A plant chitinase controls cortical infection thread progression and nitrogen-fixing symbiosis

Morphogens provide positional information and their concentration is key to the organized development of multicellular organisms. Nitrogen-fixing root nodules are unique organs induced by Nod factor-producing bacteria. Localized production of Nod factors establishes a developmental field within the root where plant cells are reprogrammed to form infection threads and primordia. We found that regulation of Nod factor levels by Lotus japonicus is required for the formation of nitrogen-fixing organs, determining the fate of this induced developmental program. Our analysis of plant and bacterial mutants shows that a host chitinase modulates Nod factor levels possibly in a structure-dependent manner. In Lotus, this is required for maintaining Nod factor signalling in parallel with the elongation of infection threads within the nodule cortex, while root hair infection and primordia formation are not influenced. Our study shows that infected nodules require balanced levels of Nod factors for completing their transition to functional, nitrogen-fixing organs.

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Epidermal LysM receptor ensures robust symbiotic signalling in Lotus japonicus

eLife ◽

10.7554/elife.33506 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 18

Author(s):

Eiichi Murakami ◽

Jeryl Cheng ◽

Kira Gysel ◽

Zoltan Bozsoki ◽

Yasuyuki Kawaharada ◽

...

Keyword(s):

Root System ◽

Lotus Japonicus ◽

Transcriptional Response ◽

Epidermal Cells ◽

Nitrogen Fixing ◽

Nod Factors ◽

Nod Factor ◽

Large Families ◽

Calcium Spiking ◽

Spatio Temporal

Recognition of Nod factors by LysM receptors is crucial for nitrogen-fixing symbiosis in most legumes. The large families of LysM receptors in legumes suggest concerted functions, yet only NFR1 and NFR5 and their closest homologs are known to be required. Here we show that an epidermal LysM receptor (NFRe), ensures robust signalling in L. japonicus. Mutants of Nfre react to Nod factors with increased calcium spiking interval, reduced transcriptional response and fewer nodules in the presence of rhizobia. NFRe has an active kinase capable of phosphorylating NFR5, which in turn, controls NFRe downstream signalling. Our findings provide evidence for a more complex Nod factor signalling mechanism than previously anticipated. The spatio-temporal interplay between Nfre and Nfr1, and their divergent signalling through distinct kinases suggests the presence of an NFRe-mediated idling state keeping the epidermal cells of the expanding root system attuned to rhizobia.

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ALotus japonicuscytoplasmic kinase connects Nod factor perception by the NFR5 LysM receptor to nodulation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1815425116 ◽

2019 ◽

Vol 116 (28) ◽

pp. 14339-14348 ◽

Cited By ~ 4

Author(s):

Jaslyn E. M. M. Wong ◽

Marcin Nadzieja ◽

Lene H. Madsen ◽

Christoph A. Bücherl ◽

Svend Dam ◽

...

Keyword(s):

Root Nodules ◽

Lotus Japonicus ◽

Root Hairs ◽

Cellular Level ◽

Dependent Manner ◽

Nod Factor ◽

Associated Proteins ◽

Signaling Process ◽

Insertion Mutants

The establishment of nitrogen-fixing root nodules in legume–rhizobia symbiosis requires an intricate communication between the host plant and its symbiont. We are, however, limited in our understanding of the symbiosis signaling process. In particular, how membrane-localized receptors of legumes activate signal transduction following perception of rhizobial signaling molecules has mostly remained elusive. To address this, we performed a coimmunoprecipitation-based proteomics screen to identify proteins associated with Nod factor receptor 5 (NFR5) inLotus japonicus.Out of 51 NFR5-associated proteins, we focused on a receptor-like cytoplasmic kinase (RLCK), which we named NFR5-interacting cytoplasmic kinase 4 (NiCK4). NiCK4 associates with heterologously expressed NFR5 inNicotiana benthamiana, and directly binds and phosphorylates the cytoplasmic domains of NFR5 and NFR1 in vitro. At the cellular level,Nick4is coexpressed withNfr5in root hairs and nodule cells, and the NiCK4 protein relocates to the nucleus in an NFR5/NFR1-dependent manner upon Nod factor treatment. Phenotyping of retrotransposon insertion mutants revealed that NiCK4 promotes nodule organogenesis. Together, these results suggest that the identified RLCK, NiCK4, acts as a component of the Nod factor signaling pathway downstream of NFR5.

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Sinorhizobium fredii HH103 Invades Lotus burttii by Crack Entry in a Nod Factor–and Surface Polysaccharide–Dependent Manner

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-09-16-0195-r ◽

2016 ◽

Vol 29 (12) ◽

pp. 925-937 ◽

Cited By ~ 16

Author(s):

Sebastián Acosta-Jurado ◽

Dulce-Nombre Rodríguez-Navarro ◽

Yasuyuki Kawaharada ◽

Juan Fernández Perea ◽

Antonio Gil-Serrano ◽

...

