A receptor required for chitin perception facilitates arbuscular mycorrhizal associations and distinguishes root symbiosis from immunity.

Plants establish symbiotic associations with arbuscular mycorrhizal fungi (AMF) to facilitate nutrient uptake, particularly in nutrient-limited conditions. This partnership is rooted in the plant's ability to recognize fungal signaling molecules, such as chitooligosaccharides (chitin) and lipo-chitooligosaccharides. In the legume Medicago truncatula, chitooligosaccharides trigger both symbiotic and immune responses via the same lysin-motif-receptor-like kinases (LysM-RLKs), notably CERK1 and LYR4. The nature of plant-fungal engagement is opposite according to the outcomes of immunity or symbiosis signaling, and as such, discrimination is necessary, which is challenged by the dual roles of CERK1/LYR4 in both processes. Here, we describe a LysM-RLK, LYK8, that is functionally redundant with CERK1 for mycorrhizal colonization but is not involved in chitooligosaccharides-induced immunity. Genetic mutation of both LYK8 and CERK1 blocks chitooligosaccharides-triggered symbiosis signaling, as well as mycorrhizal colonization, but shows no further impact on immunity signaling triggered by chitooligosaccharides, compared with the mutation of CERK1 alone. LYK8 interacts with CERK1 and forms a receptor complex that appears essential for chitooligosaccharides activation of symbiosis signaling, with the lyk8/cerk1 double mutant recapitulating the impact of mutations in the symbiosis signaling pathway. We conclude that this novel receptor complex allows chitooligosaccharides activation specifically of symbiosis signaling and helps the plant to differentiate between activation of these opposing signaling processes.

CRISPR/Cas9 Vector Construction for Gene Knockout.

This protocol outlines the construction of a plant transformation plasmid to express both the Cas9 nuclease and individual guide RNA (gRNA), facilitating the induction of double-stranded breaks (DSBs) in DNA and subsequent imprecise repair via the non-homologous end-joining (NHEJ) pathway. The gRNA expression cassettes are assembled from three components. First, the Medicago truncatula U6.6 (MtU6) promoter (352 bp) and scaffold (83 bp) sequences are amplified from a pUC-based plasmid. Additionally, a third fragment, corresponding to the target sequence, is synthesized as an oligonucleotide. The three gRNA expression fragments are then loosely assembled in a ligation-free cloning reaction and used as a template for an additional PCR step to amplify a single gRNA expression construct, ready for assembly into the transformation vector. The benefits of this design include cost efficiency, as subsequent cloning reactions only require 59 oligonucleotides and standard cloning reagents. Researchers engaged in CRISPR/Cas9-mediated genome editing in plants will find this protocol a clear and resource-efficient approach to create transformation plasmids for their experiments.

Exploring the role of symbiotic modifier peptidases in the legume - rhizobium symbiosis.

Legumes can establish a mutual association with soil-derived nitrogen-fixing bacteria called 'rhizobia' forming lateral root organs called root nodules. Rhizobia inside the root nodules get transformed into 'bacteroids' that can fix atmospheric nitrogen to ammonia for host plants in return for nutrients and shelter. A substantial 200 million tons of nitrogen is fixed annually through biological nitrogen fixation. Consequently, the symbiotic mechanism of nitrogen fixation is utilized worldwide for sustainable agriculture and plays a crucial role in the Earth's ecosystem. The development of effective nitrogen-fixing symbiosis between legumes and rhizobia is very specialized and requires coordinated signaling. A plethora of plant-derived nodule-specific cysteine-rich (NCR or NCR-like) peptides get actively involved in this complex and tightly regulated signaling process of symbiosis between some legumes of the IRLC (Inverted Repeat-Lacking Clade) and Dalbergioid clades and nitrogen-fixing rhizobia. Recent progress has been made in identifying two such peptidases that actively prevent bacterial differentiation, leading to symbiotic incompatibility. In this review, we outlined the functions of NCRs and two nitrogen-fixing blocking peptidases: HrrP (host range restriction peptidase) and SapA (symbiosis-associated peptidase A). SapA was identified through an overexpression screen from the Sinorhizobium meliloti 1021 core genome, whereas HrrP is inherited extra-chromosomally. Interestingly, both peptidases affect the symbiotic outcome by degrading the NCR peptides generated from the host plants. These NCR-degrading peptidases can shed light on symbiotic incompatibility, helping to elucidate the reasons behind the inefficiency of nitrogen fixation observed in certain groups of rhizobia with specific legumes.

