A rulebook for peptide control of legume–microbe endosymbioses

Plant interactions with arbuscular mycorrhizal fungi, beneficial soil bacteria, and nutrient homeostasis are optimized by an interconnected network of peptide signals.Symbiosis-regulating signaling peptides are members of large protein families, often with a variety of functions in plant physiology and development.The mechanism of peptide-signaling specificity in the context of plant–microbe interactions, nutrient homeostasis, and cross-kingdom peptide mimicry involves antagonism and coordination between individual peptide signals.Although many of the symbiosis-associated peptide signaling pathways converge at common downstream signaling hubs and intersect with phytohormone signaling, the signaling outcomes are, at least partially, unique. Plants engage in mutually beneficial relationships with microbes, such as arbuscular mycorrhizal fungi or nitrogen-fixing rhizobia, for optimized nutrient acquisition. In return, the microbial symbionts receive photosynthetic carbon from the plant. Both symbioses are regulated by the plant nutrient status, indicating the existence of signaling pathways that allow the host to fine-tune its interactions with the beneficial microbes depending on its nutrient requirements. Peptide hormones coordinate a plethora of developmental and physiological processes and, recently, various peptide families have gained special attention as systemic and local regulators of plant–microbe interactions and nutrient homeostasis. In this review, we identify five ‘rules’ or guiding principles that govern peptide function during symbiotic plant–microbe interactions, and highlight possible points of integration with nutrient acquisition pathways. Plants engage in mutually beneficial relationships with microbes, such as arbuscular mycorrhizal fungi or nitrogen-fixing rhizobia, for optimized nutrient acquisition. In return, the microbial symbionts receive photosynthetic carbon from the plant. Both symbioses are regulated by the plant nutrient status, indicating the existence of signaling pathways that allow the host to fine-tune its interactions with the beneficial microbes depending on its nutrient requirements. Peptide hormones coordinate a plethora of developmental and physiological processes and, recently, various peptide families have gained special attention as systemic and local regulators of plant–microbe interactions and nutrient homeostasis. In this review, we identify five ‘rules’ or guiding principles that govern peptide function during symbiotic plant–microbe interactions, and highlight possible points of integration with nutrient acquisition pathways. Plants engage in mutually beneficial relationships with microbes to optimize their nutrient uptake (Box 1). Arbuscular mycorrhiza (AM) symbiosis (see Glossary) is an interaction occurring between almost 70% of all land plants and fungal endosymbionts of the subphylum Glomeromycotina, which provide the plant with mineral nutrients (e.g., phosphorus, P) and other benefits (e.g., increased resistance to biotic and abiotic stresses) [1.Müller L.M. Harrison M.J. Phytohormones, miRNAs, and peptide signals integrate plant phosphorus status with arbuscular mycorrhizal symbiosis.Curr. Opin. Plant Biol. 2019; 50: 132-139Crossref PubMed Scopus (33) Google Scholar]. Similarly, a limited number of plant groups, including legumes, can engage in root nodule (RN) symbiosis with nitrogen (N)-fixing bacteria, such as rhizobia [2.Roy S. et al.Celebrating 20 years of genetic discoveries in legume nodulation and symbiotic nitrogen fixation.Plant Cell. 2020; 32: 15-41Crossref PubMed Scopus (144) Google Scholar]. In exchange for nutrients, the host provides AM fungi or N-fixing bacteria with photosynthetically fixed carbon. The plant host initiates an interaction with symbiotic microbes to meet its nutrient demands, which requires extensive signaling at the cellular, tissue, and systemic levels to accommodate the symbiont. Peptide hormones are short-chain polypeptides, ranging from five to 60 residues, that can act as regulators of symbiosis establishment when perceived by cell surface receptors [2.Roy S. et al.Celebrating 20 years of genetic discoveries