Keyword(s):

Root Hairs ◽

Broad Host Range ◽

Capsular Polysaccharides ◽

Dependent Manner ◽

Nod Factors ◽

Sinorhizobium Fredii ◽

Nod Factor ◽

Mesorhizobium Loti ◽

Infection Threads ◽

Surface Polysaccharide

Sinorhizobium fredii HH103-Rifr, a broad host range rhizobial strain, induces nitrogen-fixing nodules in Lotus burttii but ineffective nodules in L. japonicus. Confocal microscopy studies showed that Mesorhizobium loti MAFF303099 and S. fredii HH103-Rifr invade L. burttii roots through infection threads or epidermal cracks, respectively. Infection threads in root hairs were not observed in L. burttii plants inoculated with S. fredii HH103-Rifr. A S. fredii HH103-Rifr nodA mutant failed to nodulate L. burttii, demonstrating that Nod factors are strictly necessary for this crack-entry mode, and a noeL mutant was also severely impaired in L. burttii nodulation, indicating that the presence of fucosyl residues in the Nod factor is symbiotically relevant. However, significant symbiotic impacts due to the absence of methylation or to acetylation of the fucosyl residue were not detected. In contrast S. fredii HH103-Rifr mutants showing lipopolysaccharide alterations had reduced symbiotic capacity, while mutants affected in production of either exopolysaccharides, capsular polysaccharides, or both were not impaired in nodulation. Mutants unable to produce cyclic glucans and purine or pyrimidine auxotrophic mutants formed ineffective nodules with L. burttii. Flagellin-dependent bacterial mobility was not required for crack infection, since HH103-Rifr fla mutants nodulated L. burttii. None of the S. fredii HH103-Rifr surface-polysaccharide mutants gained effective nodulation with L. japonicus.

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A Nod factor- and type III secretion system-dependent manner for Robinia pseudoacacia to establish symbiosis with Mesorhizobium amorphae CCNWGS0123

Tree Physiology ◽

10.1093/treephys/tpaa160 ◽

2020 ◽

Author(s):

Haibo Huo ◽

Xinye Wang ◽

Yao Liu ◽

Juan Chen ◽

Gehong Wei

Keyword(s):

Type Iii Secretion ◽

Root Nodules ◽

Robinia Pseudoacacia ◽

Type Iii Secretion System ◽

Secretion System ◽

Dependent Manner ◽

Nod Factor ◽

Type Iii ◽

Infection Threads ◽

Plant Genes

Abstract Under nitrogen-limiting conditions, symbiotic nodulation promotes the growth of legume plants via the fixation of atmospheric nitrogen to ammonia by rhizobia in root nodules. The rhizobial Nod factor (NF) and type III secretion system (T3SS) are two key signaling pathways for establishing the legume–rhizobium symbiosis. However, whether NF signaling is involved in the nodulation of Robinia pseudoacacia and Mesorhizobium amorphae CCNWGS0123, and its symbiotic differences compared to T3SS signaling remain unclear. Therefore, to elucidate the function of NF signaling in nodulation, we mutated nodC in M. amorphae CCNWGS0123, which aborted NF synthesis. Compared to the plants inoculated with the wild type strain, the plants inoculated with the NF-deficient strain exhibited shorter shoots with etiolated leaves. These phenotypic characteristics were similar to those of the plants inoculated with the T3SS-deficient strain, which served as a nod− (non-effective nodulation) control. Both the plants inoculated with the NF- and T3SS-deficient strains formed massive root hair swellings, but no normal infection threads were detected. Sections of the nodules showed that inoculation with the NF- and T3SS-deficient strains induced small, white bumps without any rhizobia inside. Analyzing the accumulation of six plant hormones and the expression of ten plant genes indicated that the NF- and T3SS-deficient strains activated plant defense reactions while suppressing plant symbiotic signaling during the perception and nodulation processes. The requirement for NF signaling appeared to be conserved in two other leguminous trees that can establish symbiosis with M. amorphae CCNWGS0123. In contrast, the function of the T3SS might differ among species, even within the same subfamily (Faboideae). Overall, this work demonstrated that nodulation of R. pseudoacacia and M. amorphae CCNWGS0123 was both NF and T3SS dependent.

