 18th, 2022) Mentored by outgoing Wagner lab postdoc on best practices for microbial culture (summer 2022) Natalie Ford, technician in the Wagner lab (paid from other sources, but contributing to this project) Mentored by Dr. Wagner on protocols for DNA extraction and amplicon sequencing library preparation Carmen Rodriguez, undergraduate student researcher in the Wagner lab (paid from other sources, but contributing to this project) Presented a research poster at the International Phytobiomes Conference, Denver, CO (September 2022) Presented a research poster at the KU Summer Research Symposium, Lawrence, KS (July 2022) Received mentoring from Dr. Wagner and other lab members to strengthen her applications to Ph.D. programs (fall 2022) Anna Garrell, Graduate Student in the Kleiner Lab (paid from other sources, but contributing to this project) First Friday Microbiome Seminar, Microbiome Core, UNC Chapel Hill, NC International Phytobiomes Conference, Denver, CO Virtual: Genetics of Maize Microbe Interactions Seminar. Andrea Ward, Undergraduate Student in the Kleiner Lab (paid from other sources, but contributing to this project) NC ASM Conference, Boone, NC Two undergraduate students in the Kleiner Lab have been paid on this grant and involved in the relevant research on plant-microbe interactions (Aisha Mahmood and Nathan Ring) How have the results been disseminated to communities of interest?Results have been presented at multiple conferences including conferences focusing on industry stakeholders Poster presentations: Rodriguez C. M., Ginnan N., Tso F., Wagner M. (2022) Exploring microbial drought adaptations and microbially-mediated plant drought tolerance. KU Research Experience for Undergraduates Symposium, Lawrence, KS Rodriguez C. M., Ginnan N., Tso F., Wagner M. (2022) Exploring microbial drought adaptations and microbially-mediated plant drought tolerance. International Phytobiomes Conference, Denver, CO Conference and seminar presentations: M. Wagner. (2022, invited conference talk) "Adaptation to stress in plant-associated and free-living microbiomes ". Plant-Microbe Interactions Conference, Copenhagen, Denmark M. Kleiner. (2022, Conference talk). "Understanding key microbial functions related to maize-microbe interactions using metaproteomics". Plant-Microbe Interactions Conference, Copenhagen, Denmark M. Kleiner. (2022, Invited Virtual Seminar Talk). "Synthetic microbial communities (SynComs) to study plant-microbe interactions". DSMZ (German Collection of Microorganisms and Cell Cultures) M. Kleiner. (2022, invited stakeholder conference talk). "Maize-microbiome interactions" Microbiome Movement AgBiotech Conference, Durham, NC Garrell, A. G., S. Vintila, A. E. Beck, M. Kleiner (2022, invited talk). Identifying key microbial functions related to maize-microbe interactions using metaproteomics. First Friday Microbiome Seminar, Microbiome Core, UNC Chapel Hill, NC Ward, A.L., C.V. Tang, A. Garrell, M. Kleiner (2022). Development of media for growth and quantification of a synthetic community for plant-microbe research. NC ASM Conference, Boone, NC Garrell, A. G., S. Vintila, A. E. Beck, M. Kleiner (2022). Identifying key microbial functions related to maize-microbe interactions using metaproteomics. International Phytobiomes Conference, Denver, CO Garrell, A. and M. Kleiner (2022). Identifying key microbial functions related to maize-microbe interactions using metaproteomics. Virtual: Genetics of Maize Microbe Interactions Seminar. 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: The impacts of this project so far are in the area of creating a richer research infrastructure for plant-microbiome research focusing on corn. First, we have optimized and published an evaluation of protein extraction methods for metaproteomics of plant roots. Second, we have isolated more than 100 bacterial strains associated with maize roots grown in 7 different prairie and agricultural soils. These isolates represent a large proportion of the known diversity of microbes associated with corn roots. Currently we are submitting these isolates to a public strain repository and sequencing their complete genomes. These isolates and all associated genome information will be available to all researchers and will represent an invaluable resource to researchers studying maize-microbiome interactions to improve crop resilience. Third, we have carried out a large experiment of corn under drought and well-watered conditions in 6 soils collected from a rainfall gradient across Kansas, which allowed us to identify 16 microbial groups impacting plant growth. These 16 microbial groups will be further tested. Objective 1 close to complete - manuscript in preparation, preprint will be submitted in the near future Objective 1a/b/c: Completed. Soils from across the Kansas precipitation gradient (n = 6, both agricultural and prairie) were collected and used to inoculate maize genotypes (B73 and Mo17) in a factorial drought experiment. We assessed phenotypic and root microbiome (bacteria and fungi) response of the maize genotypes to drought stress. A shift in bacterial but not fungal community composition was observed under drought stress, with an enrichment in gram-positive and depletion in gram-negative bacteria. Inocula derived from soils, exposed to a natural precipitation gradient, did not have a large impact on root and shoot growth of drought stressed maize plants. Well-watered plants when inoculated with inocula derived from sites with no history of agricultural use (i.e., prairie soils) showed a negative association between shoot mass accumulation rate and mean annual precipitation of the site. We identified 13 bacterial and 3 fungal families that were correlated with promotion or repression of plant