Acropetal auxin transport inhibition is involved in indeterminate but not determinate nodule formation

Legumes enter into a symbiotic relationship with nitrogen-fixing rhizobia, leading to nodule development. Two main types of nodules have been widely studied, indeterminate and determinate, which differ in the location of the first cell division in the root cortex, and persistency of the nodule meristem. Here, we compared the control of auxin transport, content, and response during the early stages of indeterminate and determinate nodule development in the model legumes Medicago truncatula and Lotus japonicus, respectively, to investigate whether differences in auxin transport control could explain the differences in the location of cortical cell divisions. While auxin responses were activated in dividing cortical cells during nodulation of both nodule types, auxin (indole-3-acetic acid) content at the nodule initiation site was transiently increased in M. truncatula, but transiently reduced in L. japonicus. Root acropetal auxin transport was reduced in M. truncatula at the very start of nodule initiation, in contrast to a prolonged increase in acropetal auxin transport in L. japonicus. The auxin transport inhibitors 2,3,5-triiodobenzoic acid and 1-N-naphthylphthalamic acid (NPA) only induced pseudonodules in legume species forming indeterminate nodules, but failed to elicit such structures in a range of species forming determinate nodules. The development of these pseudonodules in M. truncatula exhibited increased auxin responses in a small primordium formed from the pericycle, endodermis, and inner cortex, similar to rhizobia-induced nodule primordia. In contrast, a diffuse cortical auxin response and no associated cortical cell divisions were found in L. japonicus. Collectively, we hypothesize that a step of acropetal auxin transport inhibition is unique to the process of indeterminate nodule development, leading to auxin responses in pericycle, endodermis, and inner cortex cells, while increased auxin responses in outer cortex cells likely require a different mechanism during the formation of determinate nodules.This project was supported by a grant from the Australian Research Council (DP150102002 to UM)

Auxin transport, metabolism, and signalling during nodule initiation: indeterminate and determinate nodules

Most legumes can form a unique type of lateral organ on their roots: root nodules. These structures host symbiotic nitrogen-fixing bacteria called rhizobia. Several different types of nodules can be found in nature, but the two best studied types are called indeterminate and determinate nodules. These two types differ with respect to the presence or absence of a persistent nodule meristem, which consistently correlates with the cortical cell layers giving rise to the nodule primordia. Similar to other plant developmental processes, auxin signalling overlaps with the site of organ initiation and meristem activity. Here, we review how auxin contributes to early nodule development. We focus on changes in auxin transport, signalling, and metabolism during nodule initiation, describing both experimental evidence and computer modelling. We discuss how indeterminate and determinate nodules may differ in their mechanisms for generating localized auxin response maxima and highlight outstanding questions for future research

Auxin transport, metabolism, and signalling during nodule initiation: indeterminate and determinate nodules

Most legumes can form a unique type of lateral organ on their roots: root nodules. These structures host symbiotic nitrogen-fixing bacteria called rhizobia. Several different types of nodules can be found in nature, but the two best-studied types are called indeterminate and determinate nodules. These two types differ with respect to the presence or absence of a persistent nodule meristem, which consistently correlates with the cortical cell layers giving rise to the nodule primordia. Similar to other plant developmental processes, auxin signalling overlaps with the site of organ initiation and meristem activity. Here, we review how auxin contributes to early nodule development. We focus on changes in auxin transport, signalling, and metabolism during nodule initiation, describing both experimental evidence and computer modelling. We discuss how indeterminate and determinate nodules may differ in their mechanisms for generating localized auxin response maxima and highlight outstanding questions for future research

The 2HA line of Medicago truncatula has characteristics of an epigenetic mutant that is weakly ethylene insensitive

BACKGROUND The Medicago truncatula 2HA seed line is highly embryogenic while the parental line Jemalong rarely produces embryos. The 2HA line was developed from one of the rare Jemalong regenerates and this method for obtaining a highly regenerable genotype in M. truncatula is readily reproducible suggesting an epigenetic mechanism. Microarray transcriptomic analysis showed down regulation of an ETHYLENE INSENSITIVE 3-like gene in 2HA callus which provided an approach to investigating epigenetic regulation of genes related to ethylene signalling and the 2HA phenotype. Ethylene is involved in many developmental processes including somatic embryogenesis (SE) and is associated with stress responses. RESULTS Microarray transcriptomic analysis showed a significant number of up-regulated transcripts in 2HA tissue culture, including nodule and embryo specific genes and transposon-like genes, while only a few genes were down-regulated, including an EIN3-like gene we called MtEIL1. This reduced expression was associated with ethylene insensitivity of 2HA plants that was further investigated. The weak ethylene insensitivity affected root and nodule development. Sequencing of MtEIL1 found no difference between 2HA and wild-type plants. DNA methylation analysis of MtEIL1 revealed significant difference between 2HA and wild-type plants. Tiling arrays demonstrated an elevated level of miRNA in 2HA plants that hybridised to the antisense strand of the MtEIL1 gene. AFLP-like methylation profiling revealed more differences in DNA methylation between 2HA and wild-type. Segregation analysis demonstrated the recessive nature of the eil1 phenotype and the dominant nature of the SE trait. CONCLUSIONS We have demonstrated that EIL1 of Medicago truncatula (MtEIL1) is epigenetically silenced in the 2HA seed line. The possible cause is an elevated level of miRNA that targets its 3'UTR and is also associated with DNA methylation of MtEIL1. Down regulation of MtEIL1 makes it possible to form nodules in the presence of ethylene and affects root growth under normal conditions. Segregation analysis showed no association between MtEIL1 expression and SE in culture but the role and mechanism of ethylene signalling in the process of plant regeneration through SE requires further investigation. The work also suggests that epigenetic changes to a particular gene induced in culture can be fixed in regenerated plants.This work was funded by the Australian Research Council (CEO348212) through the ARC Centre of Excellence for Integrative Legume Research (CILR)

