Sinorhizobium meliloti succinylated high‐molecular‐weight succinoglycan and the Medicago truncatula LysM receptor‐like kinase MtLYK10 participate independently in symbiotic infection

The formation of nitrogen‐fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen‐fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin‐motif receptor‐like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild‐type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild‐type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan‐defective strains was achieved by in trans rescue with a Nod factor‐deficient S. meliloti mutant. While the Nod factor‐deficient strain was always more abundant inside nodules, the succinoglycan‐deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules.     

Spatiotemporal cytokinin response imaging and ISOPENTENYLTRANSFERASE 3 function in Medicago nodule development

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.

Precise spatial and temporal characterization of cytokinin (CK) responses reveals the function of the CK biosynthesis gene ISOPENTENYLTRANSFERASE 3 during nodule development in Medicago truncatula.     

The AGC Kinase MtIRE: A Link to Phospholipid Signaling During Nodulation?

The development of nitrogen fixing root nodules is complex and involves an interplay of signaling processes. During maturation of plant host cells and their endocytosed rhizobia in symbiosomes, host cells and symbiosomes expand. This expansion is accompanied by a large quantity of membrane biogenesis. We recently characterized an AGC kinase gene, MtIRE, that could play a role in this expansion. MtIRE''s expression coincides with host cell and symbiosome expansion in the proximal side of the invasion zone in developing Medicago truncatula nodules. MtIRE''s closest homolog is the Arabidopsis AGC kinase family IRE gene, which regulates root hair elongation. AGC kinases are regulated by phospholipid signaling in animals and fungi as well as in the several instances where they have been studied in plants. Here we suggest that a phospholipid signaling pathway may also activate MtIRE activity and propose possible upstream activators of MtIRE protein''s presumed AGC kinase activity.Key Words: AGC kinase, nitrogen fixation, nodulation, Medicago truncatula, Sinorhizobium meliloti, infection zone, 3-phosphoinositide-dependent kinase, root hair elongationDuring symbiotic nitrogen-fixing nodule development, both plant cells and rhizobia undergo cell division and expansion.^1–3 In legume roots, nodule organogenesis is triggered by rhizobial Nod factor at the emerging root hair zone. In the indeterminate Medicago-Sinorhizobium symbiosis, inner cortical cell divisions form nodule primordia which emerge from the root and differentiate into complex nodule structures. Rhizobia enter the nodules through plant derived conduits, the infection threads (ITs). ITs begin in curled root hairs, grow through several cell layers and end at nodule primordia where rhizobia are deposited into host cell symbiosomes.² In mature nodules, the meristematic zone I at the nodule apex contains dividing cells. Rhizobia from ITs infect these cells as they exit zone I and enter the infection zone, zone II. The newly released rhizobia, now termed bacteroids, are rod-shaped. In the distal part of zone II, bacteroids divide along with the symbiosome membrane (also called the peribacteroid membrane) that contains them.⁴ As the plant cells with their internalized bacteroids progress toward the proximal end of zone II, bacteroid division ceases. Bacteroid elongation and expansion of the surrounding symbiosome space and membrane is a feature of the proximal side of zone II.⁴ Enormous membrane biogenesis accompanies progression through zone II. As the cells exit zone II, both host cells and bacteroids stop expanding. Interzone II-III is characterized by starch accumulation and zone III is where nitrogen fixation takes place.Members of the protein kinase AGC (for cAMP dependent, cGMP dependent, and protein kinase C) family have been shown to be important in yeast and mammalian signal transduction. The interaction of growth factors with their receptors leads to the activation of phosphatidylinositol (PtdIns) 3-kinase and the phosphorylation of PtdIns species.⁵ These then activate PDK1 enzymes, 3-phosphoinositide-dependent kinases, also AGC kinases,⁵ which then phosphorylate and activate downstream AGC kinases. Several plant AGC kinases have important roles in development and defense,^6–8 although most plant AGC kinases'' functions are still to be discovered.⁹ Two Arabidopsis AGC kinases, IRE and AGC2-1 have been shown to have roles regulating root hair elongation.