Progress 05/01/19 to 04/30/24
Outputs
Target Audience:This project provided scientific training to several young and developing scientists (postdoc, graduate students, and undergraduate students). In addition, the target audiences reached by this project include the international community of scientists in academia, industry, and the private sector in the agricultural and environmental sciences through presentations at conferences and the undergraduate and graduate students in two courses on plant-bacterial interactions through formal classroom instruction. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided hands-on scientific training for a total of six undergraduate students, two graduate students, one postdoc, and one research scientist. The specific training activities included training in microbiological culturing from environmental samples (for four undergraduates and one graduate student), in plant physiology (for one undergraduate and one graduate student) in molecular biology (for two undergraduates and one graduate student), and in bioinformatics, computational biology, statistical analysis, and data visualization, particularly with big datasets (for two graduate students and one research scientist). In addition, the project provided all of these scientists with training in the experimental probing of plant-microbe interactions, interdisciplinary collaboration, critical thinking, data interpretation, and oral and written science communication skills. The project also provided professional development skills in the form of professional presentations at conferences (for three undergraduate and one graduate student). How have the results been disseminated to communities of interest?Over the life of the project, the results were disseminated to other undergraduate students in STEM fields at Iowa State University and the University of Iowa via oral and poster presentations by three undergraduate students. They were also disseminated to academic and industrial research scientists via invited talks at one international and one national symposium and via poster presentations at one international, one national, and one regional symposium. The project was highlighted in formal course curricula on plant-bacterial interactions, reaching 35 undergraduate and ten graduate students. Lastly, the personnel on the project were engaged in outreach activities to secondary school students interested in STEM by leading multiple two-hour workshops on microbial impacts on plants. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Water insecurity is a major threat to sustained agricultural productivity and food production worldwide. Droughts, which dramatically reduce crop yields, are increasing in frequency, duration, and intensity. Plant-associated microbes can be key players in enhancing the tolerance of crop plants to environmental stresses. These microbes may be incorporated into new strategies for increasing crop resilience to drought, thus complementing existing approaches such as improving crop germplasm and altering irrigation and land management practices. A major knowledge gap in designing such microbe-based crop protection strategies is in understanding how plants assemble microbial communities when plants are stressed. This project explored the selective forces involved in the assembly of these microbial communities, or microbiomes, on soybean roots during drought. The approach taken in the project was first to characterize the nature of the impact of drought on the microbial communities at a high taxonomic resolution, thus identifying the major microbial players influenced by environmental stress. Three bacteria genera accounted for the majority of the favored microbes in soybean roots under drought, accounting for 1% in water-rich conditions but nearly 35% under drought conditions. Second, the hypothesis that the accumulation of extracellular reactive oxygen species (ROS) in plants during drought serves as a major driver of this shift in the microbiome composition was experimentally tested. Multiple lines of evidence were found to support the hypothesis, indicating that environmentally-induced systemic oxidative stress in soybean, and likely other crops, is a major driver of microbiome shifts in their roots. This finding identifies a mechanism that could be exploited in strategies to promote microbe-based crop protection, including screening microbial inoculants for those that either produce antioxidants or induce antioxidant activities in crops and improving the performance of existing agricultural microbials such as soybean rhizobial inoculants by enhancing their resiliency to ROS. Aim 1. Characterize the molecular mechanisms driving assembly patterns of soybean root-associated microbiomes in response to drought. Expanding on previous reports that drought enriched for bacteria in the phylum Actinomycetota, fine-scale taxonomic changes were performed that identified the enrichment of a single Actinomycetota class, the Actinobacteria, and three specific genera within this class, Nocardioides, Glycomyces, and Streptomyces. These genera comprised nearly a third of the endosphere community under drought. They were enriched in both relative and absolute abundance and based