CLAVATA3/EMBRYO SURROUNDING REGION Research Articles

The LaCLE35 peptide modifies rootlet density and length in cluster roots of white lupin.

White lupin (lupinus albus L.) forms special bottlebrush-like root structures called cluster roots (CR) when phosphorus is low, to remobilise sparingly soluble phosphates in the soil. The molecular mechanisms that control the CR formation remain unknown. Root development in other plants is regulated by CLE (CLAVATA3/ EMBRYO SURROUNDING REGION (ESR)-RELATED) peptides, which provide more precise control mechanisms than common phytohormones. This makes these peptides interesting candidates to be involved in CR formation, where fine tuning to environmental factors is required. In this study we present an analysis of CLE peptides in white lupin. The peptides LaCLE35 (RGVHy PSGANPLHN) and LaCLE55 (RRVHy PSCHy PDPLHN) reduced root growth and altered CR in hydroponically cultured white lupins. We demonstrate that rootlet density and rootlet length were locally, but not systemically, impaired by exogenously applied CLE35. The peptide was identified in the xylem sap. The inhibitory effect of CLE35 on root growth was attributed to arrested cell elongation in root tips. Taken together, CLE peptides affect both rootlet density and rootlet length, which are two critical factors for CR formation, and may be involved in fine tuning this peculiar root structure that is present in a few crops and many Proteaceae species, under low phosphorus availability.

CRISPR/Cas9-Mediated Knock-Out of the MtCLE35 Gene Highlights Its Key Role in the Control of Symbiotic Nodule Numbers under High-Nitrate Conditions

Legume plants have the ability to establish a symbiotic relationship with soil bacteria known as rhizobia. The legume–rhizobium symbiosis results in the formation of symbiotic root nodules, where rhizobia fix atmospheric nitrogen. A host plant controls the number of symbiotic nodules to meet its nitrogen demands. CLE (CLAVATA3/EMBRYO SURROUNDING REGION) peptides produced in the root in response to rhizobial inoculation and/or nitrate have been shown to control the number of symbiotic nodules. Previously, the MtCLE35 gene was found to be upregulated by rhizobia and nitrate treatment in Medicago truncatula, which systemically inhibited nodulation when overexpressed. In this study, we obtained several knock-out lines in which the MtCLE35 gene was mutated using the CRISPR/Cas9-mediated system. M. truncatula lines with the MtCLE35 gene knocked out produced increased numbers of nodules in the presence of nitrate in comparison to wild-type plants. Moreover, in the presence of nitrate, the expression levels of two other nodulation-related MtCLE genes, MtCLE12 and MtCLE13, were reduced in rhizobia-inoculated roots, whereas no significant difference in MtCLE35 gene expression was observed between nitrate-treated and rhizobia-inoculated control roots. Together, these findings suggest the key role of MtCLE35 in the number of nodule numbers under high-nitrate conditions, under which the expression levels of other nodulation-related MtCLE genes are reduced.

Genome-Wide Identification and Characterization of CLAVATA3/EMBRYO SURROUNDING REGION (CLE) Gene Family in Foxtail Millet (Setaria italica L.).