The GARP family transcription factor MtHHO3 negatively regulates salt tolerance in Medicago truncatula.

High salinity is one of the detrimental environmental factors restricting plant growth and crop production throughout the world. This study demonstrated that the GARP family transcription factor MtHHO3 is involved in response to salt stress and abscisic acid (ABA) signaling in Medicago truncatula. The transcription of MtHHO3 was repressed by salt, osmotic stress, and ABA treatment. The seed germination assay showed that, overexpression of MtHHO3 in Arabidopsis thaliana caused hypersensitivity to salt and osmotic stress, but increased resistance to ABA inhibition. Overexpression of MtHHO3 in M. truncatula resulted in decreased tolerance of salinity, while loss-of-function mutants mthho3-1 and mthho3-2 were more resistant to salt stress compared with wild-type plants. qRT-PCR analyses showed that MtHHO3 downregulated the expression of genes in stress and ABA responsive pathways. We further demonstrated that MtHHO3 repressed the transcription of the pathogenesis-related gene MtPR2 by binding to its promoter. Overall, these results indicate that MtHHO3 negatively regulates salt stress response in plants and deepen our understanding of the role of the GARP subfamily transcription factors in modulating salt stress and ABA signaling.

Autophagy and Symbiosis: Membranes, ER, and Speculations.

The interaction of plants and soil bacteria rhizobia leads to the formation of root nodule symbiosis. The intracellular form of rhizobia, the symbiosomes, are able to perform the nitrogen fixation by converting atmospheric dinitrogen into ammonia, which is available for plants. The symbiosis involves the resource sharing between two partners, but this exchange does not include equivalence, which can lead to resource scarcity and stress responses of one of the partners. In this review, we analyze the possible involvement of the autophagy pathway in the process of the maintenance of the nitrogen-fixing bacteria intracellular colony and the changes in the endomembrane system of the host cell. According to in silico expression analysis, ATG genes of all groups were expressed in the root nodule, and the expression was developmental zone dependent. The analysis of expression of genes involved in the response to carbon or nitrogen deficiency has shown a suboptimal access to sugars and nitrogen in the nodule tissue. The upregulation of several ER stress genes was also detected. Hence, the root nodule cells are under heavy bacterial infection, carbon deprivation, and insufficient nitrogen supply, making nodule cells prone to autophagy. We speculate that the membrane formation around the intracellular rhizobia may be quite similar to the phagophore formation, and the induction of autophagy and ER stress are essential to the success of this process.

The long noncoding RNA LAL contributes to salinity tolerance by modulating LHCB1s' expression in Medicago truncatula.

Long non-coding RNAs (lncRNAs) are abundant in plants, however, their regulatory roles remain unclear in most biological processes, such as response in salinity stress which is harm to plant production. Here we show a lncRNA in Medicago truncatula identified from salt-treated Medicago truncatula is important for salinity tolerance. We name the lncRNA LAL, LncRNA ANTISENSE to M. truncatula LIGHT-HARVESTING CHLOROPHYLL A/B BINDING (MtLHCB) genes. LAL is an antisense to four consecutive MtLHCB genes on chromosome 6. In salt-treated M. truncatula, LAL is suppressed in an early stage but induced later; this pattern is opposite to that of the four MtLHCBs. The lal mutants show enhanced salinity tolerance, while overexpressing LAL disrupts this superior tolerance in the lal background, which indicates its regulatory role in salinity response. The regulatory role of LAL on MtLHCB1.4 is further verified by transient co-expression of LAL and MtLHCB1.4-GFP in tobacco leaves, in which the cleavage of MtLHCB1.4 and production of secondary interfering RNA is identified. This work demonstrates a lncRNA, LAL, functioning as a regulator that fine-tunes salinity tolerance via regulating MtLHCB1s' expression in M. truncatula.

