Chitin Fragments Research Articles

Plankton and marine aggregates as transmission vectors for V. aestuarianus 02/041 infecting the pacific oyster Crassostrea gigas.

Vibrio aestuarianus is a bacterium related to mass mortality outbreaks of the Pacific oyster, Crassostrea gigas in Europe. In this study, the role of different planktonic substrates (phytoplankton cells, marine aggregates and chitin fragments) in mediating V. aestuarianus 02/041 infection of oysters was evaluated by controlled infection experiments. It was shown that phytoplankton cells and, to a greater extent, marine aggregates, significantly promote V. aestuarianus 02/041 intake by C. gigas maintained under stressful conditions in the laboratory. Such intake is associated with higher concentration of the pathogen in the bivalve hemolymph and compromised health status of infected oysters. In contrast, chitin particles do not play a significant role as transmission vector for V. aestuarianus 02/041 infecting its bivalve host. Interestingly, incorporation into marine aggregates foster extracellular proteases (ECPs) activity and a higher expression of bacterial virulence genes, that are potentially involved in bivalve infection. Results from this study contribute to elucidate transmission patterns of V. aestuarianus 02/041 to C. gigas that may be useful for the development of efficient measures to prevent and control oyster disease outbreaks.

LysM-mediated signaling in Marchantia polymorpha highlights the conservation of pattern-triggered immunity in land plants

Pattern-recognition receptor (PRR)-triggered immunity (PTI) wards off a wide range of pathogenic microbes, playing a pivotal role in angiosperms. The model liverwort Marchantia polymorpha triggers defense-related gene expression upon sensing components of bacterial and fungal extracts, suggesting the existence of PTI in this plant model. However, the molecular components of the putative PTI in M.polymorpha and the significance of PTI in bryophytes have not yet been described. We here show that M.polymorpha has four lysin motif (LysM)-domain-containing receptor homologs, two of which, LysM-receptor-like kinase (LYK) MpLYK1 and LYK-related (LYR) MpLYR, are responsible for sensing chitin and peptidoglycan fragments, triggering a series of characteristic immune responses. Comprehensive phosphoproteomic analysis of M.polymorpha in response to chitin treatment identified regulatory proteins that potentially shape LysM-mediated PTI. The identified proteins included homologs of well-described PTI components in angiosperms as well as proteins whose roles in PTI are not yet determined, including the blue-light receptor phototropin MpPHOT. We revealed that MpPHOT is required for negative feedback of defense-related gene expression during PTI. Taken together, this study outlines the basic framework of LysM-mediated PTI in M.polymorpha and highlights conserved elements and new aspects of pattern-triggered immunity in land plants.

Structural and functional analysis of the cerato-platanin-like protein Cpl1 suggests diverging functions in smut fungi.

Plant-pathogenic fungi are causative agents of the majority of plant diseases and can lead to severe crop loss in infected populations. Fungal colonization is achieved by combining different strategies, such as avoiding and counteracting the plant immune system and manipulating the host metabolome. Of major importance are virulence factors secreted by fungi, which fulfil diverse functions to support the infection process. Most of these proteins are highly specialized, with structural and biochemical information often absent. Here, we present the atomic structures of the cerato-platanin-like protein Cpl1 from Ustilago maydis and its homologue Uvi2 from Ustilago hordei. Both proteins adopt a double-Ψβ-barrel architecture reminiscent of cerato-platanin proteins, a class so far not described in smut fungi. Our structure-function analysis shows that Cpl1 binds to soluble chitin fragments via two extended grooves at the dimer interface of the two monomer molecules. This carbohydrate-binding mode has not been observed previously and expands the repertoire of chitin-binding proteins. Cpl1 localizes to the cell wall of U. maydis and might synergize with cell wall-degrading and decorating proteins during maize infection. The architecture of Cpl1 harbouring four surface-exposed loop regions supports the idea that it might play a role in the spatial coordination of these proteins. While deletion of cpl1 has only mild effects on the virulence of U. maydis, a recent study showed that deletion of uvi2 strongly impairs U. hordei virulence. Our structural comparison between Cpl1 and Uvi2 reveals sequence variations in the loop regions that might explain a diverging function.