The CLAVATA3/EMBRYO-SURROUNDING REGION (CLE) genes encode signaling peptides that play important roles in various developmental and physiological processes. However, the systematic identification and characterization of CLE genes in foxtail millet (Setaria italica L.) remain limited. In this study, we identified and characterized 41 SiCLE genes in the foxtail millet genome. These genes were distributed across nine chromosomes and classified into four groups, with five pairs resulting from gene duplication events. SiCLE genes within the same phylogenetic group shared similar gene structure and motif patterns, while 34 genes were found to be single-exon genes. All SiCLE peptides harbored the conserved C-terminal CLE domain, with highly conserved positions in the CLE core sequences shared among foxtail millet, Arabidopsis, rice, and maize. The SiCLE genes contained various cis-elements, including five plant hormone-responsive elements. Notably, 34 SiCLE genes possessed more than three types of phytohormone-responsive elements on their promoters. Comparative analysis revealed higher collinearity between CLE genes in maize and foxtail millet, which may be because they are both C4 plants. Tissue-specific expression patterns were observed, with genes within the same group exhibiting similar and specific expression profiles. SiCLE32 and SiCLE41, classified in Group D, displayed relatively high expression levels in all tissues except panicles. Most SiCLE genes exhibited low expression levels in young panicles, while SiCLE6, SiCLE24, SiCLE25, and SiCLE34 showed higher expression in young panicles, with SiCLE24 down-regulated during later panicle development. Greater numbers of SiCLE genes exhibited higher expression in roots, with SiCLE7, SiCLE22, and SiCLE36 showing the highest levels and SiCLE36 significantly down-regulated after abscisic acid (ABA) treatment. Following treatments with ABA, 6-benzylaminopurine (6-BA), and gibberellic acid 3 (GA3), most SiCLE genes displayed down-regulation followed by subsequent recovery, while jasmonic acid (JA) and indole-3-acetic acid (IAA) treatments led to upregulation at 30 min in leaves. Moreover, identical hormone treatments elicited different expression patterns of the same genes in leaves and stems. This comprehensive study enhances our understanding of the SiCLE gene family and provides a foundation for further investigations into the functions and evolution of SiCLE genes in foxtail millet.

Systemic control of symbiotic nodulation in legume plants: genetic engineering in functional studies of key regulators

Legumes are important suppliers of vegetable protein. In symbiotic interactions with soil bacteria rhizobia, legume plants form nitrogen-fixing nodules on their roots, where molecular nitrogen is fixed and incorporated into organic compounds. A host plant controls the number of symbiotic nodules to meet its nitrogen demands. The presence of high amount of nitrate in the soil suppresses the formation of nodules. CLE (CLAVATA3/EMBRYO SURROUNDING REGION) peptides produced in the root in response to rhizobial inoculation and/or nitrate were shown to control the number of symbiotic nodules. Previously, we have identified the MtCLE35 gene upregulated by the rhizobia and the nitrate treatment in Medicago truncatula, which systemically inhibited nodulation when overexpressed. Using genetic engineering approaches we increased its transcriptional activity in transgenic roots, which almost completely prevented the formation of symbiotic nodules. Moreover, we obtained stable transgenic lines overproducing the MtCLE35 peptide, and found that their had lower shoot and root biomass in comparison to the wild-type plants. The study of metabolome of MtCLE35-overproducing plants revealed the increased level of amino acids in the roots, suggesting the stimulating effect of MtCLE35 on amino acid synthetic pathways.
 In addition, we obtained several knock-out lines, where the MtCLE35 gene was edited using the CRISPR/Cas9-mediated system. The detailed analysis of these plants will allow us to understand the mechanisms of MtCLE35 action in the regulation of plant nitrogen status and symbiotic interaction with rhizobia.
 This work was supported by the grant from Saint Petersburg State University ID 93020341 and by the Russian Foundation for Basic Research project 20-016-00129.

CLAVATA3/EMBRYO SURROUNDING REGION Genes Involved in Symbiotic Nodulation in Pisum sativum

CLE (CLAVATA3/Embryo Surrounding Region) peptides regulate different aspects of plant development. In legumes, CLE peptides are known as key components of autoregulation of nodulation (AON), which systemically controls the number of nitrogen-fixing nodules formed on the root upon symbiotic interaction with soil bacteria rhizobia. CLE peptides are produced in the root in response to rhizobia inoculation and are transported via xylem to the shoot, where they are recognized by a specific receptor. As a result, a subsequent nodule development is suppressed by a negative feedback mechanism. In addition, nitrate-induced CLE genes have been identified in model legumes, which mediate nitrate-dependent inhibition of nodulation. However, little is known about the functions of nodulation-related CLE peptides, which have not been studied in Pisum sativum. Here, we studied four homologues of CLE genes in Pisum sativum, which are closely related to nodulation-suppressing CLEs from other legumes. The expression levels of these genes were increased in developing nodules. Among them, PsCLE13, PsCLE12, and the PsNIC-like genes were upregulated in response to nitrate treatment. Moreover, we found that overexpression of the PsCLE13 and PsCLE12 genes resulted in the decreased nodule number on transgenic roots. The expression levels of pea homologues of the TOO MUCH LOVE (TML) genes were upregulated in PsCLE13- and PsCLE12-overexpressing roots in comparison with the control (GUS-overexpressing) roots, suggesting that inhibitory effect of PsCLE13 and PsCLE12 is mediated through the induction of the PsTML genes.