Genome assembly of Medicago truncatula accession SA27063 provides insight into spring black stem and leaf spot disease resistance.

Medicago truncatula, model legume and alfalfa relative, has served as an essential resource for advancing our understanding of legume physiology, functional genetics, and crop improvement traits. Necrotrophic fungus, Ascochyta medicaginicola, the causal agent of spring black stem (SBS) and leaf spot is a devasting foliar disease of alfalfa affecting stand survival, yield, and forage quality. Host resistance to SBS disease is poorly understood, and control methods rely on cultural practices. Resistance has been observed in M. truncatula accession SA27063 (HM078) with two recessively inherited quantitative-trait loci (QTL), rnpm1 and rnpm2, previously reported. To shed light on host resistance, we carried out a de novo genome assembly of HM078. The genome, referred to as MtHM078 v1.0, is comprised of 23 contigs totaling 481.19 Mbp. Notably, this assembly contains a substantial amount of novel centromere-related repeat sequences due to deep long-read sequencing. Genome annotation resulted in 98.4% of BUSCO fabales proteins being complete. The assembly enabled sequence-level analysis of rnpm1 and rnpm2 for gene content, synteny, and structural variation between SBS-resistant accession SA27063 (HM078) and SBS-susceptible accession A17 (HM101). Fourteen candidate genes were identified, and some have been implicated in resistance to necrotrophic fungi. Especially interesting candidates include loss-of-function events in HM078 because they fit the inverse gene-for-gene model, where resistance is recessively inherited. In rnpm1, these include a loss-of-function in a disease resistance gene due to a premature stop codon, and a 10.85 kbp retrotransposon-like insertion disrupting a ubiquitin conjugating E2. In rnpm2, we identified a frameshift mutation causing a loss-of-function in a glycosidase, as well as a missense and frameshift mutation altering an F-box family protein. This study generated a high-quality genome of HM078 and has identified promising candidates, that once validated, could be further studied in alfalfa to enhance disease resistance.

The single-cell transcriptome program of nodule development cellular lineages in Medicago truncatula.

Legumes establish a symbiotic relationship with nitrogen-fixing rhizobia by developing nodules. Nodules are modified lateral roots that undergo changes in their cellular development in response to bacteria, but the transcriptional reprogramming that occurs in these root cells remains largely uncharacterized. Here, we describe the cell-type-specific transcriptome response of Medicago truncatula roots to rhizobia during early nodule development in the wild-type genotype Jemalong A17, complemented with a hypernodulating mutant (sunn-4) to expand the cell population responding to infection and subsequent biological inferences. The analysis identifies epidermal root hair and stele sub-cell types associated with a symbiotic response to infection and regulation of nodule proliferation. Trajectory inference shows cortex-derived cell lineages differentiating to form the nodule primordia and, posteriorly, its meristem, while modulating the regulation of phytohormone-related genes. Gene regulatory analysis of the cell transcriptomes identifies new regulators of nodulation, including STYLISH 4, for which the function is validated.

VapC10 toxin of the legume symbiont Sinorhizobium meliloti targets tRNASer and controls intracellular lifestyle.

The soil bacterium Sinorhizobium meliloti can establish a nitrogen-fixing symbiosis with the model legume Medicago truncatula. The rhizobia induce the formation of a specialized root organ called nodule, where they differentiate into bacteroids and reduce atmospheric nitrogen into ammonia. Little is known on the mechanisms involved in nodule senescence onset and in bacteroid survival inside the infected plant cells. Although toxin-antitoxin (TA) systems have been shown to promote intracellular survival within host cells in human pathogenic bacteria, their role in symbiotic bacteria was rarely investigated. S. meliloti encodes several TA systems, mainly of the VapBC family. Here we present the functional characterization, through a multidisciplinary approach, of the VapBC10 TA system of S. meliloti. Following a mapping by overexpression of an RNase in Escherichia coli (MORE) RNA-seq analysis, we demonstrated that the VapC10 toxin is an RNase that cleaves the anticodon loop of two tRNASer. Thereafter, a bioinformatics approach was used to predict VapC10 targets in bacteroids. This analysis suggests that toxin activation triggers a specific proteome reprogramming that could limit nitrogen fixation capability and viability of bacteroids. Accordingly, a vapC10 mutant induces a delayed senescence in nodules, associated to an enhanced bacteroid survival. VapBC10 TA system could contribute to S. meliloti adaptation to symbiotic lifestyle, in response to plant nitrogen status.