The role of FIBCD1 in response to Aspergillus fumigatus in lung epithelial cells.

Chitin, a polysaccharide, is ubiquitously found in nature and has been known to be an active immunogen in mammals, and interacts with Toll-like, mannose and glucan receptors, to induce cytokine and chemokine secretions. FIBCD1 is a tetrameric type II transmembrane endocytic vertebrate receptor that binds chitin, is found in human lung epithelium and modulates lung epithelial inflammatory responses to A. fumigatus cell wall polysaccharides. We previously reported the detrimental role of FIBCD1 in a murine model of pulmonary invasive aspergillosis. However, the effect that chitin and chitin-containing A. fumigatus conidia exerts on lung epithelium following exposure through FIBCD1 is not yet fully explored. Using both in vitro and in vivo strategies, we examined how lung and lung epithelial gene expression are modified after exposure to fungal conidia or chitin fragments in the presence or absence of FIBCD1. FIBCD1 expression was associated with a decrease in inflammatory cytokines with increasing size of chitin (dimer-oligomer). Thus, our results demonstrate that FIBCD1 expression modulates cytokine and chemokine expression in response to A. fumigatus conidia that is modified by the presence of chitin particles.

Bioefficacy of fungal chitin oligomers in the control of postharvest decay in tomato fruit

Tomato is one of the most commercialised and consumed fruits worldwide. However, tomatoes are highly susceptible to Alternaria rot. Among the safe strategies proposed to control Alternaria rot is the induction of defence mechanisms through biological elicitors, such as chitin. Chitin and its oligosaccharides are an activate plant defence mechanisms, but studies of fruits exposed to fungal chitin fragments are scarce. Therefore, the present work aimed to obtain and partially characterise chitin oligomers of Alternaria alternata, and evaluate their effect on the defence mechanism of tomato fruits and their tolerance to Alternaria rot. The chitin oligomers obtained had a molecular weight of ≤ 1 kDa, 12% N-acetyl-glucosamine, 0.2% residual protein, and were 94% acetylated. These oligomers markedly increased the enzymatic activity of chitinase and β-1,3-glucanase in tomato fruits, and the development of Alternaria rot was inhibited by 78%. Chitin oligomers of A. alternata represent a promising alternative to attenuate Alternaria rot in tomato fruits through an enzymatic defence mechanism.

Surface sulfation of crab chitin for anisotropic swelling and nanodispersion

Crab chitin was sulfated under heterogeneous conditions using sulfamic acid in N’N-dimethylformamide to selectively sulfate the microfibril surface. The degree of sulfation followed a first order kinetics assuming a limited available reaction sites on the surface, and leveled off at a bulk degree-of-substitution of 0.4, corresponding to 2 mol/kg of sulfate groups. The reaction rate was proportional to the square of sulfamic acid concentration, suggesting involvement of two sulfamic acid molecules in a reaction. When washed with water after sulfation, the crab shell chitin fragments swelled anisotropically in the helicoidal axis direction, revealing regular alternating birefringence under optical microscope. Further mechanical treatment with high-pressure homogenizer led to slender nanofibrils, whose diameters were of the order of 6 nm according to turbidimetric analysis, in agreement with the Scherrer size estimated from X-ray diffraction line broadening. Both atomic force microscopy and transmission electron microscopy measurement showed presence of further smaller fragments with diameter of 3–4 nm and contour length of 0.5–1 \(\upmu \hbox {m}\) with a few kinks. The current approach presents a rapid and efficient modification to chitin, and a strategy for the preparation of stable nanochitin suspension.