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MIR2111-5 locus and shoot-accumulated mature miR2111 systemically enhance nodulation depending on HAR1 in Lotus japonicus

Nature Communications ◽

10.1038/s41467-020-19037-9 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Nao Okuma ◽

Takashi Soyano ◽

Takuya Suzaki ◽

Masayoshi Kawaguchi

Keyword(s):

Root Nodules ◽

Lotus Japonicus ◽

Dependent Manner ◽

Rna Seq ◽

Signaling System ◽

Nodule Number ◽

Knockout Mutants ◽

Mutualistic Relationship ◽

Dose Dependent

Abstract Legumes utilize a shoot-mediated signaling system to maintain a mutualistic relationship with nitrogen-fixing bacteria in root nodules. In Lotus japonicus, shoot-to-root transfer of microRNA miR2111 that targets TOO MUCH LOVE, a nodulation suppressor in roots, has been proposed to explain the mechanism underlying nodulation control from shoots. However, the role of shoot-accumulating miR2111s for the systemic regulation of nodulation was not clearly shown. Here, we find L. japonicus has seven miR2111 loci, including those mapped through RNA-seq. MIR2111-5 expression in leaves is the highest among miR2111 loci and repressed after rhizobial infection depending on a shoot-acting HYPERNODULATION ABERRANT ROOT FORMATION1 (HAR1) receptor. MIR2111-5 knockout mutants show significantly decreased nodule numbers and miR2111 levels. Furthermore, grafting experiments using transformants demonstrate scions with altered miR2111 levels influence nodule numbers in rootstocks in a dose-dependent manner. Therefore, miR2111 accumulation in leaves through MIR2111-5 expression is required for HAR1-dependent systemic optimization of nodule number.

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The rhizobial autotransporter determines the symbiotic nitrogen fixation activity of Lotus japonicus in a host-specific manner

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1913349117 ◽

2020 ◽

Vol 117 (3) ◽

pp. 1806-1815 ◽

Cited By ~ 2

Author(s):

Yoshikazu Shimoda ◽

Yuki Nishigaya ◽

Hiroko Yamaya-Ito ◽

Noritoshi Inagaki ◽

Yosuke Umehara ◽

...

Keyword(s):

Nitrogen Fixation ◽

Host Plant ◽

Symbiotic Nitrogen Fixation ◽

Root Nodules ◽

Lotus Japonicus ◽

Dependent Manner ◽

Strain Specificity ◽

Passenger Domain ◽

Aspartic Peptidase ◽

Specific Manner

Leguminous plants establish endosymbiotic associations with rhizobia and form root nodules in which the rhizobia fix atmospheric nitrogen. The host plant and intracellular rhizobia strictly control this symbiotic nitrogen fixation. We recently reported a Lotus japonicus Fix− mutant, apn1 (aspartic peptidase nodule-induced 1), that impairs symbiotic nitrogen fixation. APN1 encodes a nodule-specific aspartic peptidase involved in the Fix− phenotype in a rhizobial strain-specific manner. This host-strain specificity implies that some molecular interactions between host plant APN1 and rhizobial factors are required, although the biological function of APN1 in nodules and the mechanisms governing the interactions are unknown. To clarify how rhizobial factors are involved in strain-specific nitrogen fixation, we explored transposon mutants of Mesorhizobium loti strain TONO, which normally form Fix− nodules on apn1 roots, and identified TONO mutants that formed Fix+ nodules on apn1. The identified causal gene encodes an autotransporter, part of a protein secretion system of Gram-negative bacteria. Expression of the autotransporter gene in M. loti strain MAFF3030399, which normally forms Fix+ nodules on apn1 roots, resulted in Fix− nodules. The autotransporter of TONO functions to secrete a part of its own protein (a passenger domain) into extracellular spaces, and the recombinant APN1 protein cleaved the passenger protein in vitro. The M. loti autotransporter showed the activity to induce the genes involved in nodule senescence in a dose-dependent manner. Therefore, we conclude that the nodule-specific aspartic peptidase, APN1, suppresses negative effects of the rhizobial autotransporter in order to maintain effective symbiotic nitrogen fixation in root nodules.

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Differential Effects of Combined N Sources on Early Steps of the Nod Factor–Dependent Transduction Pathway in Lotus japonicus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-8-0994 ◽

2007 ◽

Vol 20 (8) ◽

pp. 994-1003 ◽

Cited By ~ 50

Author(s):

Ani Barbulova ◽

Alessandra Rogato ◽

Enrica D'Apuzzo ◽

Selim Omrane ◽

Maurizio Chiurazzi

Keyword(s):