The STF/WOX1 MD is required for physical interaction with MtWOX9 and leaf blade outgrowth in Medicago truncatula.

Plant-specific WUSCHEL-related homeobox (WOX) family transcription factors play critical roles in maintaining meristems and lateral organ development. The WUS clade member STF/LAM1 physically interacts with the intermediate clade member WOX9. This interaction contributes to their antagonistical functions on leaf blade outgrowth by competing for the same cis-elements in the promoter of their common target in M. truncatula and N. sylvestris. Here, we identified the main interaction domains of STF and MtWOX9 in Medicago, shedding light on the mechanism of WOX gene function. The middle domain of STF and MtWOX9 are both critical for the interaction, while the conserved motif of STF in the C-terminal domain is also required. Deletion of the middle domain of STF partially rescued the leaf blade phenotypes of the stf null mutant, indicating that the middle domain plays an essential role during leaf blade expansion. This finding provides a new insight that the versatility of WOX function is not only caused by the conserved DNA binding and repression domains but also by the middle domain that recruits different partners.

Light-sensitive short hypocotyl genes confer symbiotic nodule identity in the legume Medicago truncatula.

Legumes produce specialized root nodules that are distinct from lateral roots in morphology and function, with nodules intracellularly hosting nitrogen-fixing bacteria. We have previously shown that a lateral root program underpins nodule initiation, but there must be additional developmental regulators that confer nodule identity. Here, we show two members of the LIGHT-SENSITIVE SHORT HYPOCOTYL (LSH) transcription factor family, predominantly known to define shoot meristem complexity and organ boundaries, function as regulators of nodule organ identity. In parallel to the root initiation program, LSH1/LSH2 recruit a program into the root cortex that mediates the divergence into nodules, in particular with cell divisions in the mid-cortex. This includes regulation of auxin and cytokinin, promotion of NODULE ROOT1/2 and Nuclear Factor YA1, and suppression of the lateral root program. A principal outcome of LSH1/LSH2 function is the production of cells able to accommodate nitrogen-fixing bacteria, a key feature unique to nodules.

A genome-wide study of the lipoxygenase gene families in Medicago truncatula and Medicago sativa reveals that MtLOX24 participates in the methyl jasmonate response.