A Sinorhizobium meliloti-specific N-acyl homoserine lactone quorum-sensing signal increases nodule numbers in Medicago truncatula independent of autoregulation

N-acyl homoserine lactones (AHLs) act as quorum sensing signals that regulate cell-density dependent behaviors in many gram-negative bacteria, in particular those important for plant-microbe interactions. AHLs can also be recognized by plants, and this may influence their interactions with bacteria. Here we tested whether the exposure to AHLs affects the nodule-forming symbiosis between legume hosts and rhizobia. We treated roots of the model legume, Medicago truncatula, with a range of AHLs either from its specific symbiont, Sinorhizobium meliloti, or from the potential pathogens, Pseudomonas aeruginosa and Agrobacterium vitis. We found increased numbers of nodules formed on root systems treated with the S. meliloti-specific AHL, 3-oxo-C14-homoserine lactone, at a concentration of 1 μM, while the other AHLs did not result in significant changes to nodule numbers. We did not find any evidence for altered nodule invasion by the rhizobia. Quantification of flavonoids that could act as nod gene inducers in S. meliloti did not show any correlation with increased nodule numbers. The effects of AHLs were specific for an increase in nodule numbers, but not lateral root numbers or root length. Increased nodule numbers following 3-oxo-C14-homoserine lactone treatment were under control of autoregulation of nodulation and were still observed in the autoregulation mutant, sunn4 (super numeric nodules4). However, increases in nodule numbers by 3-oxo-C14-homoserine lactone were not found in the ethylene-insensitive sickle mutant. A comparison between M. truncatula with M. sativa (alfalfa) and Trifolium repens (white clover) showed that the observed effects of AHLs on nodule numbers were specific to M. truncatula, despite M. sativa nodulating with the same symbiont. We conclude that plant perception of the S. meliloti-specific 3-oxo-C14-homoserine lactone influences nodule numbers in M. truncatula via an ethylene-dependent, but autoregulation-independent mechanism.Debora F. Veliz-Vallejos was supported by a PhD scholarship from Becas Chile; Ulrike Mathesius was supported by a Future Fellowship (FT100100669) from the Australian Research Council

Infection of Medicago truncatula by the root-knot nematode Meloidogyne javanica does not require early nodulation genes

Because of the developmental similarities between root nodules induced by symbiotic rhizobia and root galls formed by parasitic nematodes, we investigated the involvement of nodulation genes in the infection of Medicago truncatula by the root knot nematode (RKN), Meloidogyne javanica. We found that gall formation, including giant cell formation, pericycle and cortical cell division, as well as egg laying, occurred successfully in the non-nodulating mutants nfp1 (nod factor perception1), nin1 (nodule inception1) and nsp2 (nodulation signaling pathway2) and the cytokinin perception mutant cre1 (cytokinin receptor1). Gall and egg formation were significantly reduced in the ethylene insensitive, hypernodulating mutant skl (sickle), and to a lesser extent, in the low nodulation, abscisic acid insensitive mutant latd/nip (lateral root-organ defective/numerous infections and polyphenolics). Despite its supernodulation phenotype, the sunn4 (super numeric nodules4) mutant, which has lost the ability to autoregulate nodule numbers, did not form excessive numbers of galls. Co-inoculation of roots with nematodes and rhizobia significantly reduced nodule numbers compared to rhizobia-only inoculated roots, but only in the hypernodulation mutant skl. Thus, this effect is likely to be influenced by ethylene signaling, but is not likely explained by resource competition between galls and nodules. Co-inoculation with rhizobia also reduced gall numbers compared to nematode-only infected roots, but only in the wild type. Therefore, the protective effect of rhizobia on nematode infection does not clearly depend on nodule number or on Nod factor signaling. Our study demonstrates that early nodulation genes that are essential for successful nodule development are not necessary for nematode-induced gall formation, that gall formation is not under autoregulation of nodulation control, and that ethylene signaling plays a positive role in successful RKN parasitism in M. truncatula.SRC was funded by a post-doc grant (SFRH/BPD/26496/2006) supported by the Portuguese Foundation for Science and Technology (FCT)