Root Nodules ◽

Lotus Japonicus ◽

Small Scale ◽

Nodule Formation ◽

Inhibitory Effects ◽

Nod Factor ◽

Mesorhizobium Loti ◽

N Sources ◽

N Starvation ◽

And Function

The development of nitrogen-fixing nodules in legumes is induced by perception of lipochitin-oligosaccharide signals secreted by a bacterial symbiont. Nitrogen (N) starvation is a prerequisite for the formation, development, and function of root nodules, and high levels of combined N in the form of nitrate or ammonium can completely abolish nodule formation. We distinguished between nitrate and ammonium inhibitory effects by identifying when and where these combined N sources interfere with the Nod-factor-induced pathway. Furthermore, we present a small-scale analysis of the expression profile, under different N conditions, of recently identified genes involved in the Nod-factor-induced pathway. In the presence of high levels of nitrate or ammonium, the NIN gene fails to be induced 24 h after the addition of Nod factor compared with plants grown under N-free conditions. This induction is restored in the hypernodulating nitrate-tolerant har1-3 mutant only in the presence of 10 and 20 mM KNO3. These results were confirmed in Lotus plants inoculated with Mesorhizobium loti. NIN plays a key role in the nodule organogenesis program and its downregulation may represent a crucial event in the nitrate-dependent pathway leading to the inhibition of nodule organogenesis.

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Translocation of NopP by Sinorhizobium fredii USDA257 into Vigna unguiculata Root Nodules

Applied and Environmental Microbiology ◽

10.1128/aem.03122-09 ◽

2010 ◽

Vol 76 (11) ◽

pp. 3758-3761 ◽

Cited By ~ 25

Author(s):

Lisa M. Schechter ◽

Jeanette Guenther ◽

Elizabeth A. Olcay ◽

Sungchan Jang ◽

Hari B. Krishnan

Keyword(s):

Vigna Unguiculata ◽

Type Iii Secretion ◽

Root Nodules ◽

Type Iii Secretion System ◽

Secretion System ◽

Dependent Manner ◽

Nitrogen Fixing ◽

Sinorhizobium Fredii ◽

Type Iii

ABSTRACT Sinorhizobium fredii is a nitrogen-fixing legume symbiont that stimulates the formation of root nodules. S. fredii nodulation of roots is influenced by Nop proteins, which are secreted through a type III secretion system (T3SS). We demonstrate that S. fredii injects NopP into Vigna unguiculata nodules in a T3SS-dependent manner.

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Biological activity of Nod factors

Acta Biochimica Polonica ◽

10.18388/abp.2020_5353 ◽

2020 ◽

Author(s):

Dominika Kidaj ◽

Mikolaj Krysa ◽

Katarzyna Susniak ◽

Joanna Matys ◽

Iwona Komaniecka ◽

...

Keyword(s):

Molecular Level ◽

Root Nodules ◽

Cortical Cell ◽

Plant Tissues ◽

Nod Factors ◽

Nod Factor ◽

Expression Of Genes ◽

Root Hair Deformation ◽

Low Concentrations ◽

Genes Encoding

Chemically, the Nod factors (NFs) are lipochitooligosaccharides, produced mainly by bacteria of the Rhizobium genus. They are the main signaling molecules involved in the initiation of symbiosis between rhizobia and legume plants. Nod factors affect plant tissues at very low concentrations, even as low as 10–12 mol/L. They induce root hair deformation, cortical cell division, and root nodules’ formation in the host plant. At the molecular level, the cytoskeleton is reorganized and expression of genes encoding proteins called nodulins is induced in response to Nod factors in the cell. Action of Nod factors is highly specific because it depends on the structure of a particular Nod factor involved, as well as the plant receptor reacting with it.

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Rhizobium-Induced Calcium Spiking in Lotus japonicus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2003.16.4.335 ◽

2003 ◽

Vol 16 (4) ◽

pp. 335-341 ◽

Cited By ~ 38

Author(s):

Jeanne M. Harris ◽

Rebecca Wais ◽

Sharon R. Long

Keyword(s):

Host Plant ◽

Lotus Japonicus ◽

Developmental Changes ◽

Nodule Development ◽

Cytoplasmic Calcium ◽

Nod Factors ◽

Developmental Pathway ◽

Nod Factor ◽

Calcium Spiking ◽

General Component

Legumes and rhizobium bacteria form a symbiosis that results in the development of nitrogen-fixing nodules on the root of the host plant. The earliest plant developmental changes are triggered by bacterially produced nodulation (Nod) factors. Within minutes of exposure to Nod factors, sharp oscillations in cytoplasmic calcium levels (calcium spiking) occur in epidermal cells of several closely related legumes. We found that Lotus japonicus, a legume that follows an alternate developmental pathway, responds to both its bacterial partner and to the purified bacterial signal with calcium spiking. Thus, calcium spiking is not restricted to a particular pathway of nodule development and may be a general component of the response of host legumes to their bacterial partner. Using Nod factor-induced calcium spiking as a tool to identify mutants blocked early in the response to Nod factor, we show that the L. japonicus Ljsym22-1 mutant but not the Ljsym30 mutant fails to respond to Nod factor with calcium spiking.

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