in legume nodulation and symbiotic nitrogen fixation.Plant Cell. 2020; 32: 15-41Crossref PubMed Scopus (144) Google Scholar,3.Tavormina P. et al.The plant peptidome: an expanding repertoire of structural features and biological functions.Plant Cell. 2015; 27: 2095-2118Crossref PubMed Scopus (167) Google Scholar] (Box 2). Numerous discoveries in recent years resulted in an enormous expansion of our understanding of peptide function during AM or RN signaling and its integration with P and N homeostasis, respectively. Here, we synthesize five emerging principles that govern the interconnected, peptide-mediated signaling pathways regulating plant–microbe symbioses and nutrient acquisition.Box 1Plant symbioses with beneficial microbesTo optimize their nutrient uptake, many land plants engage in mutually beneficial interactions with soil microbes. While ~70% of all terrestrial plant species interact with Glomeromycotina fungi to engage in AM symbiosis, root nodule (RN) symbiosis between plants and beneficial soil bacteria is evolutionarily younger and restricted to four plant orders (Fabales, Fagales, Cucurbitales, and Rosales) [128.Parniske M. Uptake of bacteria into living plant cells, the unifying and distinct feature of the nitrogen-fixing root nodule symbiosis.Curr. Opin. Plant Biol. 2018; 44: 164-174Crossref PubMed Scopus (27) Google Scholar]. Since both AM and RN symbiosis depend on the microbe residing inside host tissue, these relationships are referred to as ‘endosymbiosis’. During RN symbiosis, rhizobia are harbored in specialized root organs (nodules), where they ‘fix’ atmospheric nitrogen (N2) to plant-usable ammonia in an energy-requiring reaction catalyzed by nitrogenase for their plant host in exchange for carbon derived from photosynthesis [2.Roy S. et al.Celebrating 20 years of genetic discoveries in legume nodulation and symbiotic nitrogen fixation.Plant Cell. 2020; 32: 15-41Crossref PubMed Scopus (144) Google Scholar]. By contrast, no specialized organs are required for AM symbiosis. Instead, AM fungi invade the root cortex cells, where they form intricately branched hyphal structures (arbuscules) that function in bidirectional nutrient exchange. In exchange for carbon provided by the host plant, AM fungi supply the plant with various mineral nutrients [predominantly phosphorus (P), but also N, potassium, sulfur, and zinc] taken up from the soil via their vast extraradical hyphal network [129.Smith S.E. Reid D. Mycorrhizal Symbiosis. Academic Press, 2008Google Scholar].Both RN and AM symbiosis are heavily regulated by nutrients and suppressed when plants can meet their nutrient demands without symbiotic microbes; for example, in the presence of a high exogenous N supply, RN symbiosis is inhibited [130.Streeter J. Wong P.P. Inhibition of legume nodule formation and N2 fixation by nitrate.CRC Crit. Rev. Plant Sci. 1988; 7: 1-23Crossref Scopus (0) Google Scholar]. Similarly, AM symbiosis is strongly suppressed by exogenous P supply [131.Menge J.A. et al.Phosphorus concentrations in plants responsible for inhibition of mycorrhizal infection.New Phytol. 1978; 80: 575-578Crossref Scopus (226) Google Scholar], but other nutrients, including N, also have a role in its regulation [31.Javot H. et al.Medicago truncatula mtpt4 mutants reveal a role for nitrogen in the regulation of arbuscule degeneration in arbuscular mycorrhizal symbiosis.Plant J. 2011; 68: 954-965Crossref PubMed Scopus (82) Google Scholar,132.Chambers C.A. et al.Effects of ammonium and nitrate ions on mycorrhizal infection, nodulation and growth of Trifolium subterraneum.New Phytol. 1980; 85: 47-62Crossref Scopus (93) Google Scholar,133.Nouri E. et al.Correction: phosphorus and nitrogen regulate arbuscular mycorrhizal symbiosis in Petunia hybrida.PLoS ONE. 2015; 10e0127472Crossref Scopus (10) Google Scholar]. AM and RN symbiosis initiation is regulated by a crosstalk between hosts and microbes. Development of both symbioses depends on an early dialog between the host and microbes followed by activation of the ‘common symbiosis’ signaling pathway [134.Oldroyd G.E.D. Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants.Nat. Rev. Microbiol. 