Location: Plant Gene Expression Center
2020 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.
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
Progress was made on all three objectives, all of which fall under National Program 301, Component 3A, Crop Biological and Molecular Processes.
For Objective 1, research continued on identifying 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. A full field-scale GWAS experiment was conducted following last year’s pilot using the plant microbiome as a phenotype, and microbiomes of roots and rhizospheres were assessed. Final analyses of the data suggest that rhizosphere microbiomes of sorghum will work as the optimal sample type for a successful GWAS. The generated data has been analyzed and a manuscript is in preparation.
For Objective 2, research continued on testing the impact of specific sorghum metabolism on recruitment of Actinobacteria to the root microbiome, and to promote drought tolerance. As in the previous year, suberin mutants in Arabidopsis of three distinct genotypes were found not to substantially alter microbiome composition. We have selected alternative targets involved in production of glycerol-3-phosphate, a glycolysis intermediate and strongly drought induced root metabolite, and constructs have been generated for deletion in sorghum germplasm. The CRISPR/Cas9 protocol has been piloted in the lab for use in sorghum, and adjustments to transformation strategy have been implemented to improve efficiency in collaboration with scientists at the University of California at Berkeley.
For Objective 3, research continued on identifying the physiological and molecular consequences of colonization by endophytic isolates on gnotobiotically-grown sorghum through phenotypic and transcriptional analysis in the plant root. Full scale inoculation experiments using eight distinct synthetic communities from our established culture collection have been performed under drought and control conditions, and sorghum plants have been phenotyped. The data has been analyzed and specific communities have been found to significantly improve root and shoot growth. Specific isolates belonging to the Streptomyces family have been selected for future full-scale experiments, including full-length genomic sequencing for comparative genomics. All selected isolates have been characterized taxonomically using full-length 16S rRNA sequencing.
Accomplishments
1. Successful implementation of genome wide association study using the crop microbiome. Lack of knowledge regarding specific host genes that control microbiome recruitment has been a major challenge to implementing methods of microbiome control and prediction for improving agriculture. ARS researchers at Albany, California, have identified specific loci that control the abundances of bacterial lineages in the rhizosphere of sorghum using a genome-wide association-based approach. This represents one of the first successful microbiome genome- wide association studies to be performed in a crop microbiome and is the first to validate its findings using an independent set of sorghum lines. This strategy provides a new method for identifying plant genetics that control microbiome composition and activity, and is a valuable breakthrough for understanding plant microbiome interactions in the rhizosphere.
Review Publications
Varoquaux, N., Cole, B., Gao, C., Pierroz, G., Baker, C., Patel, D., Madera, M., Jeffers, T., Hollingsworth, J., Sievert, J., Yoshinaga, Y., Owiti, J., Singan, V., Degraaf, S., Xu, L., Blow, M., Harrison, M., Visel, A., Jansson, C., Niyogi, K., Hutmacher, R., Coleman-Derr, D.A., O'Malley, R., Taylor, J., Dahlberg, J., Vogel, J., Lemaux, P., Purdom, E. 2019. Transcriptomic analysis of field-droughted sorghum from seeding to maturity reveals biotic and metabolic responses. Proceedings of the National Academy of Sciences. 116(52):27124–27132. https://doi.org/10.1073/pnas.1907500116.
Deng, S., Wipf, H., Pierroz, G., Raab, T.K., Khanna, R., Coleman-Derr, D.A. 2019. A plant growth-promoting microbial soil amendment dynamically alters the strawberry root bacterial microbiome. Scientific Reports. 9:17677. https://doi.org/10.1038/s41598-019-53623-2.
Gao, C., Montoya, L., Xu, L., Purdom, E., Hollingsworth, J., Singan, V., Vogel, J., Hutmacher, R., Dahlberg, J., Coleman-Derr, D.A., Lemaux, P., Taylor, J. 2020. Fungal community assembly in drought-stressed sorghum shows stochasticity, selection and universal ecological dynamics. Nature Communications. 11:34. https://doi.org/10.1038/s41467-019-13913-9.
Simmons, T., Styer, A., Pierroz, G., Goncalves, A., Pasicha, R., Hazra, A., Bubner, P., Coleman-Derr, D.A. 2020. Drought drives spatial variation in the millet root microbiome. Frontiers in Plant Science. 11. https://doi.org/10.3389/fpls.2020.00599.
