Gibberellin controls the nodulation signaling pathway in Lotus japonicus

Root nodule formation is regulated by several plant hormones, but the details of the regulation of the nodulation signaling pathway are largely unknown. In this study, the role of gibberellin (GA) in the control of root nodule symbiosis was investigated at the physiological and genetic levels in Lotus japonicus . Exogenous application of biologically active GA, GA₃, inhibited the formation of infection threads and nodules, which was counteracted by the application of a biosynthesis inhibitor of GA, Uniconazole P. Nod factor-induced root hair deformation was severely blocked in the presence of GA, which was phenocopied by nsp2 mutants. The number of spontaneous nodules triggered by the gain-of-function mutation of calcium/calmodulin-dependent kinase (CCaMK) or the lotus histidine kinase 1 (LHK1) was decreased upon the addition of GA; moreover, the overexpression of the gain-of-function mutation of L. japonicus , SLEEPY1, a positive regulator of GA signaling, resulted in a reduced nodule number, without other aspects of root development being affected. These results indicate that higher GA signaling levels specifically inhibit the nodulation signaling pathway. Nod factor-dependent induction of NSP2 and NIN was inhibited by exogenous GA. Furthermore, the cytokinin-dependent induction of NIN was suppressed by GA. From these results, we conclude that GA inhibits the nodulation signaling pathway downstream of cytokinin, possibly at NSP2, which is required for Nod factor-dependent NIN expression. These results clarify the roles of GA in the nodulation signaling pathway, and in relation to the cytokinin signaling pathway for nodulation in L. japonicus .     

LysM domains mediate lipochitin-oligosaccharide recognition and Nfr genes extend the symbiotic host range

Legume-Rhizobium symbiosis is an example of selective cell recognition controlled by host/non-host determinants. Individual bacterial strains have a distinct host range enabling nodulation of a limited set of legume species and vice versa. We show here that expression of Lotus japonicus Nfr1 and Nfr5 Nod-factor receptor genes in Medicago truncatula and L. filicaulis, extends their host range to include bacterial strains, Mesorhizobium loti or DZL, normally infecting L. japonicus. As a result, the symbiotic program is induced, nodules develop and infection threads are formed. Using L. japonicus mutants and domain swaps between L. japonicus and L. filicaulis NFR1 and NFR5, we further demonstrate that LysM domains of the NFR1 and NFR5 receptors mediate perception of the bacterial Nod-factor signal and that recognition depends on the structure of the lipochitin-oligosaccharide Nod-factor. We show that a single amino-acid variation in the LysM2 domain of NFR5 changes recognition of the Nod-factor synthesized by the DZL strain and suggests a possible binding site for bacterial lipochitin-oligosaccharide signal molecules.     

In Rhizobium meliloti, the operon associated with the nod box n5 comprises nodL, noeA and noeB, three host-range genes specifically required for the nodulation of particular Medicago species

In Rhizobium meliloti , the genes required for nodulation of legume hosts are under the control of DNA regulatory sequences called nod boxes. In this paper, we have characterized three host-specific nodulation genes, which form a flavonoid-inducible operon down-stream of the nod box n5. The first gene of this operon is identical to the nodL gene identified by Baev and Kondorosi (1992) in R. meliloti strain AK631. The product of the second gene, NoeA, presents some homology with a methyl transferase. nodL mutants synthesize Nod factors lacking the O -acetate substituent. In contrast, in strains carrying a mutation in either noeA or noeB , no modification in Nod-factor structure or production could be detected. On particular hosts, such as Medicago littoralis , mutants of the n5 operon showed a very weak nodule-forming ability, associated with a drastic decrease in the number of infection threads, while nodulation of Medicago truncatula or Melilotus alba was not affected. Thus, nodL , noeA and noeB are host-specific nodulation genes. By using a gain-of-function approach, we showed that the presence of nodL , and hence of O -acetylated Nod factors, is a major prerequisite for confering the ability to nodulate alfalfa upon the heterologous bacterium Rhizobium tropici .     

Natural variation in host-specific nodulation of pea is associated with a haplotype of the SYM37 LysM-type receptor-like kinase

