Periodic Reporting for period 1 - DryCoAdapt (Unravelling the molecular evolution of plant-microbiome interactions in drylands)
Período documentado: 2023-01-01 hasta 2025-06-30
This grant focuses on uncovering the mechanisms of plant-microbiome co-adaptation to aridity. We are employing a combination of large-scale common garden experiments and microbiome re- and deconstruction approaches.
Aim 1: Identify drought-associated species and genes within the desert plant microbiome. We will analyze the microbiomes of the 16 Brassicaceae species in a common garden experiment under either well watered or water-limiting conditions. Then we will use this dataset to understand which microbial taxa and functions are specifically recruited by desert-adapted plant species and to identify core and host-specific microbial mechanisms of drought adaptation.
Aim 2: Elucidate the genetic basis of host-microbe co-adaptation to drought in plant-associated bacteria. We will curate and sequence a multi-kingdom microbial culture collection and use it to build SynComs that mimic the taxonomic distribution found in in desert plant roots. We will perform taxon drop-out and gene knock-out assays within complex SynComs in order to (i) identify drought-protective taxa and genes and (ii) understand the roles that aridity-specific microbial taxa and genes play in plant-microbe mutualisms under drought.
Aim 3: Identify signatures of drought-adaptation in plant transcriptomes and root exudates. We will compare plant transcriptomes and changes in root exudation in response to drought among plants adapted or not adapted to aridity. We will perform exudate transplantation experiments and test the role of specific exudate compounds in protecting plants from water limitation and whether these exudates can serve as common goods among plants.
Aim 1: Identify drought-associated species and genes within the desert plant microbiome. We will analyze the microbiomes of the 16 Brassicaceae species in a common garden experiment under either well watered or water-limiting conditions. Then we will use this dataset to understand which microbial taxa and functions are specifically recruited by desert-adapted plant species and to identify core and host-specific microbial mechanisms of drought adaptation.
Aim 2: Elucidate the genetic basis of host-microbe co-adaptation to drought in plant-associated bacteria. We will curate and sequence a multi-kingdom microbial culture collection and use it to build SynComs that mimic the taxonomic distribution found in in desert plant roots. We will perform taxon drop-out and gene knock-out assays within complex SynComs in order to (i) identify drought-protective taxa and genes and (ii) understand the roles that aridity-specific microbial taxa and genes play in plant-microbe mutualisms under drought.
Aim 3: Identify signatures of drought-adaptation in plant transcriptomes and root exudates. We will compare plant transcriptomes and changes in root exudation in response to drought among plants adapted or not adapted to aridity. We will perform exudate transplantation experiments and test the role of specific exudate compounds in protecting plants from water limitation and whether these exudates can serve as common goods among plants.
Aim 1.1 – Physiological common garden experiment
We established three plant growth systems for our common garden experiments: an irrigated field plot in the Negev desert, pots, including a specialized soil mixture, and sand tubes, for exudate collection. We found that desert plants indeed differ from their Mediterranean relatives in response to drought conditions.
Aim 1.2 – Common garden experiment
We’ve set several iterations of common garden experiments. The first iteration was performed in a controlled field plot in the Negev region of Israel. We planted six species: S. erysimoides, S. officinale, M. chia, M. Africana, D. harra and D. erucoides. Endosphere, rhizosphere and soil clustered separately. In the endosphere, the effect of the drought treatment was statistically significant.
Aim 2.1 –culture collection
We plated ground roots on six different media: ½ LB, 1/10 LB, ½ TSB, 1/10 TSB, R2A and YEM, obtaining 464 isolates and applied a pre-screening approach, using a Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometer (MALDI-TOF). We clustered spectra by similarity using Cosine clustering to identify redundant isolates. Finally, 130 representative strains were chosen, and these were Sanger sequenced.
Aim 1.3 – Metagenomic data analysis
We developed a read-based annotation pipeline, in order to generate a feature table that we could use. We generated functional feature tables, and compared the metagenomes. Mean annual precipitation at the sampling site significantly affected microbiome composition at the site, for both soil and rhizosphere samples, supporting our hypothesis.
Aim 2.2: Community deconstruction for trait discovery
We are applying a new screening strategy for ecologically relevant traits within the microbiome, that we term ‘top-down community deconstruction. We constructed a closed model system consisting of Arabidopsis thaliana Col-0 plants grown in gamma-irradiated coconut coir within magenta boxes, covered with a transparent breathable membrane. We replaced the destroyed microbiota with a 150-member synthetic community (SynCom). We infected leaves of SynCom-inoculated plants with a lux-tagged Pseudomonas syringae strain (Pto), and measured the resulting population size of Pto per milligram of leaf tissue, six days post-infection.
Aims 3.1-3.2 Genomic adaptation to aridity
We grow Isatis lusitanica and Isatis microcarpa in 30 ml syringe barrels, with filter paper in the bottom, and irrigate with measured quantities of water. At the end of the experiment, we flush the sand, to collect root exudates. Samples were analyzed in an untargeted fashion, with both gas chromatography/mass spectrometry (GC/MS), using a reference library of organic acids, amino acids and sugars, and liquid chromatography–mass spectrometry (LC-MS), with a large reference library of natural products. Remarkably, the GC/MS analysis suggests a surprisingly strong response to drought conditions, in both plant species, reflected in a much higher level of exudation compared with the plants grown under full irrigation. Drought triggered the release of the sugars trehalose, sucrose and glucose, as well as several amino acids and other organic acids. LC-MS analysis also revealed a pronounced drought effect in exudation profiles. Interestingly, this effect is stronger in the case of I. macrocarpa, the desert species.