BACKGROUND: Lipoxygenase (LOX) is a multifunctional enzyme that is primarily related to plant organ growth and development, biotic and abiotic stress responses, and production of flavor-associated metabolites. In higher plants, the LOX family encompasses several isozymes with varying expression patterns between tissues and developmental stages. These affect processes including seed germination, seed storage, seedling growth, fruit ripening, and leaf senescence. LOX family genes have multiple functions in response to hormones such as methyl jasmonate (MeJA) and salicylic acid. RESULTS: In this study, we identified 30 and 95 LOX homologs in Medicago truncatula and Medicago sativa, respectively. These genes were characterized with analyses of their basic physical and chemical properties, structures, chromosomal distributions, and phylogenetic relationships to understand structural variations and their physical locations. Phylogenetic analysis was conducted for members of the three LOX subfamilies (9-LOX, type I 13-LOX, and type II 13-LOX) in Arabidopsis thaliana, Glycine max, M. truncatula, and M. sativa. Analysis of predicted promoter elements revealed several relevant cis-acting elements in MtLOX and MsLOX genes, including abscisic acid (ABA) response elements (ABREs), MeJA response elements (CGTCA-motifs), and antioxidant response elements (AREs). Cis-element data combined with transcriptomic data demonstrated that LOX gene family members in these species were most likely related to abiotic stress responses, hormone responses, and plant development. Gene expression patterns were confirmed via quantitative reverse transcription PCR. Several MtLOX genes (namely MtLOX15, MtLOX16, MtLOX20, and MtLOX24) belonging to the type I 13-LOX subfamily and other LOX genes (MtLOX7, MtLOX11, MsLOX23, MsLOX87, MsLOX90, and MsLOX94) showed significantly different expression levels in the flower tissue, suggesting roles in reproductive growth. Type I 13-LOXs (MtLOX16, MtLOX20, MtLOX21, MtLOX24, MsLOX57, MsLOX84, MsLOX85, and MsLOX94) and type II 13-LOXs (MtLOX5, MtLOX6, MtLOX9, MtLOX10, MsLOX18, MsLOX23, and MsLOX30) were MeJA-inducible and were predicted to function in the jasmonic acid signaling pathway. Furthermore, exogenous MtLOX24 expression in Arabidopsis verified that MtLOX24 was involved in MeJA responses, which may be related to insect-induced abiotic stress. CONCLUSIONS: We identified six and four LOX genes specifically expressed in the flowers of M. truncatula and M. sativa, respectively. Eight and seven LOX genes were induced by MeJA in M. truncatula and M. sativa, and the LOX genes identified were mainly distributed in the type I and type II 13-LOX subfamilies. MtLOX24 was up-regulated at 8 h after MeJA induction, and exogenous expression in Arabidopsis demonstrated that MtLOX24 promoted resistance to MeJA-induced stress. This study provides valuable new information regarding the evolutionary history and functions of LOX genes in the genus Medicago.

Nodulation Signaling Pathway 1 and 2 Modulate Vanadium Accumulation and Tolerance of Legumes.

Vanadium (V) pollution potentially threatens human health. Here, it is found that nsp1 and nsp2, Rhizobium symbiosis defective mutants of Medicago truncatula, are sensitive to V. Concentrations of phosphorus (P), iron (Fe), and sulfur (S) with V are negatively correlated in the shoots of wild-type R108, but not in mutant nsp1 and nsp2 shoots. Mutations in the P transporter PHT1, PHO1, and VPT families, Fe transporter IRT1, and S transporter SULTR1/3/4 family confer varying degrees of V tolerance on plants. Among these gene families, MtPT1, MtZIP6, MtZIP9, and MtSULTR1; 1 in R108 roots are significantly inhibited by V stress, while MtPHO1; 2, MtVPT2, and MtVPT3 are significantly induced. Overexpression of Arabidopsis thaliana VPT1 or M. truncatula MtVPT3 increases plant V tolerance. However, the response of these genes to V is weakened in nsp1 or nsp2 and influenced by soil microorganisms. Mutations in NSPs reduce rhizobacterial diversity under V stress and simplify the V-responsive operational taxonomic unit modules in co-occurrence networks. Furthermore, R108 recruits more beneficial rhizobacteria related to V, P, Fe, and S than does nsp1 or nsp2. Thus, NSPs can modulate the accumulation and tolerance of legumes to V through P, Fe, and S transporters, ion homeostasis, and rhizobacterial community responses.

Nodule-specific Cu+ -chaperone NCC1 is required for symbiotic nitrogen fixation in Medicago truncatula root nodules.

Cu+ -chaperones are a diverse group of proteins that allocate Cu+ ions to specific copper proteins, creating different copper pools targeted to specific physiological processes. Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper, for example for energy delivery via respiration, for which targeted copper deliver systems would be required. MtNCC1 is a nodule-specific Cu+ -chaperone encoded in the Medicago truncatula genome, with a N-terminus Atx1-like domain that can bind Cu+ with picomolar affinities. MtNCC1 is able to interact with nodule-specific Cu+ -importer MtCOPT1. MtNCC1 is expressed primarily from the late infection zone to the early fixation zone and is located in the cytosol, associated with plasma and symbiosome membranes, and within nuclei. Consistent with its key role in nitrogen fixation, ncc1 mutants have a severe reduction in nitrogenase activity and a 50% reduction in copper-dependent cytochrome c oxidase activity. A subset of the copper proteome is also affected in the ncc1 mutant nodules. Many of these proteins can be pulled down when using a Cu+ -loaded N-terminal MtNCC1 moiety as a bait, indicating a role in nodule copper homeostasis and in copper-dependent physiological processes. Overall, these data suggest a pleiotropic role of MtNCC1 in copper delivery for symbiotic nitrogen fixation.

