Periodic Reporting for period 3 - SIREM (Systemic Induced Root Exudation of Metabolites: A Multimodal Approach to Uncover Root Signaling Mechanisms and the Chemical Language used by Plants to Shape the Rhizosphere Microbiome)
Período documentado: 2023-05-01 hasta 2024-10-31
The prosperity of human beings is dependent on the outer layer of soil that makes up our planet’s shell. Curiously, a significant portion of plants' most precious elements, carbon and nitrogen, is secreted by roots into soil in the form of chemically-rich exudates. This is not merely 'dumping' of waste but rather the chemical language of plants, used in their underground communication with billions of detrimental and beneficial microorganisms. Yet, an important question remains to date: How do plants control and manipulate root metabolism and exudation in time and space to fine-tune this complex underground web of interactions to their benefit? The proposed project takes on this challenge and aims to decipher the newly discovered process we term 'SIREM', for 'Systemic Induced Root Exudation of Metabolites'. SIREM is a fundamental feature of rhizosphere interactions, in which local biotic stimuli induce systemic exudation in parts of the root, modifying the rhizosphere environment to maintain plant fitness. SIREM objectives include: (i) dissecting the SIREM signaling pathways, focusing primarily on the mobile signal(s) and receiving proteins at the systemic, exuding root; (ii) discovery of the exudation machinery and its genetic control; and (iii) establishing the role of SIREM signaling and exudation-metabolites in shaping the rhizosphere microbiome. The unconventional integration of approaches in SIREM underscores the unique combination of our team's expertise in plant metabolism, computational biology, microbiome exploration and the application of cutting-edge analytical and molecular technologies for high-resolution spatial-temporal profiling. Outcomes of the project will have wide-ranging impacts on understanding systemic signaling, metabolic and transport systems in plants and are anticipated to drive the new biotechnological concept of 'Exudation Agriculture'.
In the reporting period we have been working on achieving the three main objectives of SIREM. (i) dissecting the SIREM signaling pathways: we performed transcriptome analysis of SIREM by treating the local side of the root system with microbiome obtained from tomato root rhizosphere and analyzing the systemic root side for gene expression in time. As a result of this work we identified a dozen regulatory genes that are induced in SIREM. These are currently characterized for their function in controlling the quantity and composition of exudation in SIREM. To identify the possible mobile signal in SIREM that induces systemic exudation we have generated tomato plants that are either mutated in the FMO1 gene involved in N-hydroxy pipecolic acid (NHP) metabolite production and NHP over-producing plants. These plants will be examined for SIREM activity in the near future. In parallel, we set-up the system to purify individual components of tomato root exudation (that includes hundreds of metabolites) in order to examine their function and impact on tomato rhizosphere bacterial and fungal communities. A different technology has be established (for the first time in plants!) that captures proteins that are bound by metabolites and applied it to discover NHP-binding proteins. At this stage candidate proteins identified through this approach are being characterized. (ii) discovery of the exudation machinery and its genetic control: our major focus here was to identify transporter proteins that mediate the exudation of different classes of metabolites from the root to the rhizosphere. We employed various genetic and molecular approaches and identified more than 50 candidate transporter genes; a dozen of these are currently under thorough investigation to determine their function in SIREM exudation. As part of studying the exudation machinery we discovered that tomato roots secrete extracellular vesicles (EVs) and now examining the contribution of EVs in the transfer of small molecules and proteins as part of the SIREM exudation machinery, (iii) establishing the role of SIREM signaling and exudation-metabolites in shaping the rhizosphere microbiome: In the past one and a half years we have been working on the establishment of a tomato root bacterial synthetic community (SynCom). We cultivated 1500 isolates from tomato plant growing soil and conduced DNA sequencing in order to determine their identify. This unique SynCom will be highly valuable in examining the effect of particular bacterial species on exudate metabolite composition in SIREM and the impact of individual or class of metabolites exuded by tomato root on the plant rhizosphere microbiome community. In parallel we have established the methodology for collecting rhizosphere samples from mature plants grown outdoors in natural settings as plants altered in SIREM will be examined for the microbial community that colonizes their root system.
The SIREM project progressed beyond the state of the art both in term of new discoveries as well as technologies. Up to date only a few transporter and regulatory proteins have been identified that are associated with root exudation. In the course of the project we identified a set of proteins that are likely involved in the process of root metabolite exudation in tomato. The discovery of extracellular vesicles release from plant roots to the rhizosphere are a very significant achievement and a breakthrough that advances the entire field of plant interactions in the rhizosphere. It is a major step towards answering the question regarding the way by which metabolites and proteins are released from roots to the environment. At the technological level we developed an array of protocols, methodologies and approaches that include among others the establishment of bacterial synthetic communities from plant roots, isolation of extracellular vesicles, and the detailed characterization of genome edited plants grown in natural settings.