on both RNA- and DNA-derived rRNA amplicon data, indicating that their increased abundance was due to growth rather than, or in addition to, selective survival. Several lines of evidence were generated to support the hypothesis that one mechanism driving this enrichment was a drought-mediated increase in extracellular (apoplastic) oxidative stress. First, the microbiome compositional shifts were recapitulated by exposure of soybean to other stresses known to induce oxidative stress, namely salinity and heavy metal stress. Second, the effects of these stresses were observed in both the root interior (the endosphere), and although muted, in the root exterior (the rhizosphere). Third, these effects were observed on roots of plants that were spatially separated from the roots exposed to the treatments, thus these effects involved systemic signaling in the plant. Fourth, the effects were dose-dependent, with strong shifts associated with an increasing gradient of plant stress. These findings provide the first evidence that systemic oxidative stress, likely involving the accumulation of ROS in the apoplast, is a driver of microbiome shifts during drought. The hypothesis that drought-mediated microbiome shifts were due to soybean-driven changes in the production of antimicrobial compounds by Actinobacteria was evaluated. A protocol using colony screening with Actinobacteria-specific primers was used to generate an isolate collection rich in Actinobacteria from stressed and non-stressed soybean roots. Inhibition assays with this collection highlighted the known antimicrobial activities of Streptomycesbut provided no evidence for similar high-level antimicrobial activities in the other stress-enriched genera. These findings provide evidence against this proposed mechanistic driver of microbiome shifts. Beyond these increases in knowledge, this project provided several tools for plant-microbiome research, including (i) a split-root growth system for propagating plants under stressful and non-stressful conditions, which is critical for differentiating between local and systemic stress effects, (ii) an effective method to separate microbes from root cells based on a discontinuous gradient centrifugation protocol, which greatly enhances the recovery of microbial biomaterials from inside plant roots, (iii) protocols for the addition and analysis of spike-in standards to generate absolute and relative abundance measures of the microbial taxa in root microbiomes, (iv) a pipeline for high-resolution taxonomic analysis of microbiomes using long-read amplicon sequencing, and (v) a soybean root isolate collection that is distinct from other plant isolate collections its richness of Actinobacteria, having at least 21 genera beyond the commonly isolated Streptomyces genus. Aim 2. Identify the functional microbiome traits associated with feedback on soybean physiology and fitness under drought conditions. Root microbiome assembly is influenced not only by abiotic stress but also by root tissue and plant development, with early assemblages potentially influencing later assemblages. This project generated a fine-scale temporal and spatial map of the soybean root microbiome of plants grown in the absence of drought. Specific taxa were repeatably recruited to the root tips of distinct plants, indicating a highly consistent recruitment process. The number and diversity of taxa that were recruited increased during the V1 (vegetative) plant growth stage, and the core microbiome completely turned over between the V1 and V3 growth stages on all of the root tissues. Several lines of evidence suggested that the microbes recruited to the root tips of older plants were influenced by previous colonization activities, indicating that the microbial pool for recruitment on newly forming roots changes as plants grow. These findings provide the first detailed look at root microbiome assembly processes in soybeans and highlight the consistency of the assembly process. Biometric data were collected from soybean plants grown in split-root pots, with one root section exposed to environmental stress and the other root section exposed to microbe-rich soil. Measurements of 14 aspects of plant growth were subjected to principal component analysis and dimensionality reduction to generate a single vector that was used as a unified index of plant stress. This index showed that the metabolic and morphological changes in aerial tissues were the physiological traits most strongly impacted by environmental stress when examined across a stress gradient. Linear regression analyses based on this index were used to identify how increasing systemic stress impacts individual microbial taxa, again highlighting that, among all genera in the root microbiome, specific genera within the Actinobacteria most strongly correlate with environmental stress. This project thus developed a powerful, quantitative approach for correlating physiological traits in a plant with specific microbiome traits in the root microbiome. This quantitative approach is a tool that can be used by the wider microbiome research community in its efforts to identify the host or environmental factors most strongly associated with and potentially driving shifts in a microbiome.