Phosphate Record in Pleistocene-Holocene Sediments from Denisova Cave: Formation Mechanisms and Archaeological Implications

The distribution of authigenic phosphates in the sedimentary sequence of prehistoric Denisova Cave (Altai, South Siberia) has important archeological implications. The sampled Late Pleistocene–Early Holocene sedimentary sequence in the East Chamber of the cave consists of argilo-sandy-phosphatic sediments intercalated with guano layers of insectivorous bats. The sediments bear partially degraded N-rich organic matter (OM); chitin fragments enriched in S, P, Zn, and Cu; and a set of phosphates. The guano layers record at least three prolonged episodes of cave occupation by colonies of insectivorous bats between 10 kyr and 5 kyr BP, after people had left the cave or visited it rarely in small groups. The formation of phosphates follows the OM biodegradation pathways, with acidic leaching and gradual neutralization of P-rich solutions. The depth profile of authigenic phosphates shows a suite of mineral assemblages that mark a trend from acidic to slightly alkaline pH conditions of guano degradation (from top to bottom): ardealite, taranakite, and leucophosphite corresponding to acidic environments; whitlockite, brushite, and hydroxylapatite, which are stable under slightly acidic and neutral conditions; and hydroxylapatite in coexistence with calcite and stable at the bottom of the leaching profile under alkaline conditions. Authigenic phosphates can be used as reliable indicators of human non-occupation (abandonment) periods of Denisova Cave. Acidic leaching is responsible for disturbance and/or elimination of archaeological and paleontological materials in Late Pleistocene–Early Holocene sediments that were exposed to at least three “acidic waves”.

Identification of fresh and burnt bat guano and pigeon droppings in Eastern Mediterranean karstic cave sites based on micromorphological and chemical characteristics

Karstic caves in the Eastern Mediterranean region hold significant archaeological importance as they served as an important dwelling place for prehistoric hominins. Yet, such caves were used at the same time as a habitat for birds and bats. As a result, bat guano and bird droppings are not only a major source of cave deposits, but also trigger significant chemical alterations (diagenesis) due to their decomposition that can greatly affect the preservation of archaeological materials as reported from many Palaeolithic cave sites. While the diagenetic processes associated with bat guano degradation are quite well understood, the identification of partially-preserved or burnt guano residues in archaeological cave sites has not received much attention. Here we present a micromorphological, chemical and mineralogical study of modern guano of insectivorous bats, frugivorous bats and pigeons, collected from karstic caves in Israel. Each of these guano types was studied in fresh, charred and ashed forms. Based on the patterns obtained we present guidelines that allow identification of partially-degraded and burnt bat and bird guano remains. These include (1) high phosphorous and calcium associated with high quantities of chitin fragments in insectivorous bat guano; (2) low phosphorous and calcium associated with high quantities of seed coats, fibrous organic matter and red oval organic particles in frugivorous bat and pigeon guano. We found that faecal spherulites are present in bat guano and absent in pigeon droppings. Blackening and rounding of the edges of organic components characterise guano upon charring, while ashed guano is characterised by a micritic calcite groundmass in the case of fruit bat and pigeon guano without any pseudomorphs after calcium oxalate, unlike wood ash. We further show that ashed insectivorous bat guano is characterised by a micritic groundmass that includes phosphatic particles and potassium sulphate. These data indicate that while the combustion of guano presents similarities to other organic-based burnt deposits found in archaeological sites, a careful micromorphological study, aided by certain elemental and mineralogical signatures, can reveal whether or not burnt deposits originate from guano. Last, we studied the micromorphology of in situ degraded frugivorous and insectivorous bat guano in caves, observing that a bedded and laminated structure characterises fresh deposits while, as degradation progresses the proportion of authigenic minerals, appearing as nodules and veins, increases, disrupting the laminated structure. Importantly, chitin fragments in insectivorous bat guano and seed coats and some organic fibers in frugivorous bat guano preserve within degraded bat guano deposits and can serve as indicators for the presence and feeding habits of bats. Applying the guidelines given here to archaeological studies at prehistoric cave sites may produce new insights regarding human behavior, human–bat interactions, as well as for the formation processes at these sites.

