Location: Plant Gene Expression Center
2023 Annual Report
Objectives
The goal of this research is to generate a better understanding of the interrelationship between crop plant genetics and plant microbiomes. Collectively, the experiments described in the Objectives below will provide for direct testing of a priori hypotheses regarding the genetic pathways associated with plant microbiome recruitment (Objective 2), which we define in this proposal as the assembly and maintenance of plant-associated microbiome, as well as unbiased genome wide analyses that may capture genes previously unsuspected to be involved in this phenotype (Objectives 1 and 3). Integration of the results from this multifaceted approach may allow for corroboration of key findings from individual Objectives. For instance, loci identified in Objective 1 from the association-based analysis of host genes will be cross-referenced with the genes identified as part of the transcriptional analyses described in Objective 3. In Objective 2, genes confirmed through mutant analysis to have direct effects on plant microbiome phenotypes can potentially serve as positive controls for analyses carried out in Objective 1. While the Objectives differ in terms of experimental methodology, as well as the precise question being addressed, the broader goal of each is the identification of elements involved in host control of the microbiome, and we anticipate that this diversified approach in a single host system and by a single investigative team will provide valuable insight addressing this key knowledge gap in the field of plant-biotic interactions.
Objective 1: Identify the host genetic elements (genes and other functional sequences) associated with microbiome recruitment through genome wide association studies in Sorghum bicolor using microbial community analysis as the scoreable phenotype.
Objective 2: Create sorghum mutants in cell wall biosynthesis pathway genes, and other target plant processes, that are critical for the recruitment of microbes during drought stress; characterize the microbiomes and abiotic stress tolerance of these lines.
Objective 3. Use an established microbial culture collection for sorghum to test the changes in the sorghum root following exposure to root endophytes and abiotic stress to establish causality with stress tolerance.
Objective 4: Utilize host-mediated microbiome engineering under drought to guide the development of assembled communities capable of improving crop performance under drought.
Approach
Obj. 1: We aim to identify loci that contribute to the plant bacterial microbiome phenotype in sorghum through Genome-wide association studies (GWAS) analysis using a mapping panel of diverse sorghum lines. GWAS represent a powerful approach to detect variants at genomic loci associated with complex traits in a genetically diverse population. We will use the Sorghum Association Panel (SAP), grown in a randomized block design. Root samples will be harvested and separated into root endosphere and rhizosphere samples and bacterial communities for target plant samples will be assessed via amplicon-based next-generation, and the relative abundance of specific bacterial taxa showing large differential abundance across our genotypes, or overall microbial community composition using individual component axes of principal coordinate analyses with the broader dataset, will be used as phenotypic inputs in the GWAS. Should our analysis of the root endosphere microbiome be unsuccessful, alternate sample types including rhizospheres and leaf epispheres, and alternate phenotypes, can be investigated.
Obj. 2: We aim to test whether suberin deposition within roots of sorghum during drought stress acts to recruit and support specific bacterial taxa, namely Actinobacteria, to the root microbiome, and to promote drought tolerance. We will develop a protocol for CRISPR/Cas9 delivery into sorghum roots via Agrobacterium rhizogenes, and utilize this system to create engineered lines with alterations in the suberin biosynthesis pathway. The engineered genotypes along with wildtype will be grown in the greenhouse in field soil under two watering regimes, and sampled, phenotyped, and assayed for root suberin content via fluorescence microscopy with a suberin specific stain and subjected to 16S rRNA and ITS2 metagenomic analysis to investigate changes in the bacterial and fungal microbiome. Should the construction of CRISPR lines prove challenging, we will obtain mutants from a TILLING mutant population in sorghum.
Obj. 3: We aim to identify the physiological and molecular consequences of colonization by endophytic isolates on gnotobiotically-grown sorghum through phenotypic and transcriptional analysis in the plant root. Sorghum roots will be grown in a sterile system filled with autoclaved soil medium. Select Actinobacteria and non-Actinobacterial isolates from our collection will be introduced individually to the roots of 7-day-old sorghum plants. One week following inoculation, we will induce drought stress in half of the gnotobiotic systems, and collect roots of inoculated and control plants. Plant samples will be phenotyped and used to isolate high-quality total RNA for preparation of RNA-Seq libraries for Illumina sequencing. An analysis of the datasets will determine differentially expressed genes between drought and control treatments, inoculated and uninoculated roots, and between the individual microbial treatments used in our experiment. Should the induction of drought prove challenging, we have an alternative gnotobiotic system that will allow for longer periods of sorghum growth, which has been achieved in other grass systems by our collaborators.
