Performing Department
(N/A)
Non Technical Summary
Wheat cultivars and their associated microbial communities, or microbiomes,represent a versatile and compact toolbox to enhance yield and improve resilience to abiotic and biotic stressors. Determining host genotype impact on the plant microbiome is crucial, as host genotype can be easily changed in a field setting, whereas other factors that influence plant microbiomes are less easily manipulated. Wheat, due to its self-pollination and trait stability, serves as an excellent candidate for examination of the rhizosphere microbiome in the context of drought stress. The overarching goal of this project is to increase basic knowledge of how host genotype and phenotype influence the root associatedmicrobial community under drought stress. The proposed research will utilize drought susceptible and tolerant wheat cultivars bred in the Pacific Northwest to provide useful contrast of drought tolerance phenotypes. By utilizing a genotype-by-environment framework, we aim to determine how host genotype and drought stress impactroot associatedmicrobial communities of wheat in the context of individual cultivars and cultivar mixtures of contrasting drought phenotypes. Our overarching goal is to evaluate potential translational solutions for improving drought tolerance of currently available wheat cultivars.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
0%
Goals / Objectives
The overarching goal of this project is to increase basic knowledge of how wheat genotype and phenotype influence the rhizosphere microbial community under drought stress. The proposed research will utilize drought susceptible and tolerant wheat cultivars bred in the Pacific Northwest to provide useful contrast of drought tolerance phenotypes. By utilizing a genotype-by-environment framework, we aim to determine how host genotype and drought stress impact the structure and functional influence of rhizosphere microbial communities of wheat in the context of individual cultivars and cultivar mixtures of contrasting drought phenotypes. Our overarching goal is to evaluate potential translational solutions for improving drought tolerance of currently available wheat cultivars. My proposed research seeks to evaluate how interactions among wheat genotypes and the rhizosphere microbiome give rise to drought tolerance. My proposed research seeks to evaluate how interactions among wheat genotypes and the rhizosphere microbiome give rise to drought tolerance (Fig. 1). The specific aims include:Aim 1. To determine how host genotype and drought stress impact the structure and functional influence of rhizosphere microbial communities of wheat.Aim 2. To determine how genotype mixtures impact the structure and functional influence of rhizosphere microbial communities of wheat.
Project Methods
Aim 1) The experimental design for this aim will include 3 drought tolerant and 3 susceptible cultivars. Each cultivar will be planted in 54 pots filled with autoclaved potting soil, with 2 seeds initially planted per pot and thinned to one plant per pot after emergence. Three pots will form a replicate, with 3 replicates (9 pots) per treatment. Field soil slurry sourced from fields with a history of wheat cultivation (Pendleton, OR) will be added to 2 selection treatments (drought tolerance and random) to provide microbial inoculum. In addition to a sterile control treatment (autoclaved potting soil inoculated with autoclaved soil slurry), all 4 treatments will be subjected to 2 water stress conditions (drought and well-watered). Inoculated treatments will undergo drought tolerance or random selection to identify a subset of plants that will be processed to obtain rhizosphere soil and generate the subsequent enrichment's soil slurry. After each enrichment, all plants will be assessed for wilting, leaf transpiration, chlorophyll content, in addition to above and below ground biomass. Six successive enrichments at minimum will be employed to amplify host genotype effects on the rhizosphere. After the final enrichment cycle, we will plant to a common, drought susceptible genotype, seeking to "cure" the susceptible phenotype with the enriched microbial communities. The autoclaved soil controlallows for comparisons of drought tolerance phenotypes in control vs. enriched soil, which will shed light on host genotype vs. host genotype and rhizosphere enrichment effect on drought tolerance. We expect that a minimum of 6 cycles of enrichment will be necessary to detect differences in the rhizosphere by host genotype/phenotype, but will implement up to 9 cycles if necessary. To evaluate the rhizosphere communities associated with the different genotypes under varying selection and water stress conditions, we will collect rhizosphere samples from each replicate prior to enrichment and post each enrichment cycle, for a total of 108 samples per enrichment and ~650 samples per experiment. DNA will be extracted from rhizosphere samples using DNEasy PowerSoil kit (Qiagen), and amplicon sequencing of the 16S V4 region and ITS1 region will be accomplished using the Illumina Nextseq platform at OSU's sequencing facility to allow for sufficient sequencing depth of roughly 1300 rhizosphere samples for the