Publications
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2023
Citation:
Delp, Drew. 2023. Abiotic stress-induced changes in the structure and activity of root-associated bacterial communities at high taxonomic resolution. (Publication No. 30420114) M.S. Thesis, Iowa State University. ProQuest Dissertations and Theses Global.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Delp, D., A. Welty, Y.B. Jun, D. Nettleton and G.A. Beattie. 2023. Oxidative stress as a driver of root microbiome composition. Interdisciplinary Plant Group 2023 Symposium: Redox Regulation of Plant Stress and Development, May 23-26, 2023, Columbia, MO (oral presentation)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Delp, D. and G.A. Beattie. Plant-derived reactive oxygen species as a driver of root-associated bacterial community shifts during abiotic stress. Plant Health 2023, Aug 12-16, 2023, Denver, CO. (poster presentation)
|
Progress 05/01/22 to 04/30/23
Outputs
Target Audience:The target audience for the scientific findings of the project is the community of scientists in academia, industry, and the private sector in the agricultural and environmental sciences. The target audience for the training is the postdoctoral research fellow, graduate student, and several undergraduates at Iowa State University who have been directly involved in the research. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In this reporting period, the project provided opportunities for training a graduate student and an undergraduate student. How have the results been disseminated to communities of interest?They were disseminated in one oral presentation at an international conference and two poster presentations at another international conference, with the latter attended by academics, industry, and growers. What do you plan to do during the next reporting period to accomplish the goals?We have evidence that several abiotic stresses induce conserved shifts in the root microbiome of soybean, and these stresses are known to be associated with the generation of reactive oxygen species (ROS) in the plant apoplast. We will perform a final set of experiments aimed at (1) generating more robust evidence for the role of ROS as a driver of root microbiome shifts in response to abiotic stress and (2) identifying the mechanisms by which abiotic stress favors only the selected Actinomycetota genera. For (1), we will generate and isolate endosphere communities from roots exposed to a stress gradient controlled by salinity stress. For this, we will use several tools we have already developed, including the soybean split-root system, a plant stress index for stress validation, and a gradient centrifugation protocol for endosphere microbiome isolation. Following the protocol in our pilot test evaluating the impact of a single factor, oxidative stress, on microbial community shifts, we will expose these endosphere communities (recovered en masse without cultivation) to several levels of oxidative stress in vitro. We will use the magnitude of the taxonomic shifts in the endosphere microbiome to evaluate whether a cause-and-effect relationship exists between oxidative stress and the changes in the microbiome. The quantitative nature of this experiment, which is based on using communities exposed to a range of stress levels in planta and in vitro, will support a robust evaluation of the role of ROS as a driver of microbiome assembly dynamics. We will complement these studies with experiments using our collection of microbial isolates from the rhizosphere, endosphere, and soil surrounding soybean plants. We will identify the isolates that are as-yet unclassified by sequencing the 16S rRNA gene of each, and if key stress-favored genera are not represented, we will use targeted isolation strategies to obtain members of these genera. We will complete three lines of experiments to address the mechanisms by which abiotic stress favors only selected Actinomycetota genera using selected isolates to represent genera that were and were not favored in the endosphere of stressed plants. These experiments will evaluate three specific mechanisms of stress-mediated enrichment. The first mechanism is that the genera that are enriched are simply more resilient to the toxic effects of ROS and thus exhibit higher survival and more rapid recovery after exposure. We will assess this based on dose-response assessments of individual isolates. The second mechanism is that the genera that are enriched are induced by ROS to produce antimicrobial compounds that selectively favor the microbial producers. We will assess this based on the temporal separation between ROS exposure and classic antagonism assays to genera that exhibit stress-mediated depletion in the endosphere. The third mechanism is not associated with ROS, but rather is that the genera that are enriched exhibit superior growth on drought-induced compounds in soybean rhizodeposits. We will assess this based on growth, or growth rate, assessments using compounds reported to accumulate in soybean rhizodeposits under drought.
Impacts
What was accomplished under these goals?
