Periodic Reporting for period 4 - 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)
Reporting period: 2024-11-01 to 2025-04-30
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? In this project we took on this challenge and aimed to decipher the 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. Outcomes of the project 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 this project we uncovered key components of the SIREM signaling pathway, linking local root responses to systemic plant regulation. We identified transcriptional regulators, specific transporters, and extracellular vesicles (EVs) as central elements controlling metabolite secretion and defense. The discovery that EVs carry bioactive metabolites such as acylsugars and contribute to pathogen resistance represents a major conceptual advance in understanding root exudation. We also established a synthetic tomato root microbiome (SynCom) and new field-based phenotyping systems that bridge laboratory findings with real-world conditions. Together, these achievements provide a comprehensive framework for improving plant–microbe interactions and crop resilience through targeted manipulation of root metabolic signaling. Based on the fundamental insights gained through SIREM, two start-up companies are being established to develop innovative applications of root exudation research in the food, cosmetics, and pharmaceutical sectors.
In the reporting period we have been working on achieving the three main objectives of SIREM. Our first goal was to uncover how the SIREM signaling pathway works, focusing on identifying the mobile signal that connects local and systemic responses in roots. By analyzing gene activity in tomato roots exposed to soil microbes, we found 12 transcription factors (TFs) that likely regulate metabolite release. Using CRISPR-based screening of 80 additional TFs, we discovered several key regulators of acylsugar biosynthesis, including ARF10/16 and WRKY72a/b, as well as a new metabolic gene cluster containing acylsugar-related enzymes and regulatory genes. We also investigated N-hydroxy-pipecolic acid (NHP), a strong candidate for the SIREM mobile signal. Using proteomics, we identified several NHP-binding proteins and generated tomato lines that either overproduce or lack FMO1, a key NHP enzyme. Field testing of these lines is underway. A major discovery was a set of NAC transcription factors acting as negative regulators of systemic signaling, the first such regulators identified for SIREM. We also found that roots may release metabolites not only through transporters but also via extracellular vesicles (EVs), small membrane-bound particles. We developed a method to isolate EVs from tomato roots and identified around 100 proteins enriched within them, many linked to plant immunity. Notably, EVs containing a protein kinase inhibited bacterial growth, and plants lacking EV-associated proteins TET8/TET9 were more sensitive to the fungal pathogen Fusarium oxysporum. These findings suggest that EVs carry defensive compounds, including acylsugars, and play a major role in plant protection. Our second major objective was to study the root exudation machinery by transporter proteins (TPs). We identified 51 candidate TPs potentially regulated by SIREM. Using a fast CRISPR-based hairy root system, we tested their roles and found three with strong effects: TP-36 exports hydroxycinnamic acid amides, TP-7 exports coumarins, and TP-20 likely transports steroidal glycoalkaloids. Plants lacking TP-36 or TP-7 accumulated these metabolites in roots but not in exudates, showing they are essential for secretion. Together, these transporters reveal how SIREM controls the flow of chemical defenses from roots into the soil, helping shape plant–microbe interactions. In the third objective we investigated the impact of SIREM Metabolites on the root microbiome. We created a synthetic tomato root microbiome (SynCom) of 270 well-characterized bacterial strains to study how root-secreted metabolites influence microbial communities. This resource is now used in multiple greenhouse and field experiments. To connect lab results with natural conditions, we developed field phenotyping and mobile lab methods to monitor plant performance, root microbiomes, and insect activity. Edited tomato lines, including NHP deficient and acylsugar mutants, were successfully tested outdoors in the field demonstrating the importance of these secreted metabolites to the underground interactions of plants with other organisms.
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.