Structural and functional analysis of Chitinase-IV of Brassica juncea: molecular modeling and dynamic simulation study

Brassica juncea is an important oil seed crop. The productivity of this plant, however, is known to be low due to the attack of plant pathogens. The plant chitinase-IV is known to hydrolyse the chitin present in the cell walls of the plant pathogens and thus enhance the plant defense systems. In this connection, studies were carried out by us on the prediction and characterization of the 3D structure of chitinase-IV, the structural changes that take place when the protein is in complex with Allosamidin and the chitin fragments (Tri-oligosaccharide and N-acetyl glucosamine) that act as elicitors to induce plant innate immunity against the invading pathogens, and molecular dynamic simulation studies on the stability of these complexes. These studies are expected to give us an insight into the chitin-binding domain and information on the dynamics and energetics of the protein, which is not possible to obtain by experimental methods. The predicted 3D structure of the protein should give us a better understanding of the molecular function of the chitinase gene in Brassica juncea for devising better methods of biocontrol against fungal phytopathogens and harmful insects so as to increase the crop yield.

Chitin and Chitosan Fragments Responsible for Plant Elicitor and Growth Stimulator.

Chitin and chitosan are natural polysaccharides with huge application potential in agriculture, such as promoting plant growth, eliciting plant resistance against biotic and abiotic stress, and activating symbiotic signaling between plants and beneficial microorganisms. Chitin and chitosan offer a sustainable alternative for future crop production. The bioactivities of chitin and chitosan closely depend on their structural factors, including molecular size, degree of acetylation, and pattern of acetylation. It is of great significance to identify the key fragments in chitin and chitosan chains that are responsible for these agricultural bioactivities. Herein, we review the recent progress in the structure-function relationship of chitin and chitosan in the field of agriculture application. The preparation of chitin and chitosan fragments and their action mode for plant protection and growth are also discussed.

Switching perception of friend and foe

Plant Science Lysine motif receptors in plants perceive glycans that signal the presence of pathogenic or symbiotic nitrogen-fixing microbes. Bozsoki et al. now define the portions of these receptors that create the discriminatory binding pocket (see the Perspective by Bisseling and Geurts). The motifs were conserved in receptors that initiate immune responses, reflecting the invariable nature of the chitin fragments that they sense. Conversely, the motifs in receptors that respond to symbiotic signals were more varied, reflecting the greater diversity of the lipochitooligosaccharides (Nod factors) that they sense. With domain swapping, the authors switched the Nod factor specificity of receptors from two legume species and also enabled a chitin receptor that was otherwise dedicated to the detection of pathogenic microbes to instead recognize Nod factors. Science , this issue p. [663][1]; see also p. [620][2] [1]: /lookup/doi/10.1126/science.abb3377 [2]: /lookup/doi/10.1126/science.abd3857

Ligand-recognizing motifs in plant LysM receptors are major determinants of specificity.

Plants evolved lysine motif (LysM) receptors to recognize and parse microbial elicitors and drive intracellular signaling to limit or facilitate microbial colonization. We investigated how chitin and nodulation (Nod) factor receptors of Lotus japonicus initiate differential signaling of immunity or root nodule symbiosis. Two motifs in the LysM1 domains of these receptors determine specific recognition of ligands and discriminate between their in planta functions. These motifs define the ligand-binding site and make up the most structurally divergent regions in cognate Nod factor receptors. An adjacent motif modulates the specificity for Nod factor recognition and determines the selection of compatible rhizobial symbionts in legumes. We also identified how binding specificities in LysM receptors can be altered to facilitate Nod factor recognition and signaling from a chitin receptor, advancing the prospects of engineering rhizobial symbiosis into nonlegumes.