Editorial: Evolution of Signaling in Plant Symbioses

Plants are surrounded by microbes, but only a small number of microbes have evolved an intimate, endosymbiotic association, in which they live inside host cells. Root symbioses are important sources of nutrition for plants and microbes alike, with over 80% of all terrestrial plants forming intracellular symbioses with arbuscular mycorrhizal fungi

The presence of plant-associated bacteria alters responses to N-acyl homoserine lactone quorum sensing signals that modulate nodulation

Bacteria use quorum sensing signaling for cell-to-cell communication, which is also important for their interactions with plant hosts. Quorum sensing via N-acyl-homoserine lactones (AHLs) is important for successful symbioses between legumes and nitrogen-fixing rhizobia. Previous studies have shown that plant hosts can recognize and respond to AHLs. Here, we tested whether the response of the model legume Medicago truncatula to AHLs from its symbiont and other bacteria could be modulated by the abundance and composition of plant-associated microbial communities. Temporary antibiotic treatment of the seeds removed the majority of bacterial taxa associated with M. truncatula roots and significantly altered the effect of AHLs on nodule numbers, but lateral root density, biomass, and root length responses were much less affected. The AHL 3-oxo-C14-HSL (homoserine lactone) specifically increased nodule numbers but only after the treatment of seeds with antibiotics. This increase was associated with increased expression of the early nodulation genes RIP1 and ENOD11 at 24 h after infection. A 454 pyrosequencing analysis of the plant-associated bacteria showed that antibiotic treatment had the biggest effect on bacterial community composition. However, we also found distinct effects of 3-oxo-C14-HSL on the abundance of specific bacterial taxa. Our results revealed a complex interaction between plants and their associated microbiome that could modify plant responses to AHLs

Nodulating Legumes Are Distinguished by a Sensitivity to Cytokinin in the Root Cortex Leading to Pseudonodule Development

Root nodule symbiosis (RNS) is a feature confined to a single clade of plants, the Fabids. Among Fabids capable of RNS, legumes form root cortex-based nodules in symbioses with rhizobia, while actinorhizal species form lateral root-based nodules with actinomycetes. Cytokinin has previously been shown to be sufficient for “pseudonodule” initiation in model legumes. Here, we tested whether this response correlates with the ability to nodulate across a range of plant species. We analyzed the formation of pseudonodules in 17 nodulating and non-nodulating legume species, and 11 non-legumes, including nodulating actinorhizal species, using light and fluorescence microscopy. Cytokinin-induced pseudonodules arising from cortical cell divisions occurred in all nodulating legume species, but not in any of the other species, including non-nodulating legumes. Pseudonodule formation was dependent on the CRE1 cytokinin receptor in Medicago truncatula. Inhibition of root growth by cytokinin occurred across plant groups, indicating that pseudonodule development is the result of a specific cortical cytokinin response unique to nodulating legumes. Lack of a cortical cytokinin response from the Arabidopsis thaliana cytokinin reporter TCSn::GFP supported this hypothesis. Our results suggest that the ability to form cortical cell-derived nodules was gained in nodulating legumes, and likely lost in non-nodulating legumes, due to a specific root cortical response to cytokinin

Bioinformatic analysis of the CLE signaling peptide family

Background. Plants encode a large number of leucine-rich repeat receptor-like kinases. Legumes encode several LRR-RLK linked to the process of root nodule formation, the ligands of which are unknown. To identify ligands for these receptors, we used a combination of profile hidden Markov models and position-specific iterative BLAST, allowing us to detect new members of the CLV3/ESR (CLE) protein family from publicly available sequence databases. Results. We identified 114 new members of the CLE protein family from various plant species, as well as five protein sequences containing multiple CLE domains. We were able to cluster the CLE domain proteins into 13 distinct groups based on their pairwise similarities in the primary CLE motif. In addition, we identified secondary motifs that coincide with our sequence clusters. The groupings based on the CLE motifs correlate with known biological functions of CLE signaling peptides and are analogous to groupings based on phylogenetic analysis and ectopic overexpression studies. We tested the biological function of two of the predicted CLE signaling peptides in the legume Medicago truncatula. These peptides inhibit the activity of the root apical and lateral root meristems in a manner consistent with our functional predictions based on other CLE signaling peptides clustering in the same groups. Conclusion. Our analysis provides an identification and classification of a large number of novel potential CLE signaling peptides. The additional motifs we found could lead to future discovery of recognition sites for processing peptidases as well as predictions for receptor binding specificity