The nitrate transporter-sensor MtNPF6.8 regulates the branched chain amino acid/pantothenate metabolic pathway in barrel medic (Medicago truncatula Gaertn.) root tip.

Nitrogen is the most limiting nutrient for plants, and it is preferentially absorbed in the form of nitrate by roots, which adapt to nitrate fluctuations by remodelling their architecture. Although core mechanisms of the response to nitrate availability are relatively well-known, signalling events controlling root growth and architecture have not all been identified, in particular in Legumes. However, the developmental effect of nitrate in Legumes is critical since external nitrate not only regulates root architecture but also N2-fixing nodule development. We have previously shown that in barrel medic (Medicago truncatula), the nitrate transporter MtNPF6.8 is required for nitrate sensitivity in root tip. However, uncertainty remains as to whether nitrogen metabolism itself is involved in the MtNPF6.8-mediated response. Here, we examine the metabolic effects of MtNPF6.8-dependent nitrate signalling using metabolomics and proteomics in WT and mtnpf6.8 root tips in presence or absence of nitrate. We found a reorchestration of metabolism due to the mutation, in favour of the branched chain amino acids/pantothenate metabolic pathway, and lipid catabolism via glyoxylate. That is, the mtnpf6.8 mutation was likely associated with a specific rerouting of acetyl-CoA production (glyoxylic cycle) and utilisation (pantothenate and branched chain amino acid synthesis). In agreement with our previous findings, class III peroxidases were confirmed as the main protein class responsive to nitrate, although in an MtNPF6.8-independent fashion. Our data rather suggest the involvement of other pathways within mtnpf6.8 root tips, such as Ca2+ signalling or cell wall methylation.

The Roles of the PSEUDO-RESPONSE REGULATORs in Circadian Clock and Flowering Time in Medicago truncatula.

PSEUDO-RESPONSE REGULATORs (PRRs) play key roles in the circadian rhythms and flowering in plants. Here, we identified the four members of the PRR family in Medicago truncatula, including MtPRR9a, MtPRR9b, MtPRR7 and MtPRR5, and isolated their Tnt1 retrotransposon-tagged mutants. They were expressed in different organs and were nuclear-localized. The four MtPRRs genes played important roles in normal clock rhythmicity maintenance by negatively regulating the expression of MtGI and MtLHY. Surprisingly, the four MtPRRs functioned redundantly in regulating flowering time under long-day conditions, and the quadruple mutant flowered earlier. Moreover, MtPRR can recruit the MtTPL/MtTPR corepressors and the other MtPRRs to form heterodimers to constitute the core mechanism of the circadian oscillator.

Genome-wide identification and characterization of filamentation temperature-sensitive H (FtsH) genes and expression analysis in response to multiple stresses in Medicago truncatula.

BACKGROUND: Filamentation temperature-sensitive H (FtsH) is an AAA+ ATP-dependent protease that plays a vital role in plant environmental adaption and tolerance. However, little is known about the function of the FtsH gene family in the most important legume model plant, Medicago truncatula. METHODS AND RESULTS: To identify and investigate the potential stress adaptation roles of FtsH gene family in M. truncatula, we conducted a series of genome-wide characterization and expression analyses. Totally, twenty MtFtsH genes were identified, which were unevenly distributed across eight chromosomes and classified into six evolution groups based on their phylogenetic relationships, with each group containing similar structures and motifs. Furthermore, MtFtsH genes exhibited a high degree of collinearity and homology with leguminous plants such as alfalfa and soybean. Multiple cis-elements in the upstream region of MtFtsH genes were also identified that responded to light, abiotic stress, and phytohormones. Public RNA-seq data indicated that MtFtsH genes were induced under both salt and drought stresses, and our transcript expression analysis showed that MtFtsH genes of MtFtsH1, MtFtsH2, MtFtsH4, MtFtsH9, and MtFtsH10 were up-regulated after ABA, H2O2, PEG, and NaCl treatments. These results suggest that MtFtsH genes may play a critical role in drought and high salt stress responses and the adaption processes of plants. CONCLUSIONS: This study provides a systematic analysis of FtsH gene family in M. truncatula, serving as a valuable molecular theoretical basis for future functional investigations. Our findings also extend the pool of potential candidate genes for the genetic improvement of abiotic stress tolerance in legume crops.