Rhizobium leguminosarum bv. viciae, which nodulates pea and vetch, makes a mixture of secreted nodulation signals (Nod factors) carrying either a C18:4 or a C18:1 N-linked acyl chain. Mutation of nodE blocks the formation of the C18:4 acyl chain, and nodE mutants, which produce only C18:1-containing Nod factors, are less efficient at nodulating pea. However, there is significant natural variation in the levels of nodulation of different pea cultivars by a nodE mutant of R. leguminosarum bv. viciae. Using recombinant inbred lines from two pea cultivars, one which nodulated relatively well and one very poorly by the nodE mutant, we mapped the nodE-dependent nodulation phenotype to a locus on pea linkage group I. This was close to Sym37 and PsK1, predicted to encode LysM-domain Nod-factor receptor-like proteins; the Sym2 locus that confers Nod-factor-specific nodulation is also in this region. We confirmed the map location using an introgression line carrying this region. Our data indicate that the nodE-dependent nodulation is not determined by the Sym2 locus. We identified several pea lines that are nodulated very poorly by the R. leguminosarum bv. viciae nodE mutant, sequenced the DNA of the predicted LysM-receptor domains of Sym37 and PsK1, and compared the sequences with those derived from pea cultivars that were relatively well nodulated by the nodE mutant. This revealed that one haplotype (encoding six conserved polymorphisms) of Sym37 is associated with very poor nodulation by the nodE mutant. There was no such correlation with polymorphisms at the PsK1 locus. We conclude that the natural variation in nodE-dependent nodulation in pea is most probably determined by the Sym37 haplotype.     

Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor-mediated signalling in cooperation with Nod factor receptor 5

Soil-living rhizobia secrete lipochitin oligosaccharides known as Nod factors, which in Lotus japonicus are perceived by at least two Nod-factor receptors, NFR1 and NFR5. Despite progress in identifying molecular components critical for initial legume host recognition of the microsymbiont and cloning of downstream components, little is known about the activation and signalling mechanisms of the Nod-factor receptors themselves. Here we show that both receptor proteins localize to the plasma membrane, and present evidence for heterocomplex formation initiating downstream signalling. Expression of NFR1 and NFR5 in Nicotiana benthamiana and Allium ampeloprasum (leek) cells caused a rapid cell-death response. The signalling leading to cell death was abrogated using a kinase-inactive variant of NFR1. In these surviving cells, a clear interaction between NFR1 and NFR5 was detected in vivo through bimolecular fluorescence complementation (BiFC). To analyse the inter- and intramolecular phosphorylation events of the kinase complex, the cytoplasmic part of NFR1 was assayed for in vitro kinase activity, and autophosphorylation on 24 amino acid residues, including three tyrosine residues, was found by mass spectrometry. Substitution of the phosphorylated amino acids of NFR1 identified a single phosphorylation site to be essential for NFR1 Nod-factor signalling in vivo and kinase activity in vitro. In contrast to NFR1, no in vitro kinase activity of the cytoplasmic domain of NFR5 was detected. This is further supported by the fact that a mutagenized NFR5 construct, substituting an amino acid essential for ATP binding, restored nodulation of nfr5 mutant roots.     

豆科植物共生结瘤的分子基础和调控研究进展

豆科植物与根瘤菌共生互作的结果导致了一个新的植物器官――根瘤的形成, 根瘤菌生活在根瘤中, 它们具有将氮气转化为能被植物同化的氨的能力。该文阐述了根瘤的形成过程和类型, 并主要以模式豆科植物蒺藜苜蓿(Medicago truncatula)和日本百脉根(Lotus japonicus)为例, 对近年来共生结瘤过程中宿主植物对根瘤菌结瘤因子的识别和信号传递、侵入线形成和固氮的分子基础, 以及宿主植物对根瘤形成的自主调控机制、环境中氮素营养对结瘤的影响研究进行了综述, 指出当前豆科植物与根瘤菌共生互作研究存在的问题, 并对今后的研究方向作了分析与展望。     

A member of the ALOG gene family has a novel role in regulating nodulation in Lotus japonicus

Legumes can control the number of symbiotic nodules that form on their roots, thus balancing nitrogen assimilation and energy consumption. Two major pathways participate in nodulation: the Nod factor(NF)signaling pathway which involves recognition of rhizobial bacteria by root cells and promotion of nodulation, and the autoregulation of nodulation(AON) pathway which involves long-distance negative feedback between roots and shoots. Although a handful of genes have a clear role in the maintenance of nodule number, additional unknown factors may also be involved in this process. Here, we identify a novel function for a Lotus japonicus ALOG(Arabidopsis LSH1 and Oryza G1) family member, LjALOG1,involved in positively regulating nodulation. LjALOG1 expression increased substantially after inoculation with rhizobia, with high levels of expression in whole nodule primordia and in the base of developing nodules. The ljalog1 mutants, which have an insertion of the LORE1 retroelement in LjALOG1, had significantly fewer nodules compared with wild type, along with increased expression of LjCLE-RS1(L. japonicus CLE Root Signal 1), which encodes a nodulation suppressor in the AON pathway. In summary,our findings identified a novel factor that participates in controlling nodulation, possibly by suppressing the AON pathway.     

