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The unique evolutionary adaptation of legumes for nitrogen-fixing symbiosis leading to nodulation is tightly regulated by the host plant. The autoregulation of nodulation (AON) pathway negatively regulates the number of nodules formed in response to the carbon/nitrogen metabolic status of the shoot and root by long-distance signaling to and from the shoot and root. Central to AON signaling in the shoots ofMedicago truncatulais SUNN, a leucine-rich repeat receptor-like kinase with high sequence similarity with CLAVATA1 (CLV1), part of a class of receptors inArabidopsisinvolved in regulating stem cell populations in the root and shoot. This class of receptors inArabidopsisincludes the BARELY ANY MERISTEM family, which, like CLV1, binds to CLE peptides and interacts with CLV1 to regulate meristem development.M. truncatulacontains five members of theBAMfamily, but onlyMtBAM1andMtBAM2are highly expressed in the nodules 48 hours after inoculation. Plants carry mutations in individualMtBAMs, and several doubleBAMmutant combinations all displayed wild-type nodule number phenotypes. However,Mtbam2suppressed thesunn-5hypernodulation phenotype and partially rescued the short root length phenotype ofsunn-5 when present in asunn-5background. Grafting determined thatbam2suppresses supernodulation from the roots, regardless of theSUNNstatus of the root. Overexpression ofMtBAM2in wild-type plants increases nodule numbers, while overexpression ofMtBAM2in somesunnmutants rescues the hypernodulation phenotype, but not the hypernodulation phenotypes of AON mutantrdn1-2orcrn. Relative expression measurements of the nodule transcription factor MtWOX5 downstream of the putativebam2 sunn-5complex revealed disruption of meristem signaling; while bothbam2andbam2 sunn-5influenceMtWOX5expression, the expression changes are in different directions. We propose a genetic model wherein the specific root interactions of BAM2/SUNN are critical for signaling in nodule meristem cell homeostasis inM. truncatula. 

We report a public resource for examining the spatiotemporal RNA expression of 54,893 Medicago truncatula genes during the first 72 h of response to rhizobial inoculation. Using a methodology that allows synchronous inoculation and growth of more than 100 plants in a single media container, we harvested the same segment of each root responding to rhizobia in the initial inoculation over a time course, collected individual tissues from these segments with laser capture microdissection, and created and sequenced RNA libraries generated from these tissues. We demonstrate the utility of the resource by examining the expression patterns of a set of genes induced very early in nodule signaling, as well as two gene families (CLE peptides and nodule specific PLAT-domain proteins) and show that despite similar whole-root expression patterns, there are tissue differences in expression between the genes. Using a rhizobial response dataset generated from transcriptomics on intact root segments, we also examined differential temporal expression patterns and determined that, after nodule tissue, the epidermis and cortical cells contained the most temporally patterned genes. We circumscribed gene lists for each time and tissue examined and developed an expression pattern visualization tool. Finally, we explored transcriptomic differences between the inner cortical cells that become nodules and those that do not, confirming that the expression of 1-aminocyclopropane-1-carboxylate synthases distinguishes inner cortical cells that become nodules and provide and describe potential downstream genes involved in early nodule cell division. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .