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Legumes develop root nodules in symbiosis with nitrogen-fixing rhizobial bacteria. Rhizobia evoke cell division of differentiated cortical cells into root nodule primordia for accommodating bacterial symbionts. In this study, we show that NODULE INCEPTION (NIN), a transcription factor in Lotus japonicus that is essential for initiating cortical cell divisions during nodulation, regulates the gene ASYMMETRIC LEAVES 2-LIKE 18/LATERAL ORGAN BOUNDARIES DOMAIN 16a (ASL18/LBD16a). Orthologs of ASL18/LBD16a in nonlegume plants are required for lateral root development. Coexpression of ASL18a and the CCAAT box–binding protein Nuclear Factor-Y (NF-Y) subunits, which are also directly targeted by NIN, partially suppressed the nodulation-defective phenotype of L. japonicus daphne mutants, in which cortical expression of NIN was attenuated. Our results demonstrate that ASL18a and NF-Y together regulate nodule organogenesis. Thus, a lateral root developmental pathway is incorporated downstream of NIN to drive nodule symbiosis.

The ability of root cells to distinguish mutualistic microbes from pathogens is crucial for plants that allow symbiotic microorganisms to infect and colonize their internal root tissues. Here we show that Lotus japonicus and Medicago truncatula possess very similar LysM pattern-recognition receptors, LjLYS6/MtLYK9 and MtLYR4, enabling root cells to separate the perception of chitin oligomeric microbe-associated molecular patterns from the perception of lipochitin oligosaccharide by the LjNFR1/MtLYK3 and LjNFR5/MtNFP receptors triggering symbiosis. Inactivation of chitin-receptor genes in Ljlys6, Mtlyk9, and Mtlyr4 mutants eliminates early reactive oxygen species responses and induction of defense-response genes in roots. Ljlys6, Mtlyk9, and Mtlyr4 mutants were also more susceptible to fungal and bacterial pathogens, while infection and colonization by rhizobia and arbuscular mycorrhizal fungi was maintained. Biochemical binding studies with purified LjLYS6 ectodomains further showed that at least six GlcNAc moieties (CO6) are required for optimal binding efficiency. The 2.3-Å crystal structure of the LjLYS6 ectodomain reveals three LysM βααβ motifs similar to other LysM proteins and a conserved chitin-binding site. These results show that distinct receptor sets in legume roots respond to chitin and lipochitin oligosaccharides found in the heterogeneous mixture of chitinaceous compounds originating from soil microbes. This establishes a foundation for genetic and biochemical dissection of the perception and the downstream responses separating defense from symbiosis in the roots of the 80–90% of land plants able to develop rhizobial and/or mycorrhizal endosymbiosis.

Nitrogen fixation by legumes results from a symbiotic partnership between plant and microbes. These together elaborate nodules on the plant roots that house the bacteria. Tsikou et al. identified a microRNA made in the aboveground shoots of Lotus japonicus that translocates to the plant's roots. In the roots, the microRNA posttranscriptionally regulates a key suppressor of symbiosis, thus keeping the uninfected root susceptible to productive infection by symbiotic bacteria. Science , this issue p. 233 A microRNA made in the shoot regulates microbial nodulation in Lotus roots. Nitrogen-fixing root nodules on legumes result from two developmental processes, bacterial infection and nodule organogenesis. To balance symbiosis and plant growth, legume hosts restrict nodule numbers through an inducible autoregulatory process. Here, we present a mechanism where repression of a negative regulator ensures symbiotic susceptibility of uninfected roots of the host Lotus japonicus . We show that microRNA miR2111 undergoes shoot-to-root translocation to control rhizobial infection through posttranscriptional regulation of the symbiosis suppressor TOO MUCH LOVE in roots. miR2111 maintains a susceptible default status in uninfected hosts and functions as an activator of symbiosis downstream of LOTUS HISTIDINE KINASE1–mediated cytokinin perception in roots and HYPERNODULATION ABERRANT ROOT FORMATION1, a shoot factor in autoregulation. The miR2111- TML node ensures activation of feedback regulation to balance infection and nodulation events.