Abscisic acid coordinates nod factor and cytokinin signaling during the regulation of nodulation in Medicago truncatula

Nodulation is tightly regulated in legumes to ensure appropriate levels of nitrogen fixation without excessive depletion of carbon reserves. This balance is maintained by intimately linking nodulation and its regulation with plant hormones. It has previously been shown that ethylene and jasmonic acid (JA) are able to regulate nodulation and Nod factor signal transduction. Here, we characterize the nature of abscisic acid (ABA) regulation of nodulation. We show that application of ABA inhibits nodulation, bacterial infection, and nodulin gene expression in Medicago truncatula. ABA acts in a similar manner as JA and ethylene, regulating Nod factor signaling and affecting the nature of Nod factor-induced calcium spiking. However, this action is independent of the ethylene signal transduction pathway. We show that genetic inhibition of ABA signaling through the use of a dominant-negative allele of ABSCISIC ACID INSENSITIVE1 leads to a hypernodulation phenotype. In addition, we characterize a novel locus of M. truncatula, SENSITIVITY TO ABA, that dictates the sensitivity of the plant to ABA and, as such, impacts the regulation of nodulation. We show that ABA can suppress Nod factor signal transduction in the epidermis and can regulate cytokinin induction of the nodule primordium in the root cortex. Therefore, ABA is capable of coordinately regulating the diverse developmental pathways associated with nodule formation and can intimately dictate the nature of the plants' response to the symbiotic bacteria.     

Salicylic acid inhibits indeterminate-type nodulation but not determinate-type nodulation

LCOs (lipochitin oligosaccharides, Nod factors) produced by the rhizobial symbiote of Vicia sativa subsp. nigra (vetch, an indeterminate-type nodulating plant) are mitogenic when carrying an 18:4 acyl chain but not when carrying an 18:1 acyl chain. This suggests that the 18:4 acyl chain specifically contributes to signaling in indeterminate-type nodulation. In a working hypothesis, we speculated that the 18:4 acyl chain is involved in oxylipin signaling comparable to, for example, signaling by derivatives of the 18:3 fatty acid linolenic acid (the octadecanoid pathway). Because salicylic acid (SA) is known to interfere with oxylipin signaling, we tested whether nodulation of vetch could be affected by addition of 10(-4) M SA. This concentration completely blocked nodulation of vetch by Rhizobium leguminosarum bv. viciae and inhibited the mitogenic effect of 18:4 LCOs but did not affect LCO-induced root-hair deformation. SA did not act systemically, and only biologically active SA derivatives were capable of inhibiting nodule formation. SA also inhibited R. leguminosarum bv. viciae association with vetch roots. In contrast, addition of SA to Lotus japonicus (a determinate-type nodulating plant responding to 18:1 LCOs) did not inhibit nodulation by Mesorhizobium loti. Other indeterminate-type nodulating plants showed the same inhibiting response toward SA, whereas SA did not inhibit the nodulation of other determinate-type nodulating plants. SA may be a useful tool for studying fundamental differences between signal transduction pathways of indeterminate- and determinate-type nodulating plants.     

Long-distance control of nodulation: Molecules and models

Legume plants develop root nodules to recruit nitrogen-fixing bacteria called rhizobia. This symbiotic relationship allows the host plants to grow even under nitrogen limiting environment. Since nodule development is an energetically expensive process, the number of nodules should be tightly controlled by the host plants. For this purpose, legume plants utilize a long-distance signaling known as autoregulation of nodulation (AON). AON signaling in legumes has been extensively studied over decades but the underlying molecular mechanism had been largely unclear until recently. With the advent of the model legumes, L. japonicus and M. truncatula, we have been seeing a great progress including isolation of the AON-associated receptor kinase. Here, we summarize recent studies on AON and discuss an updated view of the long-distance control of nodulation.     

Plant responses to nodulation factors

The focus of research on signalling in Rhizobium-legume interactions has moved from understanding the structure and synthesis of rhizobially made Nod factors, towards an analysis of how they function in plants. Nod-factor-induced changes in ion fluxes across membranes, followed by establishment of an oscillation of intracellular Ca(2+) concentration, point to the involvement of a receptor-mediated signal transduction pathway. Progress towards the identification of components in this pathway is being made by identifying Nod-factor binding proteins, isolating plant mutants that are defective in signalling and analysing plant responses to Nod factors.     

Ethylene inhibits the Nod factor signal transduction pathway of Medicago truncatula

Legumes form a mutualistic symbiosis with bacteria collectively referred to as rhizobia. The bacteria induce the formation of nodules on the roots of the appropriate host plant, and this process requires the bacterial signaling molecule Nod factor. Although the interaction is beneficial to the plant, the number of nodules is tightly regulated. The gaseous plant hormone ethylene has been shown to be involved in the regulation of nodule number. The mechanism of the ethylene inhibition on nodulation is unclear, and the position at which ethylene acts in this complex developmental process is unknown. Here, we used direct and indirect ethylene application and inhibition of ethylene biosynthesis, together with comparison of wild-type plants and an ethylene-insensitive supernodulating mutant, to assess the effect of ethylene at multiple stages of this interaction in the model legume Medicago truncatula. We show that ethylene inhibited all of the early plant responses tested, including the initiation of calcium spiking. This finding suggests that ethylene acts upstream or at the point of calcium spiking in the Nod factor signal transduction pathway, either directly or through feedback from ethylene effects on downstream events. Furthermore, ethylene appears to regulate the frequency of calcium spiking, suggesting that it can modulate both the degree and the nature of Nod factor pathway activation.     