Progress Report
This is the final report for project 2030-12210-002-000D, “Discovery of Plant Genetic Mechanisms Controlling Microbial Recruitment to the Root Microbiome” which has expired in 2/24/2023, and has been replaced with project 2030-12210-003-000D, “Enhancing Crop Resilience to Biotic and Abiotic Stress Through Understanding the Immune and Microbiome Signaling Mechanisms”. For additional information, see the new project report.
In support of Objective 1, we aimed to identify loci that contribute to the plant bacterial microbiome phenotype in sorghum through Genome-wide association studies (GWAS) analysis using a mapping panel of diverse sorghum lines. GWAS represent a powerful approach to detect variants at genomic loci associated with complex traits in a genetically diverse population. We used the Sorghum Association Panel (SAP) grown in a randomized block design. Root samples were harvested and separated into root endosphere and rhizosphere samples and bacterial communities for target plant samples were assessed via amplicon-based next-generation. Next, the relative abundance of specific bacterial taxa showing large differential abundance across our genotypes, or overall microbial community composition using individual component axes of principal coordinate analyses with the broader dataset, was used as phenotypic inputs in the GWAS. The analyses identified multiple loci correlated with microbiome phenotypes, including a locus containing genes associated with plant cell wall structure and plant immunity. Our analysis of the root endosphere microbiome was ultimately successful, as a final validation step using a distinct set of lines not part of the original GWAS analysis confirmed patterns of microbial enrichment based on allele content.
In support of Objective 2, we aimed to test whether suberin deposition within roots of sorghum during drought stress acts to recruit and support specific bacterial taxa, namely Actinobacteria, to the root microbiome, and to promote drought tolerance. We developed a protocol for clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 delivery into sorghum via Agrobacterium and utilized this system to create engineered lines with alterations in the suberin biosynthesis pathway or related pathways. Multiple lines were created in several target genes, including those associated with both glycerol-3-phosphate metabolism, a metabolite that is shown to be upregulated during drought stress. Ultimately, after multiple rounds of screening, we were unsuccessful in creating lines with altered glycerol-3-phosphate content, an no detectable insertions or deletions in either of the target genes were identified. We interpret this result as evidence of the critical need for glycerol-3-phosphate for sorghum development. Future work will focus on alternate target loci for mutagenesis.
In support of Objective 3, we aimed to identify the physiological and molecular consequences of colonization by endophytic isolates on gnotobiotically-grown sorghum through phenotypic and transcriptional analysis in the plant root. Sorghum roots were grown in a sterile system filled with autoclaved soil medium. Select Actinobacteria and non-Actinobacterial isolates from our collection were introduced individually to the roots of 7-day-old sorghum plants. One week following inoculation, we induced drought stress in half of the gnotobiotic systems and collected roots of inoculated and control plants. Plant samples were phenotyped and a focus on the Actinobacterial genus Streptomyces has revealed remarkable diversity within this clade for both phenotypic performance of the host and metabolic production and isolate characteristics. An analysis of the datasets has demonstrated that differentially expressed metabolites between treatments contain resources connected to plant immunity and carbohydrate or resource transport, consistent with other research from our group.
In support of Objective 4, we have designed and completed a pilot experiment using host-mediated microbiome engineering in rice to create a community capable of improving drought tolerance in terms of plant biomass. The data demonstrate the utility of the method, and identified several taxa with correlative association with increased plant resilience under water stress conditions.
Accomplishments
1. Identification of genes connected to host control of drought-responsive microbiomes. Drought represents a significant negative determinant of crop yields and health each year, and the severity and duration of drought is anticipated to increase across the country in the coming decades. In addition to ongoing efforts to improve drought tolerance through breeding and crop engineering, there is a major need for alternative methods to mitigate drought stress in crops. ARS researchers in Albany, California, have identified specific root-associated microbes and microbial synthetic communities that can alter root and shoot traits in sorghum and other grass crops under drought stress, and genes in plants that impact the colonization of these microbes. This discovery paves the way for the development of more efficient technologies to support crop productivity under abiotic stress.
Review Publications
Caddell, D.F., Langenfeld, N.J., Zhen, S., Eckels, M., Zhen, S., Klaras, R., Mishra, L., Bugbee, B., Coleman-Derr, D.A. 2023. Photosynthesis in rice is increased by CRISPR/Cas9-mediated transformation of two truncated light-harvesting antenna. Frontiers in Plant Science. 14. Article 1050483. https://doi.org/10.3389/fpls.2023.1050483.
Elmore, J., Dexter, G., Baldino, H., Huenemann, J., Francis, R., Peabody, G.L., Martinez-Baird, J., Riley, L.A., Simmons, T., Coleman-Derr, D.A., Guss, A., Egbert, R. 2023. High-throughput genetic engineering of nonmodel and undomesticated bacteria via iterative site-specific genome integration. Science Advances. 9(10). Article eade1285. https://doi.org/10.1126/sciadv.ade1285.