Arbuscular mycorrhiza (AM) symbioses contribute to global carbon cycles as plant hosts divert up to 20% of photosynthate to the obligate biotrophic fungi. Previous studies suggested carbohydrates as the only form of carbon transferred to the fungi. However, de novo fatty acid (FA) synthesis has not been observed in AM fungi in absence of the plant. In a forward genetic approach, we identified two Lotus japonicus mutants defective in AM-specific paralogs of lipid biosynthesis genes (KASI and GPAT6). These mutants perturb fungal development and accumulation of emblematic fungal 16:1ω5 FAs. Using isotopolog profiling we demonstrate that 13C patterns of fungal FAs recapitulate those of wild-type hosts, indicating cross-kingdom lipid transfer from plants to fungi. This transfer of labelled FAs was not observed for the AM-specific lipid biosynthesis mutants. Thus, growth and development of beneficial AM fungi is not only fueled by sugars but depends on lipid transfer from plant hosts. Most land plants are able to form partnerships with certain fungi – known as arbuscular mycorrhiza fungi – that live in the soil. These fungi supply the plant with mineral nutrients, especially phosphate and nitrogen, in return for receiving carbon-based food from the plant. To exchange nutrients, the fungi grow into the roots of the plant and form highly branched structures known as arbuscules inside plant cells. Due to the difficulties of studying this partnership, it has long been believed that plants only provide sugars to the fungus. However, it has recently been discovered that these fungi lack important genes required to make molecules known as fatty acids. Fatty acids are needed to make larger fat molecules that, among other things, store energy for the organism and form the membranes that surround each of its cells. Therefore, these results raise the possibility that the plant may provide the fungus with some of the fatty acids the fungus needs to grow. Keymer, Pimprikar et al. studied how arbuscules form in a plant known as Lotus japonicus, a close relative of peas and beans. The experiments identified a set of mutant L. japonicus plants that had problems forming arbuscules. These plants had mutations in several genes involved in fat production that are only active in plant cells containing arbuscules. Further experiments revealed that certain fat molecules that are found in fungi, but not plants, were present at much lower levels in samples from mutant plants colonized with the fungus, compared to samples from normal plants colonized with the fungus. This suggests that the fungi colonizing the mutant plants may be starved of fat molecules. Using a technique called stable isotope labelling it was possible to show that fatty acids made in normal plants can move into the colonizing fungus. The findings of Keymer, Pimprikar et al. provide evidence that the plant feeds the fungus not only with sugars but also with fat molecules. The next challenge will be to find out exactly how the fat molecules are transferred from the plant cell to the fungus. Many crop plants are able to form partnerships with arbuscular mycorrhizal fungi. Therefore, a better understanding of the role of fat molecules in these relationships may help to breed crop plants that, by providing more support to their fungal partner, may grow better in the field.

Roots of different plant species are colonized by bacterial communities, that are distinct even when hosts share the same habitat. It remains unclear to what extent the host actively selects these communities and whether commensals are adapted to a specific plant species. To address this question, we assembled a sequence-indexed bacterial culture collection from roots and nodules of Lotus japonicus that contains representatives of most species previously identified using metagenomics. We analysed taxonomically paired synthetic communities from L. japonicus and Arabidopsis thaliana in a multi-species gnotobiotic system and detected signatures of host preference among commensal bacteria in a community context, but not in mono-associations. Sequential inoculation experiments revealed priority effects during root microbiota assembly, where established communities are resilient to invasion by latecomers, and that host preference of commensal bacteria confers a competitive advantage in their cognate host. Our findings show that host preference in commensal bacteria from diverse taxonomic groups is associated with their invasiveness into standing root-associated communities. Host preferences of commensal bacteria in the root microbiota are revealed using systematic analyses of synthetic bacterial communities in a gnotobiotic system.

The biological interactions between plants and their root microbiomes are essential for plant growth, and even though plant genotype (G), soil microbiome (M), and growth conditions (environment; E) are the core factors shaping root microbiome, their relationships remain unclear. In this study, we investigated the effects of G, M, and E and their interactions on the Lotus root microbiome and plant growth using an in vitro cross-inoculation approach, which reconstructed the interactions between nine Lotus accessions and four soil microbiomes under two different environmental conditions. Results suggested that a large proportion of the root microbiome composition is determined by M and E, while G-related (G, G × M, and G × E) effects were significant but small. In contrast, the interaction between G and M had a more pronounced effect on plant shoot growth than M alone. Our findings also indicated that most microbiome variations controlled by M have little effect on plant phenotypes, whereas G × M interactions have more significant effects. Plant genotype-dependent interactions with soil microbes warrant more attention to optimize crop yield and resilience.

