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Abstract BackgroundLegumes utilize a long-distance signaling feedback pathway, termed Autoregulation of Nodulation (AON), to regulate the establishment and maintenance of their symbiosis with rhizobia. Several proteins key to this pathway have been discovered, but the AON pathway is not completely understood. ResultsWe report a new hypernodulating mutant,defective in autoregulation, with disruption of a gene,DAR(Medtr2g450550/MtrunA17_Chr2g0304631), previously unknown to play a role in AON. Thedar-1mutant produces ten-fold more nodules than wild type, similar to AON mutants with disruptedSUNNgene function. As insunnmutants, suppression of nodulation by CLE peptides MtCLE12 and MtCLE13 is abolished indar. Furthermore,dar-1also shows increased root length colonization by an arbuscular mycorrhizal fungus, suggesting a role for DAR in autoregulation of mycorrhizal symbiosis (AOM). However, unlikeSUNNwhich functions in the shoot to control nodulation,DARfunctions in the root. ConclusionsDARencodes a membrane protein that is a member of a small protein family inM. truncatula. Our results suggest that DAR could be involved in the subcellular transport of signals involved in symbiosis regulation, but it is not upregulated during symbiosis.DARgene family members are also present in Arabidopsis, lycophytes, mosses, and microalgae, suggesting the AON and AOM may use pathway components common to other plants, even those that do not undergo either symbiosis. 

Two symbiotic processes, nodulation and arbuscular mycorrhiza, are primarily controlled by the plant’s need for nitrogen (N) and phosphorus (P), respectively. Autoregulation of nodulation (AON) and autoregulation of mycorrhizal symbiosis (AOM) both negatively regulate their respective processes and share multiple components—plants that make too many nodules usually have higher arbuscular mycorrhiza (AM) fungal root colonization. The protein TML (TOO MUCH LOVE) was shown to function in roots to maintain susceptibly to rhizobial infection under low N conditions and control nodule number through AON inLotus japonicus.Medicago truncatulahas two sequence homologs:MtTML1 andMtTML2. We report the generation of stable single and double mutants harboring multiple allelic variations inMtTML1andMtTML2using CRISPR–Cas9 targeted mutagenesis and screening of a transposon mutagenesis library. Plants containing single mutations inMtTML1 orMtTML2 produced two to three times the nodules of wild-type plants, whereas plants containing mutations in both genes displayed a synergistic effect, forming 20× more nodules compared to wild-type plants. Examination of expression and heterozygote effects suggests that genetic compensation may play a role in the observed synergy. Plants with mutations in bothTMLs only showed mild increases in AM fungal root colonization at later timepoints in our experiments, suggesting that these genes may also play a minor role in AM symbiosis regulation. The mutants created will be useful tools to dissect the mechanism of synergistic action ofMtTML1 andMtTML2 inM. truncatulasymbiosis with beneficial microbes. 

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 . 

Legumes are of great interest for sustainable agricultural production as they fix atmospheric nitrogen to improve the soil. Medicago truncatula is a well-established model legume, and extensive studies in fundamental molecular, physiological, and developmental biology have been undertaken to translate into trait improvements in economically important legume crops worldwide. However, M. truncatula reference genome was generated in the accession Jemalong A17, which is highly recalcitrant to transformation. M. truncatula R108 is more attractive for genetic studies due to its high transformation efficiency and Tnt1-insertion population resource for functional genomics. The need to perform accurate synteny analysis and comprehensive genome-scale comparisons necessitates a chromosome-length genome assembly for M. truncatula cv. R108. Here, we performed in situ Hi-C (48×) to anchor, order, orient scaffolds, and correct misjoins of contigs in a previously published genome assembly (R108 v1.0), resulting in an improved genome assembly containing eight chromosome-length scaffolds that span 97.62% of the sequenced bases in the input assembly. The long-range physical information data generated using Hi-C allowed us to obtain a chromosome-length ordering of the genome assembly, better validate previous draft misjoins, and provide further insights accurately predicting synteny between A17 and R108 regions corresponding to the known chromosome 4/8 translocation. Furthermore, mapping the Tnt1 insertion landscape on this reference assembly presents an important resource for M. truncatula functional genomics by supporting efficient mutant gene identification in Tnt1 insertion lines. Our data provide a much-needed foundational resource that supports functional and molecular research into the Leguminosae for sustainable agriculture and feeding the future.