Nitrogen-deprived legume plants form new root organs, the nodules, following a symbiosis with nitrogen-fixing rhizobial bacteria 1. Because this interaction is beneficial for the plant but has a high ...energetic cost, nodulation is tightly controlled by host plants through systemic pathways (acting at long distance) to promote or limit rhizobial infections and nodulation depending on earlier infections and on nitrogen availability 2. In the Medicago truncatula model legume, CLE12 (Clavata3/Embryo surrounding region 12) and CLE13 signaling peptides produced in nodulated roots act in shoots through the SUNN (Super Numeric Nodule) receptor to negatively regulate nodulation and therefore autoregulate nodule number 3–5. Conversely, CEP (C-terminally Encoded Peptide) signaling peptides produced in nitrogen-starved roots act in shoots through the CRA2 (Compact Root Architecture 2) receptor to promote nodulation already in the absence of rhizobia 6–9. We show in this study that a downstream shoot-to-root signaling effector of these systemic pathways is the shoot-produced miR2111 microRNA 10 that negatively regulates TML1 (Too Much Love 1) and TML2 11 transcripts accumulation in roots, ultimately promoting nodulation. Low nitrogen conditions and CEP1 signaling peptides induce in the absence of rhizobia the production of miR2111 depending on CRA2 activity in shoots, thus favoring root competence for nodulation. Together with the SUNN pathway negatively regulating the same miR2111 systemic effector when roots are nodulated, this allows a dynamic fine-tuning of the nodulation capacity of legume roots by nitrogen availability and rhizobial cues.
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•The miR2111 microRNA positively regulates root symbiotic nodulation•This regulation is systemic from shoots and relies on the CRA2 receptor•CRA2-related CEP1 peptides and low nitrogen conditions promote miR2111 expression•The CEP/CRA2 pathway favors root competence to nodulate under low nitrogen
Legumes form symbiotic nitrogen-fixing root nodules, whose number is controlled by systemic pathways acting from shoots. Gautrat et al. show that the miR2111 microRNA is a systemic effector acting at the crossroads of the SUNN-negative and CRA2-positive pathways, allowing a dynamic fine-tuning of nodulation by nitrogen and symbiotic rhizobia.
Abstract
Legumes tightly regulate nodule number to balance the cost of supporting symbiotic rhizobia with the benefits of nitrogen fixation. C-terminally Encoded Peptides (CEPs) and CLAVATA3-like ...(CLE) peptides positively and negatively regulate nodulation, respectively, through independent systemic pathways, but how these regulations are coordinated remains unknown. Here, we show that rhizobia, Nod Factors, and cytokinins induce a symbiosis-specific
CEP
gene,
MtCEP7
, which positively regulates rhizobial infection. Via grafting and split root studies, we reveal that MtCEP7 increases nodule number systemically through the MtCRA2 receptor.
MtCEP7
and
MtCLE13
expression in rhizobia-inoculated roots rely on the MtCRE1 cytokinin receptor and on the MtNIN transcription factor. MtNIN binds and transactivates
Mt
CEP7
and
MtCLE13
, and a NIN Binding Site (NBS) identified within the proximal
MtCEP7
promoter is required for its symbiotic activation. Overall, these results demonstrate that a cytokinin-MtCRE1-MtNIN regulatory module coordinates the expression of two antagonistic, symbiosis-related, peptide hormones from different families to fine-tune nodule number.
Legume plants form nitrogen (N)-fixing symbiotic nodules when mineral N is limiting in soils. As N fixation is energetically costly compared to mineral N acquisition, these N sources, and in ...particular nitrate, inhibit nodule formation and N fixation. Here, in the model legume Medicago truncatula, we characterized a CLAVATA3-like (CLE) signaling peptide, MtCLE35, the expression of which is upregulated locally by high-N environments and relies on the Nodule Inception-Like Protein (NLP) MtNLP1. MtCLE35 inhibits nodule formation by affecting rhizobial infections, depending on the Super Numeric Nodules (MtSUNN) receptor. In addition, high N or the ectopic expression of MtCLE35 represses the expression and accumulation of the miR2111 shoot-to-root systemic effector, thus inhibiting its positive effect on nodulation. Conversely, ectopic expression of miR2111 or downregulation of MtCLE35 by RNA interference increased miR2111 accumulation independently of the N environment, and thus partially bypasses the nodulation inhibitory action of nitrate. Overall, these results demonstrate that the MtNLP1-dependent, N-induced MtCLE35 signaling peptide acts through the MtSUNN receptor and the miR2111 systemic effector to inhibit nodulation.
A large range of microorganisms can associate with plants, resulting in neutral, friendly or hostile interactions. The ability of plants to recognize compatible and incompatible microorganisms and to ...limit or promote their colonization is therefore crucial for their survival. Elaborated communication networks determine the degree of association between the host plant and the invading microorganism. Central to these regulations of plant microbe interactions, phytohormones modulate microorganism plant associations and coordinate cellular and metabolic responses associated to the progression of microorganisms across different plant tissues. We review here hormonal regulations, focusing on auxin and cytokinin phytohormones, involved in the interactions between plant roots and soil microorganisms, including bacterial and fungi associations, either beneficial (symbiotic) or detrimental (pathogenic). The aim is to highlight similarities and differences in cytokinin/auxin functions amongst various compatible versus incompatible associations.