2013; 11: 252-263Crossref PubMed Scopus (871) Google Scholar], which comprises a shared set of core genes required for the reprogramming of the host cells before the accommodation of symbiotic microbes [134.Oldroyd G.E.D. Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants.Nat. Rev. Microbiol. 2013; 11: 252-263Crossref PubMed Scopus (871) Google Scholar]. In addition, both symbioses are governed by mechanistically similar, systemic autoregulation pathways, which restrict the formation of additional nodules or AM fungal colonization once a critical symbiosis level is reached [66.Wang C. et al.The art of self-control - autoregulation of plant-microbe symbioses.Front. Plant Sci. 2018; 9: 988Crossref PubMed Scopus (26) Google Scholar]. These negative feedback loops are thought to prevent oversequestration of carbon by the microbial symbiont.Box 2Peptide hormone characteristics and perceptionPeptide hormones, defined as mobile, proteinaceous signaling molecules of 5–60 amino acids, often derived from longer polypeptides called pre-propeptides, are encoded within the plant genome [20.de Bang T.C. et al.Genome-wide identification of Medicago peptides involved in macronutrient responses and nodulation.Plant Physiol. 2017; 175: 1669-1689Crossref PubMed Scopus (49) Google Scholar,106.Olsson V. et al.Look Closely, the beautiful may be small: precursor-derived peptides in plants.Annu. Rev. Plant Biol. 2019; 70: 153-186Crossref PubMed Scopus (50) Google Scholar,135.Hellens R.P. et al.The emerging world of small ORFs.Trends Plant Sci. 2016; 21: 317-328Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar]. These can serve as cross-kingdom signals between hosts and their microbial symbionts (see Box 3 in the main text) or act as signals within the host plant itself to mediate cell–cell signaling between neighboring cells or systemically facilitate organ–organ signaling by traveling through the vascular tissue.The function of peptide hormones depends on their perception by cognate receptor-like kinases (RLKs), which selectively bind peptide ligands with their extracellular domains [136.Chakraborty S. et al.Plant leucine-rich repeat receptor kinase (LRR-RK): structure, ligand perception, and activation mechanism.Molecules. 2019; 24: 3081Crossref Scopus (27) Google Scholar]. So far, most peptides, including CLAVATA3/ESR (CLE), C-terminally encoded peptides (CEPs), RGF, and PSK, have been found to interact with leucine-rich repeat RLKs (LRR-RLKs); however, other peptides, such as RALF, interact with RLKs of the Catharanthus roseus RLK1-like (CrRLK1L) subfamily [137.Stegmann M. et al.The receptor kinase FER is a RALF-regulated scaffold controlling plant immune signaling.Science. 2017; 355: 287-289Crossref PubMed Scopus (287) Google Scholar]. In either case, the intracellular serine/threonine kinase domain of the RLK is required for signal transduction upon ligand binding. In plants, most peptide receptors are found in LRR-RLK clades X, XI, and XIII [138.Shiu S.H. Bleecker A.B. Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10763-10768Crossref PubMed Scopus (1016) Google Scholar]. For example, legume orthologs of the arabidopsis (Arabidopsis thaliana) clade XI LRR-RLK CLAVATA1 (AtCLV1) include MtSUNN, LjHAR1, and GmNARK. In arabidopsis, CLV1-type LRR-RLKs typically act in higher order complexes as homo- or heterodimers, or in association with CLV2-type receptor proteins and CRN-type membrane kinases [60.Somssich M. et al.CLAVATA-WUSCHEL signaling in the shoot meristem.Development. 2016; 143: 3238-3248Crossref PubMed Scopus (231) Google Scholar], orthologs of which are also implicated in CLE perception during symbiosis [64.Miyazawa H. et al.The receptor-like kinase KLAVIER mediates systemic regulation of nodulation and non-symbiotic shoot development in Lotus japonicus.Development. 