^10,11We recently cloned and characterized a Medicago IRE-like AGC kinase gene MtIRE,¹² possibly orthologous to the Arabidopsis IRE gene, AtIRE.¹⁰ Because of MtIRE''s homology to AtIRE we thought it might function during infection, because infection threads can be viewed as inward root hair growth. However, MtIRE''s expression is novel. It is expressed only in nodules and flowers and not in roots or root hairs. During nodule development, its initial expression correlates with the onset of host cell and symbiosome expansion. Expression studies with nodulation mutants demonstrate that MtIRE expression correlates with mutant nodules'' abilities to support host cell and symbiosome expansion.¹² An MtIRE promoter-gusA reporter construct (Fig. 1A) shows expression in the proximal part of zone II, the site of continued host cell expansion and bacteroid and symbiosome elongation. RNA interference experiments were unfortunately inconclusive,¹² probably because of closely related more ubiquitously expressed IRE homologs.Open in a separate windowFigure 1(A) Localization of pMtIRE-gusA expression in wild-type nodulated roots. Composite M. truncatula plants with transgenic roots were grown in the presence of S. meliloti and stained with X-Gluc (blue) for the localization of MtIRE promoter activity. The arrow points to the X-Gluc staining in the proximal side of zone II in a 15 dpi nodule. The arrowhead points to root hairs in which no staining was observed. Bar = 100 µM. (B) Phospholipid signaling pathway that may activate MtIRE protein''s presumed kinase activity.We predict that MtIRE is part of a signal pathway regulating an aspect of host cell expansion or symbiosome elongation, or both. The CCS52A gene has a demonstrated role in host cell expansion, mediating endoreduplication.¹³ In contrast to MtIRE, its expression is found throughout zone II, as well as zone I, where it acts in cell division. One might expect other genes that regulate host cell expansion to also be expressed throughout zone II, which MtIRE is not. A unique feature of the region expressing MtIRE is symbiosome elongation.⁴ Because of MtIRE''s temporal and spatial expression patterns, we favor it having a role in symbiosome expansion, although we cannot rule out a role in the latter stages of host cell expansion.Signaling pathway for MtIRE activation is speculative (Fig. 1B) and based on AGC kinase signaling in other systems. AGC kinases are activated by phosphorylation by phosphoinositide-dependent kinase (PDK1) enzymes, also AGC kinases.⁹ We found 4 tentative consensus sequences (TCs) in the DFCI index (compbio.dfci.harvard.edu) that correspond to PDK1 genes of which 3, TC107355, TC94724 and TC94899, were isolated from expression libraries from roots with developing or mature nodules. PDKs are activated by interaction with lipids. The Arabidopsis PDK1 binds to several signaling lipids, including phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidic acid (PA).¹⁴ Phosphatidylinositol 3-kinase (PI3K) activity produces PtdIns3P and PI3K genes have been observed to be induced during nodule organogenesis in soybean¹⁵ and in M. truncatula.¹⁶ In soybean, two PI3K genes were identified with one specifically expressed during the early stages of nodulation when membrane biogenesis takes place. This gene''s predicted protein has potential phosphorylation sites for cAMP dependent kinases and Ca/calmodulin-dependent kinases.¹⁵ In soybean, PI3K enzymatic activity correlated with membrane proliferation during nodulation.¹⁵ More generally, PI3Ks are implicated in vesicular trafficking and cytoskeletal organization;¹⁷ both are required for host cell and symbiosome elongation. We suggest a model where MtIRE kinase activity is activated by PDK1, which is itself regulated by PI3K through the production of PtdIns3P. More speculatively, PI3K could be under the control of the Nod factor signaling pathway Ca/calmodulin-dependent kinase DMI3.^18,19 DMI3 is induced during nodulation, with highest expression levels found in the distal side of the infection zone,²⁰ before expression of MtIRE. Expression could persist to the proximal side of this zone, similar to the expression of another Nod factor signaling component, DMI2.²¹ Alternatively, MtIRE could be activated by PA in a PDK1-dependent manner similar to Arabidopsis AGC2-1.¹¹ PA can be produced by phospholipase C (PLC) or phospholipase D (PLD) pathways, both of which have been implicated in transducing Nod factor signals.^22–26 Either of these models includes Nod factor signaling in proximal zone II, which has not been well-studied. Expression of rhizobial nod genes has been observed in zone II,²⁷ making Nod factor signaling in this zone plausible. Further examination of zone II and predicted upstream regulators of MtIRE will address this model.     