Plants use their associations with microbes as a flexible form of insurance against, and adaptation to, changes in environmental conditions. Understanding how plant microbiomes assemble is central to strategies aimed at using microbes to enhance the health and productivity of crops under non-ideal growth conditions. This project is exploring the selective forces involved in the differential enrichment of specific microbes in roots during drought. In the previous years of the project, we developed (i) single-root and split-root growth systems for propagating soybean under stressful and non-stressful conditions, (ii) a discontinuous gradient centrifugation protocol to separate microbes from root cells prior to extracting nucleic acids, and (iii) protocols for the addition and analysis of spike-in standards to generate absolute and relative abundance measures of the microbial taxa in root microbiomes. We have used these tools to identify fine-scale taxonomic changes resulting from drought exposure during serial plant growth cycles. We have also used them to test for specific mechanistic drivers of these taxonomic changes by evaluating if they were recapitulated on soybean roots exposed to alternate abiotic stresses that induce similar physiological responses to drought. Moreover, by characterizing the microbiome assembly patterns across the soybean root system, we have developed a context in which to understand the root-driven recruitment and development patterns of these microbial communities with and without stress. Results from this project are providing foundational data that are critical to future microbial-based strategies for protecting crops from drought and other abiotic stresses. Aim 1. Characterize the molecular mechanisms driving assembly patterns of soybean root-associated microbiomes in response to drought. In contrast to the previously reported phylum-level changes in root microbiomes in response to drought, we characterized fine-scale taxonomic changes in these microbiomes, namely identifying preferential enrichment for a single Actinomycetota class, the Actinobacteria and three genera within this class. These genera comprised nearly a third of the endosphere community under drought. They were enriched in both relative and absolute abundance and based on both RNA- and DNA-derived rRNA amplicon data, indicating that their increased abundance was due to growth rather than, or in addition to, selective survival. These data provide genus-level resolution to the growing body of research on rhizosphere microbiomes under drought and highlight that growth of selected taxa contributes to their enrichment in the root endosphere. We postulated that one mechanism driving this enrichment is a drought-mediated increase in extracellular (apoplastic) oxidative stress. We designed experiments to evaluate if the enrichment profile was recapitulated by other abiotic stresses that are known to induce oxidative stress, namely salinity and heavy metal stress. We developed a split-root system in which we exposed one root of a split-root soybean plant to salinity or copper stress, thus generating oxidative stress within the plant, and then looked at the impact of systemically propagated stress on the microbiome of a separate root. Consistent with our hypothesis, the stress treatments induced a visible increase in reactive oxygen species (ROS) in the roots distal to the stress (based on ROS-responsive staining) and specifically enriched for members of the Actinomycetota. These findings provide the first evidence that systemic oxidative stress, likely involving accumulation of ROS in the apoplast, is a driver of microbiome shifts during drought. Aim 2. Identify the functional microbiome traits associated with feedback on soybean physiology and fitness under drought conditions. We collected biometric data on soybean plants during the split-pot experiment in Aim 1 to better understand the effect of the treatments on plant growth. We measured 14 aspects of plant growth, including biomass, leaf area, water use efficiency measurements, and photosynthetic performance, and used exploratory factor analysis to reduce the data to the most influential factors affecting plant growth. Using principal component analysis and dimensionality reduction, we generated a single vector that we used as a unified index of plant stress. Consistent with previous knowledge that abiotic stress effects often manifest first in metabolic and morphological changes in aerial tissues, the plant stress index was most strongly impacted by the effects of the treatments (drought, copper, and salinity stress) on phyllosphere measures. Collectively, our samples represented a gradient of plant stress, which enabled us to evaluate the impact of increasing systemic stress on the compositional dynamics of the root microbiomes. Despite that, the total endosphere community decreased in size with increasing plant stress, and the absolute abundance of the Actinomycetota, as a phylum, increased and was the only phylum to do so. Importantly, linear regression analysis of changes in relative abundance across the plant stress gradient provided a powerful approach to identifying the taxa most impacted by stress. Again, our findings highlighted that, within the phylum Actinomycetota, only a subset of Actinobacterial genera were strongly impacted by stress, and these genera exhibited the strongest positive correlations with stress among all genera in the community. This powerful approach for correlating the dynamics of individual taxa within communities with plant stress was effective in identifying taxa responsive to systemic plant signals initiated by any of several abiotic stresses known to induce ROS accumulation. Root microbiome assembly is influenced not only by abiotic stress but also by the root tissue and plant developmental stage, with early assemblages potentially influencing later assemblages. As part of this project, we also generated a fine-scale temporal and spatial map of the soybean endosphere microbiome on plants grown without the imposition of abiotic stress. We found using hierarchical clustering