2010; 137: 4317-4325Crossref PubMed Scopus (88) Google Scholar, 65.Crook A.D. et al.The systemic nodule number regulation kinase SUNN in Medicago truncatula interacts with MtCLV2 and MtCRN.Plant J. 2016; 88: 108-119Crossref PubMed Scopus (16) Google Scholar, 66.Wang C. et al.The art of self-control - autoregulation of plant-microbe symbioses.Front. Plant Sci. 2018; 9: 988Crossref PubMed Scopus (26) Google Scholar,139.Krusell L. et al.The Clavata2 genes of pea and Lotus japonicus affect autoregulation of nodulation.Plant J. 2011; 65: 861-871Crossref PubMed Scopus (0) Google Scholar]. In Medicago truncatula, clade XI contains 100 proteins, ~70% of which are transcriptionally regulated upon infection by rhizobia or AM fungi (Figure I) [151.Meng J. et al.Genome-wide characterization, evolution, and expression analysis of the leucine-rich repeat receptor-like protein kinase (LRR-RLK) gene family in Medicago truncatula.Life. 2020; 10: 176Crossref Scopus (7) Google Scholar]. Interestingly, when comparing clade XI of M. truncatula with that of the nonmycorrhizal and nonnodulating arabidopsis, it becomes apparent that the clade is massively expanded in the legume (Figure I), and that most of these genes have no described function. While the M. truncatula lineage experienced massive gene duplication in general [140.Young N.D. et al.The Medicago genome provides insight into the evolution of rhizobial symbioses.Nature. 2011; 480: 520-524Crossref PubMed Google Scholar], the increased number of receptors may also reflect an increased need for signaling pathways to allow the legume to deal with signals associated with different symbionts, as indicated by their heightened expression during RN and AM symbiosis (Figure I). To optimize their nutrient uptake, many land plants engage in mutually beneficial interactions with soil microbes. While ~70% of all terrestrial plant species interact with Glomeromycotina fungi to engage in AM symbiosis, root nodule (RN) symbiosis between plants and beneficial soil bacteria is evolutionarily younger and restricted to four plant orders (Fabales, Fagales, Cucurbitales, and Rosales) [128.Parniske M. Uptake of bacteria into living plant cells, the unifying and distinct feature of the nitrogen-fixing root nodule symbiosis.Curr. Opin. Plant Biol. 2018; 44: 164-174Crossref PubMed Scopus (27) Google Scholar]. Since both AM and RN symbiosis depend on the microbe residing inside host tissue, these relationships are referred to as ‘endosymbiosis’. During RN symbiosis, rhizobia are harbored in specialized root organs (nodules), where they ‘fix’ atmospheric nitrogen (N2) to plant-usable ammonia in an energy-requiring reaction catalyzed by nitrogenase for their plant host in exchange for carbon derived from photosynthesis [2.Roy S. et al.Celebrating 20 years of genetic discoveries in legume nodulation and symbiotic nitrogen fixation.Plant Cell. 2020; 32: 15-41Crossref PubMed Scopus (144) Google Scholar]. By contrast, no specialized organs are required for AM symbiosis. Instead, AM fungi invade the root cortex cells, where they form intricately branched hyphal structures (arbuscules) that function in bidirectional nutrient exchange. In exchange for carbon provided by the host plant, AM fungi supply the plant with various mineral nutrients [predominantly phosphorus (P), but also N, potassium, sulfur, and zinc] taken up from the soil via their vast extraradical hyphal network [129.Smith S.E. Reid D. Mycorrhizal Symbiosis. Academic Press, 2008Google Scholar]. Both RN and AM symbiosis are heavily regulated by nutrients and suppressed when plants can meet their nutrient demands without symbiotic microbes; for example, in the presence of a high exogenous N supply, RN symbiosis is inhibited [130.Streeter J. Wong P.P. Inhibition of legume nodule formation and N2 fixation by nitrate.CRC Crit. Rev. Plant Sci. 1988; 7: 1-23Crossref Scopus (0) Google Scholar]. Similarly, AM symbiosis is strongly suppressed by exogenous P supply [131.Menge J.A. et al.Phosphorus concentrations in plants responsible for inhibition of mycorrhizal infection.New Phytol. 1978; 80: 575-578Crossref Scopus (226) Google Scholar], but other nutrients, including N, also have a role in its regulation [31.Javot H. et al.Medicago truncatula mtpt4 mutants reveal a role for nitrogen in the regulation of arbuscule degeneration in arbuscular mycorrhizal symbiosis.Plant J. 2011; 68: 954-965Crossref PubMed Scopus (82) Google Scholar,132.Chambers C.A. et al.Effects of ammonium and nitrate ions on mycorrhizal infection, nodulation and growth of Trifolium subterraneum.New Phytol. 