Auxins upregulate nif and fix genes

In a recent publication we analyzed the global effects triggered by IAA overproduction in S. meliloti RD64 under free-living conditions by comparing the gene expression pattern of wild type 1021 with that of RD64 and 1021 treated with IAA and other four chemically or functionally related molecules. Among the genes differentially expressed in RD64 and IAA-treated 1021 cells we found two genes of pho operon, phoT and phoC. Based on this finding we examined the mechanisms for mineral P solubilization in RD64 and the potential ability of this strain to improve Medicago growth under P-starved conditions. Here, we further analyze the expression profiles obtained in microarray analysis and evaluate the specificity and the extent of overlap between all treatments. Venn diagrams indicated that IAA- and 2,4-D-regulated genes were closely related. Furthermore, most differentially expressed genes from pSymA were induced in 1021 cells treated with 2,4-D, ICA, IND and Trp as compared to the untreated 1021 cells. RT-PCR analysis was employed to analyze the differential expression patterns of nitrogen fixation genes under free-living and symbiotic conditions. Under symbiotic condition, the relative expression levels of nif and fix genes were significantly induced in Mt- RD64 plants and in Mt-1021 plants treated with IAA and 2,4-D whereas they were unchanged or repressed in Mt-1021 plants treated with the other selected compounds when compared to the untreated Mt-1021 plants.Key words: 2,4-D; IAA; Medicago truncatula; nitrogen-fixation; Sinorhizobium melilotiWe have previously shown that IAA triggered the upregulation of a central backbone of metabolism such as TCA cycle and the accumulation of PHB granules in free-living Rhizobium.¹ Under symbiotic conditions increased acetylene reduction and plant or seed dry weight production were observed for plants nodulated by IAA-overproducing strains.^1,2 More recently we showed that IAA led to an improvement of stress responses both in free-living and symbiotic conditions. It is known that plants develop a plethora of physiological, developmental and biochemical changes to deal with environmental stress conditions.^3–6 These changes require the activation of biochemical pathways that probably act additively and synergistically and depend largely on efficient nitrogen fixation in the root nodules, a sensitive target for abiotic stresses.^7,8 In this addendum, we comment our recent published data and report that Mt-RD64 plants exhibited enhanced expression of nitrogen fixation genes. The treatment of Mt-1021 plants with exogenous IAA led to similar upregulation. We speculate that this positive alteration might be of agronomic advantage: it could improve the adaptation of these plants to stressful environments as we have found for the salt-stress and P-starvation.^9,14     

Genome-Wide Identification and Expression Profiling Analysis of the Aux/IAA Gene Family in Medicago truncatula during the Early Phase of Sinorhizobium meliloti Infection

Background

Auxin/indoleacetic acid (Aux/IAA) genes, coding a family of short-lived nuclear proteins, play key roles in wide variety of plant developmental processes, including root system regulation and responses to environmental stimulus. However, how they function in auxin signaling pathway and symbiosis with rhizobial in Medicago truncatula are largely unknown. The present study aims at gaining deeper insight on distinctive expression and function features of Aux/IAA family genes in Medicago truncatula during nodule formation.

Principal Findings

Using the latest updated draft of the full Medicago truncatula genome, a comprehensive identification and analysis of IAA genes were performed. The data indicated that MtIAA family genes are distributed in all the M. truncatula chromosomes except chromosome 6. Most of MtIAA genes are responsive to exogenous auxin and express in tissues-specific manner. To understand the biological functions of MtIAA genes involved in nodule formation, quantitative real-time polymerase chain reaction (qRT-PCR) was used to test the expression profiling of MtIAA genes during the early phase of Sinorhizobium meliloti (S. meliloti) infection. The expression patterns of most MtIAA genes were down-regulated in roots and up-regulated in shoots by S. meliloti infection. The differences in expression responses between roots and shoots caused by S. meliloti infection were alleviated by 1-NOA application.

Conclusion

The genome-wide identification, evolution and expression pattern analysis of MtIAA genes were performed in this study. The data helps us to understand the roles of MtIAA-mediated auxin signaling in nodule formation during the early phase of S. meliloti infection.     