• Nodule microbiota are dominated by symbiotic nitrogen-fixing rhizobia, however, other non-rhizobial bacteria also colonise this niche. Although many of these bacteria harbour plant-growth-promoting functions, it is not clear whether these less abundant nodule colonisers impact root–nodule symbiosis. • We assessed the relationship between the nodule microbiome and nodulation as influenced by the soil microbiome, by using a metabarcoding approach to characterise the communities inside nodules of healthy and starved Lotus species. A machine learning algorithm and network analyses were used to identify nodule bacteria of interest, which were re-inoculated onto plants in controlled conditions to observe their potential functionality. • The nodule microbiome of all tested species differed according to inoculum, but only that of Lotus burttii varied with plant health. Amplicon sequence variants representative of Pseudomonas species were the most indicative non-rhizobial signatures inside healthy L. burttii nodules and negatively correlated with Rhizobium sequences. A representative Pseudomonas isolate co-colonised nodules infected with a beneficial Mesorhizobium, but not with an ineffective Rhizobium isolate and another even reduced the number of ineffective nodules induced on Lotus japonicus. • Our results show that nodule endophytes influence the overall outcome of the root–nodule symbiosis, albeit in a plant host-specific manner.

Establishment of arbuscular mycorrhizal interactions involves plant recognition of diffusible signals from the fungus, including lipochitooligosaccharides (LCOs) and chitooligosaccharides (COs). Nitrogen-fixing rhizobial bacteria that associate with leguminous plants also signal to their hosts via LCOs, the so-called Nod factors. Here, we have assessed the induction of symbiotic signaling by the arbuscular mycorrhizal (Myc) fungal-produced LCOs and COs in legumes and rice (Oryza sativa). We show that Myc-LCOs and tetra-acetyl chitotetraose (CO4) activate the common symbiosis signaling pathway, with resultant calcium oscillations in root epidermal cells of Medicago truncatula and Lotus japonicus. The nature of the calcium oscillations is similar for LCOs produced by rhizobial bacteria and by mycorrhizal fungi; however, Myc-LCOs activate distinct gene expression. Calcium oscillations were activated in rice atrichoblasts by CO4, but not the Myc-LCOs, whereas a mix of CO4 and Myc-LCOs activated calcium oscillations in rice trichoblasts. In contrast, stimulation of lateral root emergence occurred following treatment with Myc-LCOs, but not CO4, in M. truncatula, whereas both Myc-LCOs and CO4 were active in rice. Our work indicates that legumes and non-legumes differ in their perception of Myc-LCO and CO signals, suggesting that different plant species respond to different components in the mix of signals produced by arbuscular mycorrhizal fungi.

Bacterial accommodation inside living plant cells is restricted to the nitrogen-fixing root nodule symbiosis. In many legumes, bacterial uptake is mediated via tubular structures called infection threads (ITs). To identify plant genes required for successful symbiotic infection, we screened an ethyl methanesulfonate mutagenized population of Lotus japonicus for mutants defective in IT formation and cloned the responsible gene, ERN1, encoding an AP2/ERF transcription factor. We performed phenotypic analysis of two independent L. japonicus mutant alleles and investigated the regulation of ERN1 via transactivation and DNA-protein interaction assays. In ern1 mutant roots, nodule primordia formed, but most remained uninfected and bacterial entry via ITs into the root epidermis was abolished. Infected cortical nodule cells contained bacteroids, but transcellular ITs were rarely observed. A subset exhibited localized cell wall degradation and loss of cell integrity associated with bacteroid spread into neighbouring cells and the apoplast. Functional promoter studies revealed that CYCLOPS binds in a sequence-specific manner to a motif within the ERN1 promoter and in combination with CCaMK positively regulates ERN1 transcription. We conclude that the activation of ERN1 by CCaMK/CYCLOPS complex is an important step controlling IT-mediated bacterial progression into plant cells.

Bacterial infection of interior tissues of legume root nodules is controlled at the epidermal cell layer and is closely coordinated with progressing organ development. Using spontaneous nodulating Lotus japonicus plant mutants to uncouple nodule organogenesis from infection, we have determined the role of 16 genes in these two developmental processes. We show that host-encoded mechanisms control three alternative entry processes operating in the epidermis, the root cortex and at the single cell level. Single cell infection did not involve the formation of trans-cellular infection threads and was independent of host Nod-factor receptors and bacterial Nod-factor signals. In contrast, Nod-factor perception was required for epidermal root hair infection threads, whereas primary signal transduction genes preceding the secondary Ca 2+ oscillations have an indirect role. We provide support for the origin of rhizobial infection through direct intercellular epidermal invasion and subsequent evolution of crack entry and root hair invasions observed in most extant legumes. Plant and bacteria symbiosis in some species results in the coordinate formation of nitrogen fixing nodules and infection of the plant host. Using mutant Lotus japonicus plants, Madsen and colleagues have determined the role of 16 different genes in these two processes.