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Auxins can induce the formation of nodule-like structures (NLS) in plant roots even in the absence of rhizobia and nitrogen-fixing bacteria can colonize these structures. Interestingly, NLS can be induced in roots of both legumes and non-legumes. However, our understanding of NLS formation in non-legumes at a molecular level is limited. This study aims to investigate NLS formation at a developmental and molecular level in Brachypodium distachyon. We treated Brachypodium roots with the synthetic auxin, 2,4-D, to induce NLS at a high frequency (&gt; 80%) under controlled conditions. A broad base and a diffuse meristem characterized these structures. Next, we performed a comprehensive RNA-sequencing experiment to identify differentially expressed genes (DEGs) in Brachypodium roots during NLS formation. We identified 618 DEGs; several of which are promising candidates for control of NLS based on their biological and molecular functions. We validated the expression pattern of several genes via RT-PCR. Next, we compared the expression profile of Brachypodium roots with rice roots during NLS formation. We identified 76 single-copy ortholog pairs in rice and Brachypodium that are both differentially expressed during this process. Some of these genes are involved in auxin signaling, root development, and legume-rhizobia symbiosis. We established an experimental system to study NLS formation in Brachypodium at a developmental and genetic level, and used RNA-sequencing analysis to understand the molecular mechanisms controlling this root organogenesis program. Furthermore, our comparative transcriptome analysis in Brachypodium and rice identified a key set of genes for further investigating this genetic pathway in grasses.

Comparative transcriptome analysis provides key insights into gene expression pattern during the formation of nodule-like structures in Brachypodium