replicated experiments. To analyze raw sequence reads, we will utilize the DADA2 16S/ITS Pipeline Workflow (version 1.16 for 16S and 1.8 for ITS) to apply quality filtering, merge, and remove chimeras. Analysis of alpha diversity and community structure will be accomplished using the following R packages: phyloseq, vegan, metacoder, Phylosmith, NetCoMi, and RRPP. These analyses will allow us to compare species diversity and community structure across genotypes, phenotypes, water stress conditions, and selection pressure.In addition to evaluating bacterial and fungal communities in the rhizosphere using metabarcoding, we aim to observe differences in microbial responses to variation in plant secreted metabolites by using metagenomics and metatranscriptomics. To carry out functional analysis, we will flash-freeze a subset of rhizosphere samples from drought tolerant and drought susceptible cultivars at the final enrichment stage under well-watered and drought conditions. A DNA metagenomics approach using long read nanopore sequencing will be employed to construct metagenome assembled genomes (MAGs) from complex rhizosphere samples 80. Rhizosphere DNA will be extracted using Qiagen DNEasy PowerMax Soil kit. Libraries will be prepared using Rapid Barcoding kit and sequenced on Nanopore PromethION sequencing machine. Reads that map to the wheat genome will be removed prior to assembly. Kraken2 will be used to infer taxonomic assignment of raw reads. Metaflye and Medaka will be used to assemble and polish metagenome contigs. MetaBAT will be used to bin assembled contigs into MAGs, with a quality cut-off of >50% complete and <10% contamination. We will use GTDB-tk to assign taxonomy to individual MAGs. Bakta and DRAM will be used to identify open reading frames (ORFs) and annotate gene function in MAG contigs. We will compare taxonomic assignments of raw reads and MAGs within and across genotypes, phenotypes, and water stress conditions. A synergistic metatranscriptomics approach will be carried out by extracting RNA using a RNeasy PowerSoil Total RNA kit. Libraries will be prepared and submitted for Illumina sequencing on the NextSeq platform. The Trinity pipeline will be used for de novo metatranscriptomic analysis. The MetaTrans and/or SAMSA2 pipelines will also be used for comparative transcriptomics of metatranscriptome data. We will utilize KEGG classifications to compare overall transcript dynamics within and among genotypes, phenotypes, and water stress conditions. Aim 2) We aim to evaluate how mixtures of contrasting drought phenotypes influence the rhizosphere and drought tolerance of cultivars within a mixture compared to cultivars grown individually. We hypothesize that the rhizosphere community will be similar to the drought-tolerant parent under drought conditions and will be more similar to the drought-susceptible parent under well-watered conditions (H3). Additionally, we hypothesize that under drought conditions cultivar mixtures with contrasting phenotypes will exhibit higher drought tolerance compared to varieties grown individually (H4). To test these hypotheses, we will grow cultivar mixtures composed of 1 drought tolerant cultivar and 1 drought susceptible cultivar in 9 possible pair combinations across 6 cultivars evaluated in Aim 1. For example, drought tolerant TAM 105 will be paired with susceptible Omar, Rod and Stephens. To maximize rhizosphere effects on drought tolerance, we will condition these cultivar mixtures and individual cultivars for a minimum of six enrichments, similar to Aim 1. After each enrichment, all plants will be assessed for wilting, leaf transpiration, chlorophyll content, in addition to above ground biomass. After the final enrichment, we will plant to a common susceptible cultivar and evaluate drought phenotype response, as implemented in Aim 1. Rhizosphere soil from a single tray will be mixed together and sieved to generate the following enrichment's slurry. To differentiate seedlings within a mixture, we will plant seeds in a grid pattern within a single grow tray, growing seeds at a density of 1 per square inch to mimic planting density in the field, and tag each seedling to ensure that individual plants can be identified and evaluated at the end of a 30-day enrichment period. Evaluation of plants will include days to wilting, above ground biomass by cultivar, overall belowground biomass, leaf transpiration, and chlorophyll-a concentration. It is unlikely that the roots of different cultivars will be distinguishable for rhizosphere analysis.To control for soil influence on the rhizosphere microbial community, we will apply a similar approach to Aim 1, by using autoclaved potting soil and inoculated soil slurry from Pendleton, OR soil source, adjusted for the increased volume of soil in the tray compared to single plant 4" pots used in Aim 1. Three replicate trays will be included for each cultivar mixture and individual cultivar, including an inoculation control, with 2 water stress conditions, for a total of 36 cultivar mixture trays and 24 individual trays. To examine the rhizosphere community, we will employ a metabarcoding approach of the bacterial community as outlined in Aim 1 to examine both bacterial and fungal communities.