Rhizobia secrete nodulation (Nod) factors, which set in motion the formation of nitrogen-fixing root nodules on legume host plants. Nod factors induce several cellular responses in root hair cells ...within minutes, but also are essential for the formation of infection threads by which rhizobia enter the root. Based on studies using bacterial mutants, a two-receptor model was proposed, a signaling receptor that induces early responses with low requirements toward Nod factor structure and an entry receptor that controls infection with more stringent demands. Recently, putative Nod factor receptors were shown to be LysM domain receptor kinases. However, mutants in these receptors, in both Lotus japonicus (nfr1 and nfr5) and Medicago truncatula (Medicago; nfp), do not support the two-receptor model because they lack all Nod factor-induced responses. LYK3, the putative Medicago ortholog of NFR1, has only been studied by RNA interference, showing a role in infection thread formation. Medicago hair curling (hcl) mutants are unable to form curled root hairs, a step preceding infection thread formation. We identified the weak hcl-4 allele that is blocked during infection thread growth. We show that HCL encodes LYK3 and, thus, that this receptor, besides infection, also controls root hair curling. By using rhizobial mutants, we also show that HCL controls infection thread formation in a Nod factor structure-dependent manner. Therefore, LYK3 functions as the proposed entry receptor, specifically controlling infection. Finally, we show that LYK3, which regulates a subset of Nod factor-induced genes, is not required for the induction of NODULE INCEPTION.
The root nodule symbiosis between plants and nitrogen-fixing bacteria is a fascinating trait limited to several plant species. Given the agronomic potential of transferring this symbiosis to ...nonleguminous crops, the symbiosis has attracted researchers’ attention for over a century. The origins of this symbiosis can be traced back to a single ancestor, around 110 million years ago. Recent findings have uncovered that adaptations in a receptor complex and the recruitment of the transcription factor Nodule Inception (NIN) are among the first genetic adaptations that allowed this ancestor to respond to its microsymbiont. Understanding the consequences of recruiting these genes provides insights into the start of this complex genetic trait.
Red clover (Trifolium pratense L.) is a globally significant forage legume in pastoral livestock farming systems. It is an attractive component of grassland farming, because of its high yield and ...protein content, nutritional value and ability to fix atmospheric nitrogen. Enhancing its role further in sustainable agriculture requires genetic improvement of persistency, disease resistance, and tolerance to grazing. To help address these challenges, we have assembled a chromosome-scale reference genome for red clover. We observed large blocks of conserved synteny with Medicago truncatula and estimated that the two species diverged ~23 million years ago. Among the 40,868 annotated genes, we identified gene clusters involved in biochemical pathways of importance for forage quality and livestock nutrition. Genotyping by sequencing of a synthetic population of 86 genotypes show that the number of markers required for genomics-based breeding approaches is tractable, making red clover a suitable candidate for association studies and genomic selection.
Legumes host their Rhizobium spp. symbiont in novel root organs called nodules. Nodules originate from differentiated root cortical cells that dedifferentiate and subsequently form nodule primordia, ...a process controlled by cytokinin. A whole-genome duplication has occurred at the root of the legume Papilionoideae subfamily. We hypothesize that gene pairs originating from this duplication event and are conserved in distinct Papilionoideae lineages have evolved symbiotic functions. A phylogenetic strategy was applied to search for such gene pairs to identify novel regulators of nodulation, using the cytokinin phosphorelay pathway as a test case. In this way, two paralogous type-A cytokinin response regulators were identified that are involved in root nodule symbiosis. Response Regulator9 (MtRR9) and MtRR11 in medicago (Medicago truncatula) and an ortholog in lotus (Lotus japonicus) are rapidly induced upon Rhizobium spp. Nod factor signaling. Constitutive expression of MtRR9 results in arrested primordia that have emerged from cortical, endodermal, and pericycle cells. In legumes, lateral root primordia are not exclusively formed from pericycle cells but also require the involvement of the root cortical cell layer. Therefore, the MtRR9-induced foci of cell divisions show a strong resemblance to lateral root primordia, suggesting an ancestral function of MtRR9 in this process. Together, these findings provide a proof of principle for the applied phylogenetic strategy to identify genes with a symbiotic function in legumes.
Nitrogen-fixing nodules occur in ten related taxonomic lineages interspersed with lineages of non-nodulating plant species. Nodules result from an endosymbiosis between plants and diazotrophic ...bacteria; rhizobia in the case of legumes and Parasponia and Frankia in the case of actinorhizal species. Nodulating plants share a conserved set of symbiosis genes, whereas related non-nodulating sister species show pseudogenization of several key nodulation-specific genes. Signalling and cellular mechanisms critical for nodulation have been co-opted from the more ancient plant-fungal arbuscular endomycorrhizal symbiosis. Studies in legumes and actinorhizal plants uncovered a key component in symbiotic signalling, the LRR-type SYMBIOSIS RECEPTOR KINASE (SYMRK). SYMRK is essential for nodulation and arbuscular endomycorrhizal symbiosis. To our surprise, however, despite its arbuscular endomycorrhizal symbiosis capacities, we observed a seemingly critical mutation in a donor splice site in the SYMRK gene of Trema orientalis, the non-nodulating sister species of Parasponia. This led us to investigate the symbiotic functioning of SYMRK in the Trema-Parasponia lineage and to address the question of to what extent a single nucleotide polymorphism in a donor splice site affects the symbiotic functioning of SYMRK. We show that SYMRK is essential for nodulation and endomycorrhization in Parasponia andersonii. Subsequently, it is revealed that the 5'-intron donor splice site of SYMRK intron 12 is variable and, in most dicotyledon species, doesn't contain the canonical dinucleotide 'GT' signature but the much less common motif 'GC'. Strikingly, in T. orientalis, this motif is converted into a rare non-canonical 5'-intron donor splice site 'GA'. This SYMRK allele, however, is fully functional and spreads in the T. orientalis population of Malaysian Borneo. A further investigation into the occurrence of the non-canonical GA-AG splice sites confirmed that these are extremely rare. SYMRK functioning is highly conserved in legumes, actinorhizal plants, and Parasponia. The gene possesses a non-common 5'-intron GC donor splice site in intron 12, which is converted into a GA in T. orientalis accessions of Malaysian Borneo. The discovery of this functional GA-AG splice site in SYMRK highlights a gap in our understanding of splice donor sites.