that the endosphere communities cluster into four major groups, with specific taxa repeatably recruited to the root tips, more taxa recruited during the V1 growth stage, and complete displacement of the core microbiome between the V1 and V3 growth stage on all of the root tissues. The change in the endosphere root tip communities after the V3 growth stage, coupled with the similarity of these late-stage root-tip communities to the sub-crown communities, supports that late-stage root tips recruit from communities that were altered by previous colonization activities. These data provide a context in which we can understand the dynamics and role in microbiome assembly of the genera enriched by abiotic stress, above, during root colonization prior to root exposure to stress. Collectively, these activities provide foundational knowledge of how soybean root microbiomes develop in the absence of drought, as well as a foundation of tools to better understand the selective pressures conferred by drought on the microbial communities in and on roots.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Beattie, G.A. 2022. The soybean root microbiome: High resolution mapping and responses to abiotic conditions that induce oxidative stress. Intl Symposium on Microbe-assisted Crop Production (miCROPe), July 11-14, 2022, Vienna, Austria (invited talk)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Delp, D., A. Welty, and G.A. Beattie. Root-associated bacterial community changes associated with increasing plant stress. Phytobiome 2022, Sept 13-15, 2022, Denver, CO (poster presentation)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Welty, A. and G.A. Beattie. Fine-scale spatial mapping of the soybean root microbiome. Phytobiome 2022, Sept 13-15, 2022, Denver, CO (poster presentation)
|
Progress 05/01/21 to 04/30/22
Outputs
Target Audience:This project provided scientific training opportunities for several young and early-career scientists in technical skills in microbiology, molecular biology, microscopy (for oxidative stress evaluation), experimental probing of plant-microbe interactions and plant physiology, and data analysis, presentation and interpretation. Changes/Problems:The metatranscriptomic data were not collected due to insufficient recovery of RNA from the endosphere and rhizosphere, and the metabolomic data were not processed due to lack of effective comparability among samples caused by inherent drought-induced changes in plant physiology. What opportunities for training and professional development has the project provided?The project provided opportunities for training a postdoctoral research associate, a graduate student, and four undergraduate students. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?For Aim 1, we will be refining an approach for using DNA spike-in data to convert read count data to actual abundances, thus complementing our relative abundance data for each of the taxa in the DNA- and RNA-amplicon datasets for the endosphere and rhizosphere. We will also optimize approaches for identifying taxa at various taxonomic levels for which the abundances correlate negatively or positively with plant parameters that reflect plant stress. We will synthesize these results into an understanding of how plant stress affects specific taxa within the context of oxidative stress as a potential driver of compositional shifts in the microbiome. Following these analyses, the results of these plant stress experiments will be written up for publication. For Aim 2, we will be completing our synthesis of the temporal and spatial mapping data into a model of soybean root microbiome assembly, and we will write the results of these mapping experiments for publication.
Impacts
What was accomplished under these goals?
Interest is increasing in the use of microbes to enhance crop tolerance to abiotic stresses such as drought. Capturing these microbial benefits requires understanding how plants recruit and influence the assembly of plant microbiomes. We are using conserved microbial community responses to drought to help identify mechanisms driving drought-associated microbiome shifts. In this project, we profiled the microbial communities from the endosphere and rhizosphere of plants that were subjected to a gradient of plant stresses. We are correlating the responses of specific taxa with this stress gradient, as knowledge of the response of individual taxonomic groups provides clues to the mechanistic drivers of these shifts. Additionally, by characterizing the microbiome assembly patterns across the soybean root system, we are developing a context in which to understand the root-driven recruitment and development patterns of these microbial communities with and without stress. This project is providing foundational knowledge on plant microbiomes with the long-term goal of supporting microbial-based strategies to enhance the health of soybean and other crop plants. Aim 1. Characterize the molecular mechanisms driving assembly patterns of soybean root-associated microbiomes in response to drought. Oxidative stress in the intercellular spaces, or apoplast, of a plant, is widely known to increase in response to drought and other abiotic stresses like heavy metals and salinity. In this project year, we used this information to design experiments to address the hypothesis that drought-induced oxidative stress in plants is a major mechanism driving microbial taxonomic shifts in root-associated microbiomes. We used samples and methods generated in the first two project years, but discontinued efforts to evaluate the metatranscriptome due to insufficient RNA recovery from root microbes to perform either RNAseq or microarray analyses. Instead, we developed a split-root system in which we exposed one root of a split root soybean plant to copper or salinity stress, thus generating oxidative stress within the plant, and then looked at the subsequently propagated stress impact on the microbiome of a separate root. In this manner, we separated plant physiology impacts from direct treatment impacts on the root microbiome. Microbiome profiling demonstrated