Phytonematode peptide effectors exploit a host post-translational trafficking mechanism to the ER using a novel translocation signal.

Cyst nematodes induce a multicellular feeding site within roots called a syncytium. It remains unknown how root cells are primed for incorporation into the developing syncytium. Furthermore, it is unclear how CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptide effectors secreted into the cytoplasm of the initial feeding cell could have an effect on plant cells so distant from where the nematode is feeding as the syncytium expands. Here we describe a novel translocation signal within nematode CLE effectors that is recognized by plant cell secretory machinery to redirect these peptides from the cytoplasm to the apoplast of plant cells. We show that the translocation signal is functionally conserved across CLE effectors identified in nematode species spanning three genera and multiple plant species, operative across plant cell types, and can traffic other unrelated small peptides from the cytoplasm to the apoplast of host cells via a previously unknown post-translational mechanism of endoplasmic reticulum (ER) translocation. Our results uncover a mechanism of effector trafficking that is unprecedented in any plant pathogen to date, andthey illustrate how phytonematodes can deliver effector proteins into host cells and then hijack plant cellular processes for their export back out of the cell to function as external signaling molecules to distant cells.

Identification and Functional Analysis of the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) Gene Family in Wheat.

CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides are post-translationally cleaved and modified peptides from their corresponding pre-propeptides. Although they are only 12 to 13 amino acids in length, they are important ligands involved in regulating cell proliferation and differentiation in plant shoots, roots, vasculature, and other tissues. They function by interacting with their corresponding receptors. CLE peptides have been studied in many plants, but not in wheat. We identified 104 TaCLE genes in the wheat genome based on a genome-wide scan approach. Most of these genes have homologous copies distributed on sub-genomes A, B, and D. A few genes are derived from tandem duplication and segmental duplication events. Phylogenetic analysis revealed that TaCLE genes can be divided into five different groups. We obtained functional characterization of the peptides based on the evolutionary relationships among the CLE peptide families of wheat, rice, and Arabidopsis, and expression pattern analysis. Using chemically synthesized peptides (TaCLE3p and TaCLE34p), we found that TaCLE3 and TaCLE34 play important roles in regulating wheat and Arabidopsis root development, and wheat stem development. Overexpression analysis of TaCLE3 in Arabidopsis revealed that TaCLE3 not only affects the development of roots and stems, but also affects the development of leaves and fruits. These data represent the first comprehensive information on TaCLE family members.

Unraveling new molecular players involved in the autoregulation of nodulation in Medicago truncatula.

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.

CLE14 peptide signaling in Arabidopsis root hair cell fate determination

Morphological adjustment is a critical strategy for the survival of plant species in various environments. The CLE (CLAVATA3/EMBRYO SURROUNDING REGION) family of plant polypeptides is known to play important roles in various physiological and developmental processes and the relevant signaling pathways are conserved in diverse land plants. Previously, it has been suggested that overexpression of CLE14 promotes root hair cell differentiation in Arabidopsis roots. To clarify this suggested function of CLE14 peptide on root hair induction, we examined the effect of synthetic CLE14 peptide on Arabidopsis root hair development. Consistent with the results of previous overexpression analyses of CLE14, we demonstrated that application of synthetic CLE14 peptide induced excess root hair formation on CLE14-treated Arabidopsis roots. In addition, CLE14 reduced the expression of the non-hair cell fate determinant gene, GLABRA2. Our results thus indicate that CLE14 can activate the transcriptional regulatory cascade of root hair formation.