Increased production of the exopolysaccharide succinoglycan enhances Sinorhizobium meliloti 1021 symbiosis with the host plant Medicago truncatula

The nitrogen-fixing rhizobial symbiont Sinorhizobium meliloti 1021 produces acidic symbiotic exopolysaccharides that enable it to initiate and maintain infection thread formation on host legume plants. The exopolysaccharide that is most efficient in mediating this process is succinoglycan (exopolysaccharide I [EPSI]), a polysaccharide composed of octasaccharide repeating units of 1 galactose and 7 glucose residues, modified with succinyl, acetyl, and pyruvyl substituents. Previous studies had shown that S. meliloti 1021 mutants that produce increased levels of succinoglycan, such as exoR mutants, are defective in symbiosis with host plants, leading to the hypothesis that high levels of succinoglycan production might be detrimental to symbiotic development. This study demonstrates that increased succinoglycan production itself is not detrimental to symbiotic development and, in fact, enhances the symbiotic productivity of S. meliloti 1021 with the host plant Medicago truncatula cv. Jemalong A17. Increased succinoglycan production was engineered by overexpression of the exoY gene, which encodes the enzyme responsible for the first step in succinoglycan biosynthesis. These results suggest that the level of symbiotic exopolysaccharide produced by a rhizobial species is one of the factors involved in optimizing the interaction with plant hosts.     

The involvement of Medicago truncatula non-specific lipid transfer protein N5 in the control of rhizobial infection

Cysteine-rich proteins seem to play important regulatory roles in Medicago truncatula/Sinorhizobium meliloti symbiosis. In particular, a large family of nodule-specific cysteine-rich (NCR) peptides is crucial for the differentiation of nitrogen-fixing bacteroids. The Medicago truncatula N5 protein (MtN5) is currently the only reported non-specific lipid transfer protein necessary for successful rhizobial symbiosis; in addition, MtN5 shares several characteristics with NCR peptides: a small size, a conserved cysteine-rich motif, an N-terminal signal peptide for secretion and antimicrobial activity. Unlike NCR peptides, MtN5 expression is not restricted to the root nodules and is induced during the early phases of symbiosis in root hairs and nodule primordia. Recently, MtN5 was determined to be involved in the regulation of root tissue invasion; while, it was dispensable for nodule primordia formation. Here, we discuss the hypothesis that MtN5 participates in linking the progression of bacterial invasion with restricting the competence of root hairs for infection.     

Unraveling the involvement of ABA in the water deficit-induced modulation of nitrogen metabolism in Medicago truncatula seedlings

Effects of water deficit and/or abscisic acid (ABA) were investigated on early seedling growth of Medicago truncatula, and on glutamate metabolism under dark conditions. Water deficit (simulated by polyethylene glycol, PEG), ABA and their combination resulted in a reduction in growth rate of the embryo axis, and also in a synergistic increase of free amino acid (AA) content. However, the inhibition of water uptake retention induced by water deficit seemed to occur in an ABA-independent manner. Expression of several genes involved in glutamate metabolism was induced during water deficit, whereas ABA, in combination or not with PEG, repressed them. The only exception came from a gene encoding 1-pyrroline-5-carboxylate synthetase (P5CS) which appeared to be induced in an ABA-dependent manner under water deficit. Our results demonstrate clearly the involvement of an ABA-dependent and an ABA-independent regulatory system, governing growth and glutamate metabolism under water deficit.Key words: abscisic acid, amino acid metabolism, water deficit, glutamate, Medicago truncatula, seedlingsTo counter the effects of unfavorable environmental conditions, young seedlings and plants have developed complex cellular signaling mechanisms which require distinct physiological and metabolic adjustments, such as sugar, amino acid or amine accumulation¹ through different pathways. The phytohormone abscisic acid (ABA) has been reported to be rapidly produced and accumulated under different environmental stresses, and responses mediated by this hormone lead to the induction of complex tolerance mechanisms to osmotic stress.² However, it has been shown that the drought-inducible genes were governed by both ABA-dependent and ABA-independent regulatory systems,³ but it is not entirely clear how water deficit and exogenous ABA could affect and regulate plant nitrogen metabolism when applied simultaneously.