Legumes develop symbiotic interactions with rhizobial bacteria to form nitrogen-fixing nodules. Bacterial Nod factors (NFs) and plant regulatory pathways modulating NF signalling control rhizobial ...infections and nodulation efficiency. Here we show that gibberellin (GA) signalling mediated by DELLA proteins inhibits rhizobial infections and controls the NF induction of the infection marker ENOD11 in Medicago truncatula. Ectopic expression of a constitutively active DELLA protein in the epidermis is sufficient to promote ENOD11 expression in the absence of symbiotic signals. We show using heterologous systems that DELLA proteins can interact with the nodulation signalling pathway 2 (NSP2) and nuclear factor-YA1 (NF-YA1) transcription factors that are essential for the activation of NF responses. Furthermore, MtDELLA1 can bind the ERN1 (ERF required for nodulation 1) promoter and positively transactivate its expression. Overall, we propose that GA-dependent action of DELLA proteins may directly regulate the NSP1/NSP2 and NF-YA1 activation of ERN1 transcription to regulate rhizobial infections.
Phytohormonal interactions are essential to regulate plant organogenesis. In response to the presence of signals from symbiotic bacteria, the Nod factors, legume roots generate a new organ: the ...nitrogen-fixing nodule. Analysis of mutants in the Medicago truncatula CRE1 cytokinin receptor and of the MtRR4 cytokinin primary response gene expression pattern revealed that cytokinin acts in initial cortical cell divisions and later in the transition between meristematic and differentiation zones of the mature nodule. MtCRE1 signaling is required for activation of the downstream nodulation-related transcription factors MtERN1, MtNSP2 and MtNIN, as well as to regulate expression and accumulation of PIN auxin efflux carriers. Whereas the MtCRE1 pathway is required to allow the inhibition of polar auxin transport in response to rhizobia, nodulation is still negatively regulated by the MtEIN2/SICKLE-dependent ethylene pathway in cre1 mutants. Hence, MtCRE1 signaling acts as a regulatory knob, integrating positive plant and bacterial cues to control legume nodule organogenesis.
We investigated the role of KNOX genes in legume root nodule organogenesis. Class 1 KNOX homeodomain transcription factors (TFs) are involved in plant shoot development and leaf shape diversity. ...Class 2 KNOX genes are less characterized, even though an antagonistic function relative to class 1 KNOXs was recently proposed.
In silico expression data and further experimental validation identified in the Medicago truncatula model legume three class 2 KNOX genes, belonging to the KNAT3/4/5-like subclass (Mt KNAT3/4/5-like), as expressed during nodulation from early stages. RNA interference (RNAi)-mediated silencing and overexpression studies were used to unravel a function for KNOX TFs in nodule development.
Mt KNAT3/4/5-like genes encoded four highly homologous proteins showing overlapping expression patterns during nodule organogenesis, suggesting functional redundancy. Simultaneous reduction of Mt KNAT3/4/5-like genes indeed led to an increased formation of fused nodule organs, and decreased the expression of the MtEFD (Ethylene response Factor required for nodule Differentiation) TF and its direct target MtRR4, a cytokinin response gene.
Class 2 KNOX TFs therefore regulate legume nodule development, potentially through the MtEFD/MtRR4 cytokinin-related regulatory module, and may control nodule organ boundaries and shape like class 2 KNOX function in leaf development.
Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago ...truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.
Plant systemic signaling pathways allow the integration and coordination of shoot and root organ metabolism and development at the whole-plant level depending on nutrient availability. In legumes, ...two systemic pathways have been reported in the
model to regulate root nitrogen-fixing symbiotic nodulation. Both pathways involve leucine-rich repeat receptor-like kinases acting in shoots and proposed to perceive signaling peptides produced in roots depending on soil nutrient availability. In this study, we characterized in the
Jemalong A17 genotype a mutant allelic series affecting the Compact Root Architecture2 (CRA2) receptor. These analyses revealed that this pathway acts systemically from shoots to positively regulate nodulation and is required for the activity of carboxyl-terminally encoded peptides (CEPs). In addition, we generated a double mutant to test genetic interactions of the CRA2 systemic pathway with the CLAVATA3/EMBRYO SURROUNDING REGION peptide (CLE)/Super Numeric Nodule (SUNN) receptor systemic pathway negatively regulating nodule number from shoots, which revealed an intermediate nodule number phenotype close to the wild type. Finally, we showed that the nitrate inhibition of nodule numbers was observed in
mutants but not in
and
mutants. Overall, these results suggest that CEP/CRA2 and CLE/SUNN systemic pathways act independently from shoots to regulate nodule numbers.
We provide new insights into the autoregulation of nodulation that allow us to better understand how the legume Medicago truncatula restricts further nodulation once enough nodules have been formed.
...Abstract
The number of legume root nodules resulting from a symbiosis with rhizobia is tightly controlled by the plant. Certain members of the CLAVATA3/Embryo Surrounding Region (CLE) peptide family, specifically MtCLE12 and MtCLE13 in Medicago truncatula, act in the systemic autoregulation of nodulation (AON) pathway that negatively regulates the number of nodules. Little is known about the molecular pathways that operate downstream of the AON-related CLE peptides. Here, by means of a transcriptome analysis, we show that roots ectopically expressing MtCLE13 deregulate only a limited number of genes, including three down-regulated genes encoding lysin motif receptor-like kinases (LysM-RLKs), among which are the nodulation factor (NF) receptor NF Perception gene (NFP) and two up-regulated genes, MtTML1 and MtTML2, encoding Too Much Love (TML)-related Kelch-repeat containing F-box proteins. The observed deregulation was specific for the ectopic expression of nodulation-related MtCLE genes and depended on the Super Numeric Nodules (SUNN) AON RLK. Moreover, overexpression and silencing of these two MtTML genes demonstrated that they play a role in the negative regulation of nodule numbers. Hence, the identified MtTML genes are the functional counterpart of the Lotus japonicus TML gene shown to be central in the AON pathway. Additionally, we propose that the down-regulation of a subset of LysM-RLK-encoding genes, among which is NFP, might contribute to the restriction of further nodulation once the first nodules have been formed.
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