Performing Department
(N/A)
Non Technical Summary
The rhizosphere microbiome is essential for overall plant and soil health. To better understand how microbes affect their environments, it is important to understand the molecular crosstalk mediating microbial interactions in communities. Within microbial communities, each microbe responds to environmental cues, such as secondary metabolites. Secondary metabolites are small molecules produced by bacteria, fungi, and plants that are not essential to their growth. These small molecules have been largely studied for their pesticide, antibiotic, and other medicinal properties. Despite their ubiquity, little is known about the mechanisms that govern secondary metabolite production and function in the context of microbial communities in their natural habitats since they are not always produced at inhibitory concentrations. Our genetically tractable model system The Hitchhikers Of the Rhizosphere (THOR)--composed of B. cereus and its two co-isolates from field-grown soybeans and alfalfa roots, Pseudomonas koreensis and Flavobacterium johnsoniae--will allow us to dissect the effects of rhizosphere microbes on plants in a community with demonstrated relevance to their natural ecosystem. I aim to improve our understanding of rhizosphere microbial communities to enable the development of sustainable strategies to enhance agricultural production by reducing crop losses and suppressing diseases. For this I will (1) understand the chemical environment of THOR on plant roots; (2) draw from important chemical compounds from objective 1 to determine the role of secondary metabolites on growth and persistence of THOR on plant roots; (3) explore the impact of THOR on disease suppression. Together these results will allow us to identify biologically relevant interactions that affects the success of microbes in plant and soil that will benefit plant health.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
10%
Developmental
40%
Goals / Objectives
The main goal of this proposal is to understand the effects bidirectional exchange of bacterial secondary metabolite (SM) during the process of root colonization to enhance plant growth and disease protection. Rhizosphere-associated bacteria can protect plants from disease and drought. Current agricultural practices include the introduction of biocontrol agents for crop management; however, consistent, and predictable manipulation of rhizosphere communities remains a challenge.Objective 1. Elucidate the metabolomic profile of THOR on plant roots.Objective 1.1 Metametabolomics of plant roots.Objective 1.2 Metabolic network analysis.Objective 2. Determine the role of SM on growth and persistence of THOR on plant roots.Objective 3. Explore the impact of THOR on disease suppression.The results of this study will contribute to the growing interest in dissecting and manipulating microbial communities for more sustainable agricultural practices. Even though the outcomes seem specific to our system they can impact strategies used for more effective inoculation of biocontrol agents in the field across a wide variety of crops.
Project Methods
Methods per objective:1. Elucidate the metabolomic profile of THOR on plant roots. To determine the chemical composition of plant-microbe interactions I will inoculate soybean (Glycine max) roots using methods developed in our lab. Specifically, surface sterilized seeds will be placed in magenta boxes with a bottom reservoir containing plant nutrient solution (PNS). Plants will be grown in a growth chamber at 20ºC with a 12-hour photoperiod for 10 days. Roots will be collected, measured, washed, and PNS will be filtered sterilized and analyzed via Liquid Chromatography-Tandem Mass Spectrometry using a C18 chromatography column. For MS samples, MS1 data will be collected at a 70K resolution while MS2 will be collected for the seven most intense ions for each MS1 scan at 17.5 resolution at 2 m/z window. To identify molecular signatures of interest, I will analyze raw data files using Compound Discover v3.3 (Thermo Scientific). The data will be normalized to maximum peak intensity and molecular features will be identified with various databases including: ChemSpider, Natural Product Atlas, BioCyc, and Kegg. Then, I will generate a chemical network, using methods previously developed in our lab, to cluster features by molecular similarity. Each node will represent a molecular feature and the distance between nodes will suggest how similar they are. Differences within samples will suggest the importance of classes of molecules in the colonization of roots. By identifying these classes of unique molecular features, we will be able to generate lists of candidate molecules important for host colonization. Bacteria will be removed from the roots by sonication and colony forming units (CFUs) determined by serial dilution on selective media--polymyxin B selects for B. cereus, gentamicin selects for F. johnsoniae, and a mix of ampicillin and erythromycin selects for P. koreensis.2. Determine the role of secondary metabolites (SM) on growth and persistence of THOR on plant roots. Unique molecular features from Objective 1will inform mutant generation in gene clusters that are likely candidates encoding the biosynthetic machinery for the dominant metabolites. I will create mutants of THOR members in the key genes associated with these SMs to determine their importance on host-colonization. Mutants will be constructed by standard allelic exchange cloning methods and verified by sequencing. Using similar methods as Objective 1, I will inoculate soybeans with a 1:1:1 ratio of wildtype or mutant strains of THOR members. Roots will be collected, measured, washed, and dried at daily intervals and population abundance will be determined by CFUs. These results will allow dissection of genes important for colonization and their effects on plant growth. A potential limitation to this approach could be that genes responsible for metabolite synthesis are not easily mapped on the genome. In this case we will use transposon libraries of each member of THOR that the lab has generated over the years and identify mutants that are deficient in metabolites of interest based on LC/MS-MS profiles.3. Explore the impact of THOR on disease suppression. I will use Pythium ultimum as our disease model on soybean roots. Mutants that are deficient or enhanced in colonization from Objective 2 will be tested for disease suppression. I will allow THOR mutants to establish on roots before inoculation with P. ultimum. After pathogen infection plants will be incubated in a plant chamber for 5 days before assessing health. I will measure plant shoot and root weight, and bacterial populations. If the metabolites of interest are SM, I will conclude that SM are important not only for their antagonistic effects on pathogens but also for their signaling effects in nature that can modulate microbial behaviors. Lastly, I will characterize the changes in the metabolome of THOR in roots induced by P. ultimum using methods described in Objective 1. These results will help us leverage the potential benefits of SM in nature to engineer microbial communities that protect and induce plant health.The results of this work will be presented in relevantscientific conferences and publishedin an open access journal.