1980; 85: 47-62Crossref Scopus (93) Google Scholar,133.Nouri E. et al.Correction: phosphorus and nitrogen regulate arbuscular mycorrhizal symbiosis in Petunia hybrida.PLoS ONE. 2015; 10e0127472Crossref Scopus (10) Google Scholar]. AM and RN symbiosis initiation is regulated by a crosstalk between hosts and microbes. Development of both symbioses depends on an early dialog between the host and microbes followed by activation of the ‘common symbiosis’ signaling pathway [134.Oldroyd G.E.D. Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants.Nat. Rev. Microbiol. 2013; 11: 252-263Crossref PubMed Scopus (871) Google Scholar], which comprises a shared set of core genes required for the reprogramming of the host cells before the accommodation of symbiotic microbes [134.Oldroyd G.E.D. Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants.Nat. Rev. Microbiol. 2013; 11: 252-263Crossref PubMed Scopus (871) Google Scholar]. In addition, both symbioses are governed by mechanistically similar, systemic autoregulation pathways, which restrict the formation of additional nodules or AM fungal colonization once a critical symbiosis level is reached [66.Wang C. et al.The art of self-control - autoregulation of plant-microbe symbioses.Front. Plant Sci. 2018; 9: 988Crossref PubMed Scopus (26) Google Scholar]. These negative feedback loops are thought to prevent oversequestration of carbon by the microbial symbiont. Peptide hormones, defined as mobile, proteinaceous signaling molecules of 5–60 amino acids, often derived from longer polypeptides called pre-propeptides, are encoded within the plant genome [20.de Bang T.C. et al.Genome-wide identification of Medicago peptides involved in macronutrient responses and nodulation.Plant Physiol. 2017; 175: 1669-1689Crossref PubMed Scopus (49) Google Scholar,106.Olsson V. et al.Look Closely, the beautiful may be small: precursor-derived peptides in plants.Annu. Rev. Plant Biol. 2019; 70: 153-186Crossref PubMed Scopus (50) Google Scholar,135.Hellens R.P. et al.The emerging world of small ORFs.Trends Plant Sci. 2016; 21: 317-328Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar]. These can serve as cross-kingdom signals between hosts and their microbial symbionts (see Box 3 in the main text) or act as signals within the host plant itself to mediate cell–cell signaling between neighboring cells or systemically facilitate organ–organ signaling by traveling through the vascular tissue. The function of peptide hormones depends on their perception by cognate receptor-like kinases (RLKs), which selectively bind peptide ligands with their extracellular domains [136.Chakraborty S. et al.Plant leucine-rich repeat receptor kinase (LRR-RK): structure, ligand perception, and activation mechanism.Molecules. 2019; 24: 3081Crossref Scopus (27) Google Scholar]. So far, most peptides, including CLAVATA3/ESR (CLE), C-terminally encoded peptides (CEPs), RGF, and PSK, have been found to interact with leucine-rich repeat RLKs (LRR-RLKs); however, other peptides, such as RALF, interact with RLKs of the Catharanthus roseus RLK1-like (CrRLK1L) subfamily [137.Stegmann M. et al.The receptor kinase FER is a RALF-regulated scaffold controlling plant immune signaling.Science. 2017; 355: 287-289Crossref PubMed Scopus (287) Google Scholar]. In either case, the intracellular serine/threonine kinase domain of the RLK is required for signal transduction upon ligand binding. In plants, most peptide receptors are found in LRR-RLK clades X, XI, and XIII [138.Shiu S.H. Bleecker A.B. Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10763-10768Crossref PubMed Scopus (1016) Google Scholar]. For example, legume orthologs of the arabidopsis (Arabidopsis thaliana) clade XI LRR-RLK CLAVATA1 (AtCLV1) include MtSUNN, LjHAR1, and GmNARK. In arabidopsis, CLV1-type LRR-RLKs typically act in higher order complexes as homo- or heterodimers, or in association with CLV2-type receptor proteins and CRN-type membrane kinases [60.Somssich M. et al.CLAVATA-WUSCHEL signaling in the shoot meristem.Development. 2016; 143: 3238-3248Crossref PubMed Scopus (231) Google Scholar], orthologs of which are also implicated in CLE perception during symbiosis [64.Miyazawa H. et al.The receptor-like kinase KLAVIER mediates systemic regulation of nodulation and non-symbiotic shoot development in Lotus japonicus.Development. 