A Collection of Mutants for CLE-Peptide-Encoding Genes in Arabidopsis Generated by CRISPR/Cas9-Mediated Gene Targeting.

The ligand-receptor-mediated intercellular communication system plays important roles in coordinating developmental and physiological events in multicellular organisms. In plants, CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides and their cognate receptors are thought to be involved in various aspects of the plant life cycle. Although the importance of this communication is broadly recognized, most CLE peptides are yet to be functionally characterized. A major problem in research on small signaling peptide-encoding genes is the limited number of loss-of-function mutants available due to their small gene size. CRISPR/Cas9-mediated gene targeting has the potential to overcome this problem, as it can be used to generate targeted mutations in essentially any gene, regardless of size. Here we generated a series of mutants of CLE-peptide-encoding genes. Newly generated clv3 and cle40 mutants reproduced the expected mutant phenotypes in the shoot apical meristem and root meristem, respectively. Our results show that CRISPR/Cas9-mediated gene targeting is a powerful tool for genetic analyses, even of small genes. We also report a novel mutant for CLE44 [which is thought to encode a tracheary elements differentiation inhibitory factor (TDIF)] and show that CLE44 contributes to vascular development. The bioresources presented here will be a powerful tool for further characterization of CLE peptides.

Perception of root-active CLE peptides requires CORYNE function in the phloem vasculature.

Arabidopsis root development is orchestrated by signaling pathways that consist of different CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptide ligands and their cognate CLAVATA (CLV) and BARELY ANY MERISTEM (BAM) receptors. How and where different CLE peptides trigger specific morphological or physiological changes in the root is poorly understood. Here, we report that the receptor‐like protein CLAVATA 2 (CLV2) and the pseudokinase CORYNE (CRN) are necessary to fully sense root‐active CLE peptides. We uncover BAM3 as the CLE45 receptor in the root and biochemically map its peptide binding surface. In contrast to other plant peptide receptors, we found no evidence that SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) proteins act as co‐receptor kinases in CLE45 perception. CRN stabilizes BAM3 expression and thus is required for BAM3‐mediated CLE45 signaling. Moreover, protophloem‐specific CRN expression complements resistance of the crn mutant to root‐active CLE peptides, suggesting that protophloem is their principal site of action. Our work defines a genetic framework for dissecting CLE peptide signaling and CLV/BAM receptor activation in the root.

Arabidopsis MAKR5 is a positive effector of BAM3-dependent CLE45 signaling.

Receptor kinases convey diverse environmental and developmental inputs by sensing extracellular ligands. In plants, one group of receptor-like kinases (RLKs) is characterized by extracellular leucine-rich repeat (LRR) domains, which interact with various ligands that include the plant hormone brassinosteroid and peptides of the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) type. For instance, the CLE45 peptide requires the LRR-RLK BARELY ANY MERISTEM 3 (BAM3) to prevent protophloem formation in Arabidopsis root meristems. Here, we show that other proposed CLE45 receptors, the two redundantly acting LRR-RLKs STERILITY-REGULATING KINASE MEMBER 1 (SKM1) and SKM2 (which perceive CLE45 in the context of pollen tube elongation), cannot substitute for BAM3 in the root. Moreover, we identify MEMBRANE-ASSOCIATED KINASE REGULATOR 5 (MAKR5) as a post-transcriptionally regulated amplifier of the CLE45 signal that acts downstream of BAM3. MAKR5 belongs to a small protein family whose prototypical member, BRI1 KINASE INHIBITOR 1, is an essentially negative regulator of brassinosteroid signaling. By contrast, MAKR5 is a positive effector of CLE45 signaling, revealing an unexpected diversity in the conceptual roles of MAKR genes in different signaling pathways.