Translational Landscape of Medicago truncatula Seedlings under Salt Stress.

The expression of plant genes under salt stress at the transcriptional level has been extensively studied. However, less attention has been paid to gene translation regulation under salt stress. In this study, Ribo-seq and RNA-seq analyses were conducted in Medicago truncatula seedlings grown under normal and salt stress conditions. The results showed that salt stress significantly altered the gene expression at the transcriptional and translational levels, with 2755 genes showing significant changes only at the translational level. Salt stress significantly inhibited the gene translation efficiency. Small ORFs (including uORFs in the 5'UTR, dORFs in 3'UTRs, and sORFs in lncRNAs) were identified throughout the genome of M. truncatula. The efficiency of gene translation was simultaneously regulated by the uORFs, dORFs, and miRNAs. In summary, our results provide valuable information about translatomic resources and new insights into plant responses to salt stress.

The MIO1-MtKIX8 module regulates the organ size in Medicago truncatula.

Plant organ size is an important agronomic trait tightly related to crop yield. However, the molecular mechanisms underlying organ size regulation remain largely unexplored in legumes. We previously characterized a key regulator F-box protein MINI ORGAN1 (MIO1)/SMALL LEAF AND BUSHY1 (SLB1), which controls plant organ size in the model legume Medicago truncatula. In order to further dissect the molecular mechanism, MIO1 was used as the bait to screen its interacting proteins from a yeast library. Subsequently, a KIX protein, designated MtKIX8, was identified from the candidate list. The interaction between MIO1 and MtKIX8 was confirmed further by Y2H, BiFC, split-luciferase complementation and pull-down assays. Phylogenetic analyses indicated that MtKIX8 is highly homologous to Arabidopsis KIX8, which negatively regulates organ size. Moreover, loss-of-function of MtKIX8 led to enlarged leaves and seeds, while ectopic expression of MtKIX8 in Arabidopsis resulted in decreased cotyledon area and seed weight. Quantitative reverse-transcription PCR and in situ hybridization showed that MtKIX8 is expressed in most developing organs. We also found that MtKIX8 serves as a crucial molecular adaptor, facilitating interactions with BIG SEEDS1 (BS1) and MtTOPLESS (MtTPL) proteins in M. truncatula. Overall, our results suggest that the MIO1-MtKIX8 module plays a significant and conserved role in the regulation of plant organ size. This module could be a good target for molecular breeding in legume crops and forages.

Single-nucleus transcriptomes reveal spatiotemporal symbiotic perception and early response in Medicago.

Establishing legume-rhizobial symbiosis requires precise coordination of complex responses in a time- and cell type-specific manner. Encountering Rhizobium, rapid changes of gene expression levels in host plants occur in the first few hours, which prepare the plants to turn off defence and form a symbiotic relationship with the microbes. Here, we applied single-nucleus RNA sequencing to characterize the roots of Medicago truncatula at 30 min, 6 h and 24 h after nod factor treatment. We found drastic global gene expression reprogramming at 30 min in the epidermis and cortex and most of these changes were restored at 6 h. Moreover, plant defence response genes are activated at 30 min and subsequently suppressed at 6 h in non-meristem cells. Only in the cortical cells but not in other cell types, we found the flavonoid synthase genes required to recruit rhizobia are highly expressed 30 min after inoculation with nod factors. A gene module enriched for symbiotic nitrogen fixation genes showed that MtFER (MtFERONIA) and LYK3 (LysM domain receptor-like kinase 3) share similar responses to symbiotic signals. We further found that MtFER can be phosphorylated by LYK3 and it participates in rhizobial symbiosis. Our results expand our understanding of dynamic spatiotemporal symbiotic responses at the single-cell level.