We established three plant growth systems for our common garden experiments: an irrigated field plot in the Negev desert, pots, including a specialized soil mixture, and sand tubes, for exudate collection. We found that desert plants indeed differ from their Mediterranean relatives in response to drought conditions.
Aim 1.2 – Common garden experiment
We’ve set several iterations of common garden experiments. The first iteration was performed in a controlled field plot in the Negev region of Israel. We planted six species: S. erysimoides, S. officinale, M. chia, M. Africana, D. harra and D. erucoides. Endosphere, rhizosphere and soil clustered separately. In the endosphere, the effect of the drought treatment was statistically significant.
Aim 2.1 –culture collection
We plated ground roots on six different media: ½ LB, 1/10 LB, ½ TSB, 1/10 TSB, R2A and YEM, obtaining 464 isolates and applied a pre-screening approach, using a Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometer (MALDI-TOF). We clustered spectra by similarity using Cosine clustering to identify redundant isolates. Finally, 130 representative strains were chosen, and these were Sanger sequenced.
Aim 1.3 – Metagenomic data analysis
We developed a read-based annotation pipeline, in order to generate a feature table that we could use. We generated functional feature tables, and compared the metagenomes. Mean annual precipitation at the sampling site significantly affected microbiome composition at the site, for both soil and rhizosphere samples, supporting our hypothesis.
Aim 2.2: Community deconstruction for trait discovery
We are applying a new screening strategy for ecologically relevant traits within the microbiome, that we term ‘top-down community deconstruction. We constructed a closed model system consisting of Arabidopsis thaliana Col-0 plants grown in gamma-irradiated coconut coir within magenta boxes, covered with a transparent breathable membrane. We replaced the destroyed microbiota with a 150-member synthetic community (SynCom). We infected leaves of SynCom-inoculated plants with a lux-tagged Pseudomonas syringae strain (Pto), and measured the resulting population size of Pto per milligram of leaf tissue, six days post-infection.
Aims 3.1-3.2 Genomic adaptation to aridity
We grow Isatis lusitanica and Isatis microcarpa in 30 ml syringe barrels, with filter paper in the bottom, and irrigate with measured quantities of water. At the end of the experiment, we flush the sand, to collect root exudates. Samples were analyzed in an untargeted fashion, with both gas chromatography/mass spectrometry (GC/MS), using a reference library of organic acids, amino acids and sugars, and liquid chromatography–mass spectrometry (LC-MS), with a large reference library of natural products. Remarkably, the GC/MS analysis suggests a surprisingly strong response to drought conditions, in both plant species, reflected in a much higher level of exudation compared with the plants grown under full irrigation. Drought triggered the release of the sugars trehalose, sucrose and glucose, as well as several amino acids and other organic acids. LC-MS analysis also revealed a pronounced drought effect in exudation profiles. Interestingly, this effect is stronger in the case of I. macrocarpa, the desert species.
1. Our metagenomic analysis pipeline in aim 1.3. While all of the bioinformatic tools we have utilized are already published, we found that our decision to refine and apply them at a massive scale on publicly available data has revealed important hidden trends in soil microbiota. There are two major hurdles in soil microbiota analysis. One is that the reference databases do not represent the biodiversity in soil sufficiently well. The other, which our work helped uncover, is that a large and variable proportion of DNA reads from soil metagenomes is not bacterial, and is likely non-coding eukaryotic DNA. This hinders a correct normalization of count tables, leading to erroneous conclusions. We have avoided this problem by normalizing our counts to single copy marker genes, but realizing the extent of this problem led us to develop a strategy to solve it, which will be addressed in another grant.
2. The successful application of out “top-down deconstruction” strategy (Aim 2.2). This aim relied on a unique concept for screening for beneficial microbes, where instead of testing their effect one-by-one, the entire isolate library is added together, and their effectiveness is measured by sequentially removing groups of isolates. We tested the effectiveness of this approach in protecting from the model pathogen Pseudomonas syringae DC3000, with remarkable success. A manuscript reporting this success is currently being revised for resubmission.
3. Our observation of pronounced changes in root exudation under drought conditions, in particular the large difference in quantity (Aim 3.2). The effect we’ve observed, enabled by the plant growth system that we calibrated, will provide a powerful framework for studying the mechanisms behind microbiome amendment under drought.
2. The successful application of out “top-down deconstruction” strategy (Aim 2.2). This aim relied on a unique concept for screening for beneficial microbes, where instead of testing their effect one-by-one, the entire isolate library is added together, and their effectiveness is measured by sequentially removing groups of isolates. We tested the effectiveness of this approach in protecting from the model pathogen Pseudomonas syringae DC3000, with remarkable success. A manuscript reporting this success is currently being revised for resubmission.
3. Our observation of pronounced changes in root exudation under drought conditions, in particular the large difference in quantity (Aim 3.2). The effect we’ve observed, enabled by the plant growth system that we calibrated, will provide a powerful framework for studying the mechanisms behind microbiome amendment under drought.