Shared and distinct functions of the pseudokinase CORYNE (CRN) in shoot and root stem cell maintenance of Arabidopsis

Stem cell maintenance in plants depends on the activity of small secreted signaling peptides of the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) family, which, in the shoot, act through at least three kinds of receptor complexes, CLAVATA1 (CLV1) homomers, CLAVATA2 (CLV2) / CORYNE (CRN) heteromers, and CLV1/CLV2/CRN multimers. In the root, the CLV2/CRN receptor complexes function in the proximal meristem to transmit signals from the CLE peptide CLE40. While CLV1 consists of an extracellular receptor domain and an intracellular kinase domain, CLV2, a leucine-rich repeat (LRR) receptor-like protein, and CRN, a protein kinase, have to interact to form a receptor-kinase complex. The kinase domain of CRN has been reported to be catalytically inactive, and it is not yet known how the CLV2/CRN complex can relay the perceived signal into the cells, and whether the kinase domain is necessary for signal transduction at all. In this study we show that the kinase domain of CRN is actively involved in CLV3 signal transduction in the shoot apical meristem of Arabidopsis, but it is dispensable for CRN protein function in root meristem maintenance. Hence, we provide an example of a catalytically inactive pseudokinase that is involved in two homologous pathways, but functions in distinctively different ways in each of them.

Genome-wide characterization, expression and functional analysis of CLV3/ESR gene family in tomato.

BackgroundBy encoding a group of small secretory peptides, the members of the CLAVATA3/EMBRYO-SURROUNDING REGION (CLE) family play important roles in cell-to-cell communication to control the balance between stem cell proliferation and differentiation in plant development. Despite recent identification and characterization of members of this gene family in several plant species, little is known about its functional role in plants with fleshy fruits.ResultsIn total, fifteen CLE genes (SlCLE1-15) were identified from tomato (Solanum lycopersicum cv. ‘Heinz-1706’) genome and their multiple characters including phylogeny, gene structures, chromosome locations, conserved motifs and cis-elements in the promoter sequences, were analyzed. Real-time PCR analysis showed that 13 out of 15 identified SlCLE genes are transcribed and exhibit remarkably unique expression patterns among tissues and organs. In particular, SlCLE12, the homologue of Arabidopsis CLE41/44 gene, appears to be the dominant CLE gene in most of tested tissues with its maximum expression found in vascular tissues. Meanwhile, SlCLE1, 10, 13 exhibit specific but distinct expression in flower bud, root and shoot apex, respectively. More notably, several SlCLEs are dramatically regulated in their transcriptional levels during fruit development and ripening, indicating significant role these genes may potentially play in the critical physiological process. Upon the treatment with synthetic peptides corresponding to the 12-aa CLE domains of SlCLE 10, 12 and 13, tomato seedlings exhibit a clear reduction in root length to varying degrees.ConclusionsThis study provides a comprehensive genomic analysis of CLE gene family in tomato, a crop species with fleshy fruit. Differential expression patterns of various SlCLEs provide important insights into the functional divergence of CLE signaling cascade in Solanaceae species, especially their potential involvements in the regulation of fruit development and ripening.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-827) contains supplementary material, which is available to authorized users.

Bioinformatic and phylogenetic analysis of the CLAVATA3/EMBRYO-SURROUNDING REGION (CLE) and the CLE-LIKE signal peptide genes in the Pinophyta.