growth, these represent key groups to evaluate from our strain collection with further in planta testing. Objective 1d: Data generated from the factorial drought experiment has been analyzed and is currently being presented in a forthcoming manuscript. Key results and amendments to the design on the experiment and analysis are presented below. Key results: Assessed phenotypic and root microbiome (bacteria and fungi) response of maize genotypes B73 and Mo17 to drought stress. Observed a shift in bacterial but not fungal community composition under drought stress, with an enrichment in gram-positive and depletion in gram-negative bacteria. Inocula derived from soils across central Kansas, exposed to a natural precipitation gradient, did not have a large impact on root and shoot growth of drought stressed maize plants. Restricting the analysis to plants inoculated with inocula derived from sites with no history of agricultural use (i.e., native prairies), we found Well-watered plants showed a negative association between shoot mass accumulation rate and mean annual precipitation of the site. We identified 13 bacterial and 3 fungal families that were correlated with promotion or repression of plant growth, these represent key groups to evaluate from our strain collection with further in planta testing. Amendments to design: We elected to use a linear modeling framework in place of co-occurrence networks to identify key microbial groups to evaluate from our strain collection. Briefly, we extracted the residuals from a model that contained all the main and random effects, with the exception of drought treatment for each plant growth response. Then for each bacterial and fungal family, we fit the following model (traitresidual ~ Taxon abundance × Drought Treatment). This model allowed for assessing taxa with general plant growth promotion or repression correlations, from significance of the 'Taxon abundance' term, and taxa that modulated plant growth differentially depending on the watering regime via the significance of the interaction term. Objective 1e: Mostly complete. A large fraction of the core bacterial microbiome has been isolated in culture (>1000 strains). All of these strains were isolated from surface-sterilized maize roots sampled from plants that were grown in prairie soils or agricultural soils from across the state of Kansas (augmented with sterile vermiculite to prevent compaction). A smaller-scale, follow-up effort is underway with three main goals: (1) isolate root endophytes from drought-treated plants as opposed to unstressed plants; (2) use selective media to increase representation of key bacterial groups that were missing or under-represented in the original collection (e.g., Streptomyces spp., Arthrobacter spp.); (3) isolate fungal endophytes from maize roots in each soil. Objective 2a - Mostly complete. We have identified >900 of the bacterial isolates via Sanger sequencing and also recorded colony morphology. Sequencing of the remaining ~100 isolates is in progress, and the same is planned for new Actinobacteria isolates that we are targeting in a followup effort to Objective 1e. Similarly, the new fungal isolates will be identified based on full-length ITS sequence and colony morphology. Of the identified isolates, we have selected ~110 for inclusion in our curated SynCom. The selection process involved: (1) creation of a phylogenetic tree based on full-length 16S rRNA gene sequences, and selection of representatives from all major clades in the tree; (2) matching isolates' 16S sequences to amplicon sequence variants (ASVs) from the greenhouse experiment in Objective 1, and selecting families or genera that were highly prevalent (detected in more samples than expected based on their overall abundance) or associated with better performance under drought conditions; and (3) selecting additional isolates from taxa that have been reported to improve drought tolerance especially in grain crops . have made a lot of progress Objective 2b: Work has not been started yet. Awaiting completion of isolate collection. Objective 2c: In progress. Biomass for DNA extraction has been produced for >50 strains already. Sequencing will be done with a combination of Illumina and nanoPore sequencing. We have decided to increase sequencing from 40-50 strains to a total of more than 100. Objective 2d: Not started yet Objective 3a: The first part of this objective is completed (i.e. in vitro vs in vivo experiments of the species from the Niu-Kolter SynCom). We have developed a bacterial growth medium based on the plant growth MS medium to allow us to run comparisons of in vitro and in vivo growth individual microbes from the 7 member SynCom without confounding factors from widely different media composition. We have grown all 7 species individually in vitro and in planta and have generated the metaproteomic data from these experiments. Currently we are analyzing the data. For each of the microbial species we see hundreds of differentially expressed genes between the two conditions providing a large number of genes associated with growth in planta. We also observed that thousands of plant genes respond differentially to inoculation with different microbial species indicating a specific response of the plant to inoculation with different microbial species. Objectives 3b to 3d: No progress to date, awaiting completion of data analysis and microbial isolation from Obj. 1 and 2.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Salvato, F., S. Vintila, O. M. Finkel, J. L. Dangl, M. Kleiner (2022). Evaluation of protein extraction methods for metaproteomic analyses of root-associated microbes. Molecular Plant-Microbe Interactions 35(11): 977-988.
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