that phylum-level taxonomic shifts previously associated with drought stress were recapitulated by salinity, and to a lesser extent copper, consistent oxidative stress as a driving force for microbiome shifts during drought. Moreover, plant measurements indicated that our treatments collectively imposed a gradient of plant stress. We are currently characterizing how specific taxa respond to this gradient using microbiome data derived from both DNA- and RNA-based amplicon sequencing and based on relative and absolute abundance. These distinct datasets are enabling a rich understanding of how distinct microbes respond to the stress gradient, and this detailed understanding of the responses of individual taxonomic groups should provide further insights into the mechanisms driving microbiome shifts. Aim 2. Identify the functional microbiome traits associated with feedbacks on soybean physiology and fitness under drought conditions. The recruitment and assembly of root microbiomes on healthy or stressed roots likely follow an assembly process that is specific to both the root tissue and plant developmental stage, with early assemblages potentially influencing later assemblages. Knowledge of which tissues and plant growth stages direct the largest changes in microbiome composition provides key information for identifying potential microbial feedbacks on plants that influence crop growth and resilience to stress. In this project period, we modified our experimental approach from correlating specific soybean responses to drought with the abundance of specific microbial taxa, to mapping the endosphere and rhizosphere microbiomes of soybean. At present, we have nearly completed a fine-scale temporal and spatial map of the endosphere and rhizosphere microbiomes of soybean grown in a field soil. Hierarchical clustering of the microbiomes of endosphere tissues indicates that the communities of the root tissues cluster into four major groups, which collectively illustrate an interaction between plant tissues and plant development on root microbiomes. The results highlight an assembly process in which two copiotrophic genera grow rapidly on the root tips of young plants, with more taxa recruited during the V1 growth stage. Notably, the microbes making up the core microbiome changed dramatically between the V1 and V3 growth stage on all of the soybean root tissues, with the core microbes experiencing a nearly complete displacement. The change in the endosphere root tip communities on plants after the V3 growth stage coupled to the similarity of these late-stage root-tip communities to the subcrown communities suggest that, although root tip recruitment may be similar across plant development, the late-stage root tips are recruiting from communities that have been altered by earlier colonization activities. These findings contribute to a foundation of knowledge on how soybean root microbiomes develop in the absence of drought, thus providing a context for evaluating the selective pressures conferred by abiotic stresses on root microbiomes.
Publications
|
Progress 05/01/20 to 04/30/21
Outputs
Target Audience:This project provided scientific training opportunities for several young scientists in technical skills in microbiology and molecular biology, experimental probing of plant-microbe interactions and plant physiology, and data analysis, presentation and interpretation. Changes/Problems:The pandemic slowed the activities for Aim 1 as the project staff were not in the lab for many months, and it delayed the activities for Aim 2 by a year due to our inability to be in the lab and to obtain necessary resources from other labs. What opportunities for training and professional development has the project provided?The project provided opportunities for training a postdoctoral research associate, a graduate student, and an undergraduate student. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?For Aim 1, we will be extracting DNA from the rhizosphere and endosphere samples and performing DNA-based amplicon sequencing to ensure that the microbiome community shifts that we observed previously occurred in this iteration of the experiment. We will also extract RNA from these samples to perform RNA-based amplicon sequencing for evaluating the extent to which the microbial community shifts reflect active community members. We will be evaluating if protocols for the recovery of microbes from endophytic sites will provide sufficient microbes to support RNAseq-based characterization of the metatranscriptome of these microbes. If so, then following validation of the microbiome shifts, we will isolate total RNA from all of the samples and characterize the metatranscriptome of the samples by RNAseq, using metagenomic DNA sequencing of a few samples to create a reference library. If not, then we will perform metagenomic DNA sequencing of a few samples and use these sequences, or the metatranscriptome sequences from the rhizosphere, to design microarrays to test for the expression of target genes in the endosphere. Concurrently, we will complete the metabolome analysis of the soybean rhizosphere and endosphere under drought and non-drought conditions. We hope to complete the collection of these datasets in the next reporting period and begin to evaluate the probability of specific mechanistic drivers of community assembly based on the metabolites that are present and the microbial genes that are expressed. These studies will be complemented with experimental studies evaluating drought-specificity in the presence of antimicrobial root compounds. For Aim 2, we will develop protocols for measuring drought-associated soybean traits using high-throughput phenotyping in the Iowa State University Enviratron. We will measure these traits during a month-long growth period of soybean plants in high and low water content soils, and will identify the most robust measures of drought stress for monitoring in experiments employing a diverse array of microbial communities so that microbiome-associated changes in those measures can be identified.
Impacts
What was accomplished under these goals?
