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.