Diazotrophic Bradyrhizobium spp. are well known for their ability to trigger nodule formation on a variety of legume species. In nodules, Bradyrhizobium utilizes plant-derived carbohydrates in ...exchange for fixed nitrogen. The genes essential for the nodulation and nitrogen-fixation trait are clustered in a genomic region, which is known as the 'symbiotic island'. Recently, novel non-diazotrophic Bradyrhizobium spp. have been found to be highly abundant in soils, suggesting that these species can also have a 'free-living' life history. However, whether non-diazotrophic Bradyrhizobium spp. can live in association with plants remains elusive.
In this study, we show that Bradyrhizobium spp. are common root endophytes of non-legume plant species - including Arabidopsis thaliana (Arabidopsis) - grown in an ecological setting. From a single Arabidopsis root, four Bradyrhizobium sp. strains (designated MOS001 to MOS004) were isolated. Comparative genome analysis revealed that these strains were genetically and functionally highly diverse, but did not harbour the nodulation and the nitrogen fixation gene clusters. Comparative colonization experiments, with MOS strains and nitrogen-fixing symbiotic strains, revealed that all tested Bradyrhizobium spp. can colonize the root endophytic compartment of Arabidopsis.
This study provides evidence that both diazotrophic and non-diazotrophic Bradyrhizobium spp. colonize the root endophytic compartment of a wide variety of plant species, including the model species Arabidopsis. This demonstrates that plant roots form a major ecological niche for Bradyrhizobium spp., which might be ancestral to the evolution of the nodulation and nitrogen-fixation trait in this genus.
Plants that form root-nodule symbioses are within a monophyletic ‘nitrogen-fixing’ clade and associated signalling processes are shared with the arbuscular mycorrhizal symbiosis. Central to symbiotic ...signalling are nuclear-associated oscillations in calcium ions (Ca2+), occurring in the root hairs of several legume species in response to the rhizobial Nod factor signal.
In this study we expanded the species analysed for activation of Ca2+ oscillations, including nonleguminous species within the nitrogen-fixing clade.
We showed that Ca2+ oscillations are a common feature of legumes in their association with rhizobia, while Cercis, a non-nodulating legume, does not show Ca2+ oscillations in response to Nod factors from Sinorhizobium fredii NGR234. Parasponia andersonii, a nonlegume that can associate with rhizobia, showed Nod factor-induced calcium oscillations to S. fredii NGR234 Nod factors, but its non-nodulating sister species, Trema tomentosa, did not. Also within the nitrogen-fixing clade are actinorhizal species that associate with Frankia bacteria and we showed that Alnus glutinosa induces Ca2+ oscillations in root hairs in response to exudates from Frankia alni, but not to S. fredii NGR234 Nod factors.
We conclude that the ability to mount Ca2+ oscillations in response to symbiotic bacteria is a common feature of nodulating species within the nitrogen-fixing clade.
Most eukaryotic centromeres contain long arrays of tandem repeats, with unit lengths of 150-300 bp. We searched for such repeats in the functional centromeres of the model legume Medicago truncatula ...(Medicago) accession Jemalong A17. To this end three repeats, MtR1, MtR2 and MtR3, were identified in 20 Mb of a low-pass, whole genome sequencing data set generated by a random shotgun approach. The nucleotide sequence composition, genomic organization and abundance of these repeats were characterized. Fluorescent in situ hybridization of these repeats on chromosomes at meiosis I showed that only the MtR3 repeat, encompassing stretches of 450 kb to more than 1.0 Mb, is located in the functional portion of all eight centromeres. MtR1 and MtR2 occupy distinct regions in pericentromeric heterochromatin. We also studied the presence and distribution of MtRs in Medicago accession R108-1, a genotype with a genome that is 20% smaller than that of Jemalong A17. We determined that while MtR3 is also centromeric on all pachytene bivalents in R108-1, MtR1 and MtR2 are not present in the R108 genome.
Summary
We report here that the pea early nodulin genes PsENOD5 and PsENOD12A are induced during the interaction of pea roots and the endomycorrhizal fungus Gigaspora margarita. Using the pea ...nodulation mutant Sparkle‐R25, which is mutated in SYM8, it is shown that SYM8 is essential for the induction of PsENOD5 and PsENOD12Ain pea roots interacting either with Rhizobium or the endomycorrhizal fungus Gigaspora margarita. Our results suggest that mycorrhizal signals activate a signal transduction cascade sharing at least one common step with the Nod factor‐activated signal transduction cascade.
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