Microbes can help reduce the impact of environmental stresses such as drought on crop plants. Optimizing such microbial benefits on crops requires understanding how plants select for their resident microbial communities, how these microbial residents interact with the plant and each other, and even where the microbes are within the plant. In particular, microbes that are inside the host plant can form more intimate associations, and thus provide greater benefits, than those that are peripherally associated. We are using conserved microbial community responses to drought to help identify mechanisms driving drought-associated microbiome shifts. In the first year of the project, we collected samples that provide biological materials to begin to test for specific mechanistic drivers. In this second year, we have isolated these biological materials, and in doing so, optimized methods to recover microbes from the inside of the plant roots, which is critical for evaluating the activities of these intimately-associated microbial residents. We have also furthered our understanding of which microbes are successful in colonizing distinct sites of the soybean root system. This project is providing foundational knowledge on plant microbiomes with the long-term outcome to support microbial-based strategies to enhance the health of soybean and other crop plants. Aim 1. Characterize the molecular mechanisms driving assembly patterns of soybean root-associated microbiomes in response to drought. A major goal of the project is to understand how the plant's selective forces on the root microbiome changes under drought versus non-drought conditions. The genes that are expressed by these microbes under the two conditions serve as a powerful marker to understand these selective forces. Whereas identifying the genes that are expressed in microbes in the rhizosphere of drought- and non-drought-treated plants is useful, identifying the activities of the microbes within the roots would provide a much more powerful report of the selection processes. Extraction of RNA directly following homogenization of plant roots, however, cause the microbial transcripts to be completely obscured by the plant transcripts. In this project year, we optimized a discontinuous gradient centrifugation protocol to separate the microbes from plant cells prior to extracting RNA. We have now isolated DNA and RNA from the microbes in all of the rhizosphere samples collected under drought and non-drought conditions in year 1, as well as DNA and RNA from microbes following their isolation from the endosphere of these samples using this gradient approach. We also initiated metabolomic analyses on rhizosphere samples from drought and non-drought treated plants, but ran into roadblocks in generating biologically comparable metabolome analyses due to drought-induced physiological differences in the plants that dramatically impact sample collection. Microbiome studies of plant roots generally collect the microbiome from across a root system without regard to spatial differentiation of the community. However, understanding such potential spatial differentiation is critical to understanding how plants shape these root communities. In this project period, we have been characterizing the key similarities and differences among microbial communities of soybean roots based on the spatial location of these communities within the root system; moreover, we are doing this on endosphere communities collected throughout the development of the plant. Although we have thus far done this study only under non-drought conditions, the data are indicating dramatic shifts in the communities not only with plant development, but also in distinct regions of the root and are indicating some clear successional patterns. Collectively, these activities are positioning us to have a foundation of knowledge on how soybean root microbiomes develop in the absence of drought, as well a foundation of tools to better understand the selective pressures conferred by drought on the microbial communities in and on roots. Aim 2. Identify the functional microbiome traits associated with feedbacks on soybean physiology and fitness under drought conditions. Aim 2 activities will involve examining soybean responses to drought in the presence of a diverse array of soil microbial communities. During the past project period, we initiated the collection of this array of microbial communities from across the United States. We identified researchers with soybean fields representing a variety of climate conditions and soil types and obtained permission to move samples of these soils into Iowa for our experiments. Due to the pandemic, we were forced to delay requesting these soils to initiate these studies.