BackgroundThere is a rapidly growing awareness that plant peptide signalling molecules are numerous and varied and they are known to play fundamental roles in angiosperm plant growth and development. Two closely related peptide signalling molecule families are the CLAVATA3-EMBRYO-SURROUNDING REGION (CLE) and CLE-LIKE (CLEL) genes, which encode precursors of secreted peptide ligands that have roles in meristem maintenance and root gravitropism. Progress in peptide signalling molecule research in gymnosperms has lagged behind that of angiosperms. We therefore sought to identify CLE and CLEL genes in gymnosperms and conduct a comparative analysis of these gene families with angiosperms.ResultsWe undertook a meta-analysis of the GenBank/EMBL/DDBJ gymnosperm EST database and the Picea abies and P. glauca genomes and identified 93 putative CLE genes and 11 CLEL genes among eight Pinophyta species, in the genera Cryptomeria, Pinus and Picea. The predicted conifer CLE and CLEL protein sequences had close phylogenetic relationships with their homologues in Arabidopsis. Notably, perfect conservation of the active CLE dodecapeptide in presumed orthologues of the Arabidopsis CLE41/44-TRACHEARY ELEMENT DIFFERENTIATION (TDIF) protein, an inhibitor of tracheary element (xylem) differentiation, was seen in all eight conifer species. We cloned the Pinus radiata CLE41/44-TDIF orthologues. These genes were preferentially expressed in phloem in planta as expected, but unexpectedly, also in differentiating tracheary element (TE) cultures. Surprisingly, transcript abundances of these TE differentiation-inhibitors sharply increased during early TE differentiation, suggesting that some cells differentiate into phloem cells in addition to TEs in these cultures. Applied CLE13 and CLE41/44 peptides inhibited root elongation in Pinus radiata seedlings. We show evidence that two CLEL genes are alternatively spliced via 3′-terminal acceptor exons encoding separate CLEL peptides.ConclusionsThe CLE and CLEL genes are found in conifers and they exhibit at least as much sequence diversity in these species as they do in other plant species. Only one CLE peptide sequence has been 100% conserved between gymnosperms and angiosperms over 300 million years of evolutionary history, the CLE41/44-TDIF peptide and its likely conifer orthologues. The preferential expression of these vascular development-regulating genes in phloem in conifers, as they are in dicot species, suggests close parallels in the regulation of secondary growth and wood formation in gymnosperm and dicot plants. Based on our bioinformatic analysis, we predict a novel mechanism of regulation of the expression of several conifer CLEL peptides, via alternative splicing resulting in the selection of alternative C-terminal exons encoding separate CLEL peptides.

Identification of putative CLE peptide receptors involved in determinate nodulation on soybean

CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides tightly control the balance between stem cell proliferation and differentiation in several plant developmental processes. Transmission of the CLE peptide signal has been shown to be rather complex. Despite their recent identification, little is known about the receptors by which nodulation-specific CLE peptides, which were identified in soybean, are perceived. Genetic analysis has indicated that the leucine-rich repeat receptor-like kinase NARK of soybean (Glycine max) and its orthologs in other legumes are possible candidates. However, more receptors need to be identified because CLE peptides are often detected by heteromultimeric complexes. Here, we identified two additional putative CLE peptide receptor pairs in the soybean genome with a nodulation-related expression pattern, GmRLK1-GmRLK2 and GmRLK3-GmRLK4, and discuss their role in CLE peptide perception during nodulation.

CLE Peptides Control Medicago truncatula Nodulation Locally and Systemically

The CLAVATA3/embryo-surrounding region (CLE) peptides control the fine balance between proliferation and differentiation in plant development. We studied the role of CLE peptides during indeterminate nodule development and identified 25 MtCLE peptide genes in the Medicago truncatula genome, of which two genes, MtCLE12 and MtCLE13, had nodulation-related expression patterns that were linked to proliferation and differentiation. MtCLE13 expression was up-regulated early in nodule development. A high-to-low expression gradient radiated from the inner toward the outer cortical cell layers in a region defining the incipient nodule. At later stages, MtCLE12 and MtCLE13 were expressed in differentiating nodules and in the apical part of mature, elongated nodules. Functional analysis revealed a putative role for MtCLE12 and MtCLE13 in autoregulation of nodulation, a mechanism that controls the number of nodules and involves systemic signals mediated by a leucine-rich repeat receptor-like kinase, SUNN, which is active in the shoot. When MtCLE12 and MtCLE13 were ectopically expressed in transgenic roots, nodulation was abolished at the level of the nodulation factor signal transduction, and this inhibition involved long-distance signaling. In addition, composite plants with roots ectopically expressing MtCLE12 or MtCLE13 had elongated petioles. This systemic effect was not observed in transgenic roots ectopically expressing MtCLE12 and MtCLE13 in a sunn-1 mutant background, although nodulation was still strongly reduced. These results suggest multiple roles for CLE signaling in nodulation.