2010; 137: 4317-4325Crossref PubMed Scopus (88) Google Scholar, 65.Crook A.D. et al.The systemic nodule number regulation kinase SUNN in Medicago truncatula interacts with MtCLV2 and MtCRN.Plant J. 2016; 88: 108-119Crossref PubMed Scopus (16) Google Scholar, 66.Wang C. et al.The art of self-control - autoregulation of plant-microbe symbioses.Front. Plant Sci. 2018; 9: 988Crossref PubMed Scopus (26) Google Scholar,139.Krusell L. et al.The Clavata2 genes of pea and Lotus japonicus affect autoregulation of nodulation.Plant J. 2011; 65: 861-871Crossref PubMed Scopus (0) Google Scholar]. In Medicago truncatula, clade XI contains 100 proteins, ~70% of which are transcriptionally regulated upon infection by rhizobia or AM fungi (Figure I) [151.Meng J. et al.Genome-wide characterization, evolution, and expression analysis of the leucine-rich repeat receptor-like protein kinase (LRR-RLK) gene family in Medicago truncatula.Life. 2020; 10: 176Crossref Scopus (7) Google Scholar]. Interestingly, when comparing clade XI of M. truncatula with that of the nonmycorrhizal and nonnodulating arabidopsis, it becomes apparent that the clade is massively expanded in the legume (Figure I), and that most of these genes have no described function. While the M. truncatula lineage experienced massive gene duplication in general [140.Young N.D. et al.The Medicago genome provides insight into the evolution of rhizobial symbioses.Nature. 2011; 480: 520-524Crossref PubMed Google Scholar], the increased number of receptors may also reflect an increased need for signaling pathways to allow the legume to deal with signals associated with different symbionts, as indicated by their heightened expression during RN and AM symbiosis (Figure I). Given that N and P are essential for the formation of biological molecules, such as amino acids and nucleotides, their acquisition is a tightly regulated process that involves both positive and negative regulators of nutrient homeostasis [4.Ueda Y. Yanagisawa S. Perception, transduction, and integration of nitrogen and phosphorus nutritional signals in the transcriptional regulatory network in plants.J. Exp. Bot. 2019; 70: 3709-3717Crossref PubMed Scopus (21) Google Scholar]. Plants can directly take up N and P from the soil, but, in legumes, the ability to associate with rhizobia and AM fungi adds an additional layer of complexity. How do legumes distinguish and prioritize between different mechanisms of nutrient acquisition and commit to any one for optimal growth in a marginal environment? The coordination of symbiosis and N and P foraging by roots is orchestrated by a multitude of interconnected peptide signaling networks downstream of rhizobia-secreted Nod-factors or the elusive Myc-factors (Box 3). Such peptide signaling networks include root-to-shoot ‘N-hunger’ signals, such as the C-terminally encoded peptides (CEPs), which mediate enhanced uptake of N in N-poor soils and stimulate nodulation [5.Imin N. et al.The peptide-encoding CEP1 gene modulates lateral root and nodule numbers in Medicago truncatula.J. Exp. Bot. 2013; 64: 5395-5409Crossref PubMed Google Scholar, 6.Tabata R. et al.Perception of root-derived peptides by shoot LRR-RKs mediates systemic N-demand signaling.Science. 2014; 346: 343-346Crossref PubMed Scopus (318) Google Scholar, 7.Roy S. et al.Application of synthetic peptide CEP1 increases nutrient uptake rates along plant roots.Front. Plant Sci. 2021; 12793145Google Scholar]. By contrast, members of another peptide family, the CLAVATA3/ESR (CLE) peptides, limit the number of nodules that form on legume roots, possibly to balance carbon expenditure and N acquisition through symbiotic N fixation [8.Mortier V. et al.CLE peptides control Medicago truncatula nodulation locally and systemically.Plant Physiol. 2010; 153: 222-237Crossref PubMed Scopus (214) Google Scholar]; recent research revealed that AM symbiosis and P acquisition are also regulated by similar signaling mechanisms (see below) [9.Müller L.M. et al.A CLE–SUNN module regulates strigolactone content and fungal colonization in arbuscular mycorrhiza.Nat. Plants. 