Publications
|
Progress 05/01/19 to 04/30/20
Outputs
Target Audience:This project provided a scientific training opportunity for a graduate student, a postdoctoral researcher and two undergraduate students; this included training in experimental design, execution, troubleshooting, and the presentation of research plans, progress and results in both oral and written form. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided opportunities for training a postdoctoral research associate, a graduate student, and two undergraduate student researchers. How have the results been disseminated to communities of interest?The project has resulted in two posters presented at undergraduate and/or graduate student research symposia. What do you plan to do during the next reporting period to accomplish the goals?For Aim 1, we will be extracting DNA from the rhizosphere and endosphere samples and performing DNA-based amplicon sequencing to ensure that the microbiome community shifts that we observed previously occurred in this iteration of the experiment. We will also extract RNA from these samples to perform RNA-based amplicon sequencing for evaluating the extent to which the microbial community shifts reflect active community members. We will be evaluating if protocols for the recovery of microbes from endophytic sites will provide sufficient microbes to support RNAseq-based characterization of the metatranscriptome of these microbes. If so, then following validation of the microbiome shifts, we will isolate total RNA from all of the samples and characterize the metatranscriptome of the samples by RNAseq, using metagenomic DNA sequencing of a few samples to create a reference library. If not, then we will perform metagenomic DNA sequencing of a few samples and use these sequences, or the metatranscriptome sequences from the rhizosphere, to design microarrays to test for the expression of target genes in the endosphere. Concurrently, we will complete the metabolome analysis of the soybean rhizosphere and endosphere under drought and non-drought conditions. We hope to complete the collection of these datasets in the next reporting period and begin to evaluate the probability of specific mechanistic drivers of community assembly based on the metabolites that are present and the microbial genes that are expressed. These studies will be complemented with experimental studies evaluating drought-specificity in the presence of antimicrobial root compounds. For Aim 2, we will develop protocols for measuring drought-associated soybean traits using high-throughput phenotyping in the Iowa State University Enviratron. We will measure these traits during a month-long growth period of soybean plants in high and low water content soils, and will identify the most robust measures of drought stress for monitoring in experiments employing a diverse array of microbial communities so that microbiome-associated changes in those measures can be identified.
Impacts
What was accomplished under these goals?
Environmental stresses such as drought and high temperatures reduce crop yields. Whereas crop stress tolerance can be enhanced through germplasm improvement; it may also be enhanced by optimizing the partnerships between crops and their microbial residents. As static organisms, plants cannot move to evade stress; consequently, plants have evolved to exploit the benefits conferred by microbes, which are comparatively mobile components of their environment. Using microbes for crop protection, however, requires knowledge of the mechanisms by which crops promote microbial community development and microbes within those communities confer benefits. To gain this knowledge, this project is exploiting conserved microbial community responses to drought to help identify the mechanisms driving drought-associated microbiome shifts. In this first year of the project, we have collected biological materials that are required to test specific mechanisms and to link functional microbiome traits to crop traits associated with enhanced drought tolerance. The knowledge resulting from these studies will contribute to the development of drought-protective microbial inoculants and highlight targets and screening tools for breeding plants that foster the development of beneficial microbial communities. Aim 1. Characterize the molecular mechanisms driving assembly patterns of soybean root-associated microbiomes in response to drought. A major goal of Aim 1 in this project period was to collect biological materials appropriate for hypothesis testing. We obtained soils from fields in which soybeans had been grown under drought conditions, verified the absence of soybean pathogens in the soils, and optimized experimental protocols to minimize the emergence of endogenous pathogens in soybean seeds. We performed five sequential one-month plant growth cycles in these soils in the laboratory, with treatments in each cycle including high and low soil water content conditions. Replicate rhizosphere and endosphere samples were collected at the end of each cycle for the extraction of microbial DNA, microbial RNA, microbial isolates, antimicrobial metabolites, and total metabolites. These samples are currently in storage awaiting nucleic acid and metabolite extraction. We generated a collection of purified microbial isolates from the samples, and thus far have constructed a partial set of indicator organisms that exhibit susceptibility to a broad array of antimicrobial compounds for use in hypothesis-testing experiments. To identify how drought impacts soybean root metabolites, we optimized conditions for growing soybean seeds with a minimal presence of endophytes in a sterile sand matrix. We collected rhizosphere and endosphere samples weekly. We have initiated, but not yet completed, protocols for extracting metabolites from these samples for metabolome analyses. Collectively, these activities have positioned us to have the foundation of tools required to first confirm expected drought-induced microbiome shifts, and second perform both open-ended explorations and tests of predicted mechanisms underlying these shifts. Aim 2. Identify the functional microbiome traits associated with feedbacks on soybean physiology and fitness under drought conditions. Aim 2 activities will involve examining soybean responses to drought in the presence of a diverse array of soil microbial communities. During this project period, we initiated the collection of this array of microbial communities from across the United States. We identified researchers with soybean fields representing a variety of climate conditions and soil types and have obtained permission to move samples of these soils into Iowa for our experiments. Again, these biological resources will be critical to our ability to begin to relate physiological plant traits to compositional microbiome traits to help identify links that can be exploited to use microbes to manipulate plant drought tolerance.
Publications
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