<scp>S</scp> l <scp>WRI</scp> 5a and <scp>S</scp> l <scp>HY</scp> 5 co‐activate <scp>S</scp> l <scp>F</scp> at <scp>M</scp> ‐mediated fatty acid biosynthesis during arbuscular mycorrhizal symbiosis in tomato

Arbuscular mycorrhizal symbiosis (AMS) is a ubiquitous mutualistic interaction between many terrestrial plants and fungi, with lipids playing a pivotal role in nutrient exchange. However, few genetic regulators of AMS have been functionally validated in tomato. To investigate candidate genes, we employed CRISPR-Cas9 and VIGS to generate knockout and knockdown lines. A comprehensive suite of molecular biology techniques, including yeast-1/2-hybridization, BiFC, ChIP-qPCR, and RNA-sequencing, was used to elucidate the regulatory roles of SlWRI5a, SlHY5, and SlFatM in fatty acid (FA) biosynthesis and AMS in tomato. FA composition was analyzed using gas chromatography. In this study, we validated SlWRI5a and SlFatM as key regulators of 16-carbon FA biosynthesis during AMS in tomato and demonstrated physical interactions between SlWRI5a and SlHY5. SlHY5 expression was induced by AMS and promoted root FA biosynthesis. Finally, we demonstrated that SlWRI5a and SlHY5 can co-regulate SlFatM-mediated FA accumulation, thereby influencing AMF colonization efficiency in tomato. Our findings reveal the SlWRI5a/SlHY5-SlFatM regulatory module, offering new insights into lipid-mediated AMS in tomato. This work also highlights a novel role for HY5 during fungal symbiosis, underscoring its broader significance in plant-microbe interactions.