Mastoparan activates calcium spiking analogous to Nod factor-induced responses in Medicago truncatula root hair cells

The rhizobial-derived signaling molecule Nod factor is essential for the establishment of the Medicago truncatula/Sinorhizobium meliloti symbiosis. Nod factor perception and signal transduction in the plant involve calcium spiking and lead to the induction of nodulation gene expression. It has previously been shown that the heterotrimeric G-protein agonist mastoparan can activate nodulation gene expression in a manner analogous to Nod factor activation of these genes and this requires DOESN'T MAKE INFECTIONS3 (DMI3), a calcium- and calmodulin-dependent protein kinase (CCaMK) that is required for Nod factor signaling. Here we show that mastoparan activates oscillations in cytosolic calcium similar but not identical to Nod factor-induced calcium spiking. Mastoparan-induced calcium changes occur throughout the cell, whereas Nod factor-induced changes are restricted to the region associated with the nucleus. Mastoparan-induced calcium spiking occurs in plants mutated in the receptor-like kinases NOD FACTOR PERCEPTION and DMI2 and in the putative cation channel DMI1, which are all required for Nod factor induction of calcium spiking, indicating either that mastoparan functions downstream of these components or that it uses an alternative mechanism to Nod factor for activation of calcium spiking. However, both mastoparan and Nod factor-induced calcium spiking are inhibited by cyclopiazonic acid and n-butanol, suggesting some common mechanisms underpinning these two calcium agonists. The fact that mastoparan and Nod factor both activate calcium spiking and can induce nodulation gene expression in a DMI3-dependent manner strongly implicates CCaMK in the perception and transduction of the calcium signal.     

Molecular mechanisms controlling legume autoregulation of nodulation

Background

High input costs and environmental pressures to reduce nitrogen use in agriculture have increased the competitive advantage of legume crops. The symbiotic relationship that legumes form with nitrogen-fixing soil bacteria in root nodules is central to this advantage.

Scope

Understanding how legume plants maintain control of nodulation to balance the nitrogen gains with their energy needs and developmental costs will assist in increasing their productivity and relative advantage. For this reason, the regulation of nodulation has been extensively studied since the first mutants exhibiting increased nodulation were isolated almost three decades ago.

Conclusions

Nodulation is regulated primarily via a systemic mechanism known as the autoregulation of nodulation (AON), which is controlled by a CLAVATA1-like receptor kinase. Multiple components sharing homology with the CLAVATA signalling pathway that maintains control of the shoot apical meristem in arabidopsis have now been identified in AON. This includes the recent identification of several CLE peptides capable of activating nodule inhibition responses, a low molecular weight shoot signal and a role for CLAVATA2 in AON. Efforts are now being focused on directly identifying the interactions of these components and to identify the form that long-distance transport molecules take.     

The exudate from an arbuscular mycorrhizal fungus induces nitric oxide accumulation in Medicago truncatula roots

Nitric oxide (NO) is a signaling molecule involved in plant responses to abiotic and biotic stresses. While there is evidence for NO accumulation during legume nodulation, almost no information exists for arbuscular mycorrhizas (AM). Here, we investigated the occurrence of NO in the early stages of Medicago truncatula–Gigaspora margarita interaction, focusing on the plant response to fungal diffusible molecules. NO was visualized in root organ cultures and seedlings by confocal microscopy using the specific probe 4,5-diaminofluorescein diacetate. Five-minute treatment with the fungal exudate was sufficient to induce significant NO accumulation. The specificity of this response to AM fungi was confirmed by the lack of response in the AM nonhost Arabidopsis thaliana and by analyzing mutants impaired in mycorrhizal capacities. NO buildup resulted to be partially dependent on DMI1, DMI2, and DMI3 functions within the so-called common symbiotic signaling pathway which is shared between AM and nodulation. Significantly, NO accumulation was not induced by the application of purified Nod factor, while lipopolysaccharides from Escherichia coli, known to elicit defense-related NO production in plants, induced a significantly different response pattern. A slight upregulation of a nitrate reductase (NR) gene and the reduction of NO accumulation when the enzyme is inhibited by tungstate suggest NR as a possible source of NO. Genetic and cellular evidence, therefore, suggests that NO accumulation is a novel component in the signaling pathway that leads to AM symbiosis.