2019; 5: 933-939Crossref PubMed Scopus (0) Google Scholar,10.Karlo M. et al.The CLE53–SUNN genetic pathway negatively regulates arbuscular mycorrhiza root colonization in Medicago truncatula.J. Exp. Bot. 2020; 71: 4972-4984Crossref PubMed Scopus (2) Google Scholar]. Through use of loss-of-function mutants and gain-of-function approaches (Box 4), researchers found that an optimal concentration is required for the proper function of an individual peptide (Figure 1A ) [8.Mortier V. et al.CLE peptides control Medicago truncatula nodulation locally and systemically.Plant Physiol. 2010; 153: 222-237Crossref PubMed Scopus (214) Google Scholar,11.Mortier V. et al.Nodule numbers are governed by interaction between CLE peptides and cytokinin signaling.Plant J. 2012; 70: 367-376Crossref PubMed Scopus (99) Google Scholar]. These findings support a model involving partially antagonistic or additive peptide functions, which are required to balance nutrient homeostasis and symbiosis over time (Figure 1B,C) [5.Imin N. et al.The peptide-encoding CEP1 gene modulates lateral root and nodule numbers in Medicago truncatula.J. Exp. Bot. 2013; 64: 5395-5409Crossref PubMed Google Scholar,12.Moreau C. et al.Nitrate-induced CLE35 signaling peptides inhibit nodulation through the SUNN receptor and miR2111 repression.Plant Physiol. 2021; 185: 1216-1228Crossref PubMed Google Scholar, 13.Lebedeva M. et al.Nitrate-induced CLE peptide systemically inhibits nodulation in Medicago truncatula.Plants. 2020; 9: 1456Crossref Scopus (7) Google Scholar, 14.Laffont C. et al.The NIN transcription factor coordinates CEP and CLE signaling peptides that regulate nodulation antagonistically.Nat. Commun. 2020; 11: 3167Crossref PubMed Scopus (31) Google Scholar, 15.Mens C. et al.Characterisation of Medicago truncatula CLE34 and CLE35 in nitrate and rhizobia regulation of nodulation.New Phytol. 2021; 229: 2525-2534Crossref PubMed Scopus (16) Google Scholar]. As detailed in the following paragraphs, the staggered timing of peptide induction and/or their relative concentration, rather than the presence or absence of a single peptide, likely allows the plant to dynamically respond to ever-changing environmental conditions.Box 3Peptides can act as cross-kingdom signals between legumes and their microbial partnersMutually beneficial relationships are based on effective communication between both partners. In fact, the entire process is triggered when microbes, such as AM fungi or rhizobia, perceive plant-produced strigolactones (SLs) or flavonoids and, in turn, produce their own lipochitooligosacccharide (LCO) signals called Myc- or Nod- factors, respectively. Microbial symbionts also produce effector molecules, which are proteins or metabolites expressed by plant-associated microbes that enhance colonization of the host. Although best studied in plant pathogens, many symbionts produce effectors, including small, secreted peptides, to boost their infectivity [141.Ratu S.T.N. et al.Rhizobia use a pathogenic-like effector to hijack leguminous nodulation signalling.Sci. Rep. 2021; 11: 1-15Crossref PubMed Scopus (15) Google Scholar,142.Teulet A. et al.The rhizobial type III effector ErnA confers the ability to form nodules in legumes.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 21758-21768Crossref PubMed Scopus (34) Google Scholar]. For example, the AM fungus Rhizophagus irregularis produces small signaling peptides (SSPs) upon perception of SL. The SL-induced putative secreted protein, RiSIS1, was shown to positively regulate root colonization [143.Tsuzuki S. et al.Strigolactone-induced putative secreted protein 1 is required for the establishment of symbiosis by the arbuscular mycorrhizal fungus Rhizophagus irregularis.Mol. Plant-Microbe Interact. 2016; 29: 277-286Crossref PubMed Scopus (69) Google Scholar]. Mycorrhizal signaling peptides are likely post-translationally modified and/or cleaved into smaller peptides, but their mechanistic functions remain unknown [124.Pellegrin C. et al.Comparative analysis of secretomes from ectomycorrhizal fungi with an emphasis on small-secreted proteins.Front. Microbiol. 2015; 6: 1278Crossref PubMed Scopus (73) Google Scholar].Several plant peptides can also act as direct signals to the microbial symbiont. The genome o