Integrating insect frass biofertilisers into sustainable peri-urban agro-food systems

The larvae of black soldier fly (BSF) have shown great promise in transforming organic wastes into a more valuable larval biomass. Importantly, after insects have been harvested the remaining by-product, comprised of the spent substrate and frass (insect faeces), has the potential to be used as a biofertiliser. Three field-scale experiments to investigate whether frass biofertilisers (made from either poultry waste, brewery waste or green market waste) could be successfully incorporated into current small-holder farming practices were undertaken in Ghana, West Africa. In general, BSF frass biofertilisers performed as well as the local practice of amending Zaï planting pits with chicken manure, or incorporating uniformly broadcast fertilisers. For short-cycle cash crops such as chilli pepper and shallots, brewery waste biofertilisers performed better than poultry waste biofertilisers, particularly when added in combination with inorganic NPK fertilisers. For maize, green market waste biofertiliser did not significantly improve yield at applications of either 5 or 10 t/ha, even when combined with inorganic fertilisers. However, frass biofertiliser amendment did significantly reduce the loss of cowpea plants due to Fusarium wilt disease. We hypothesise that the fragments of chitin (originating from 4-5 larval moults) in frass biofertilisers can induce disease resistance in crop plants grown in biofertiliser-amended soil. The benefit of frass as a by-product of insect larvae production can increase the profitability of this burgeoning industry in developing countries, and provide employment opportunities and self-sufficiency in the nutrient supply chain by integrating organic waste management and insect farming into peri-urban agro-food systems.

Secondary types of food in the diet of a small-sized mammalian herbivore: from species ecology to habitat inferences

A macroscopic analysis of stomach contents is undertaken in the common vole, Microtus arvalis, obscurus chromosomal form in order to assess the patterns of non-herbivorous foraging activity of a herbivore distributed in different Eurasian biomes. The samples are collected in the Ural Mountains at midsummer (8 localities, 60° 38′ N to 51° 07′ N), and in winter (one locality, 56° 37′ N). Based on the frequency of occurrence and quantitative abundance in stomachs, photoassimilating parts of vascular plants are considered as the primary food category and inflorescences, roots, seeds, mycorrhizal fungi, arthropods, chitin fragments, colorless amorphous matter, and mosses are recognized as the secondary food categories. Although no alternatives to the primary food are found, some of the supplements substitute one another either geographically (fungi in the north versus mature seeds in the south) or by the vertical layers of habitats (seeds and arthropods in the herbaceous layer versus roots and fungi in the shallow layers of soil). Non-staple food consumption in different latitudinal zones at midsummer reflects spatiotemporal dynamics of food availability. From taiga to steppes of the Urals, the common vole is sporadically included in soil foraging but the consumption of mycorrhizal fungi (Glomerales) is only revealed within the zones of dark coniferous forests. The results suggest that the extents of inclusion of a herbivore in soil food webs might vary among ecosystems. In a paleoecological perspective, the analysis of stomach contents in a herbivorous micromammal might reveal fine-scale details of the habitats and provides insights into the stages of the growing season.

Possible Role of Chitin‐Like Proteins in the Etiology of Alzheimer's Disease

Chitin is a β‐linked straight chain carbohydrate matrix monopolymer prominent in invertebrates, from fungi to arthropods. Surprisingly, chitin is now documented in vertebrates, including humans, a component of vertebrate physiology that has been neglected until now. Chitin levels are elevated in Alzheimer's disease (AD) patients, not only in the central nervous system but also in the cerebrospinal fluid and plasma. Elevated levels of chitin lectin have been reported in patients with AD. Chitinase activity varies widely in the human population. Chitin levels can increase in individuals with intrinsically low chitinase activity. Elevated amounts of chitin can reflect accumulation of the small chitin fragments that remain wherever rapid hyaluronan synthesis occurs. Another source of chitin may be from remote fungal infections. Chitin can be toxic for neurons, and its accumulation may lead to the development of AD. We present new suggestions for animal models and treatment modalities that could prove useful in future research endeavors. An unexpected connection with Gaucher's disease patients and their heterozygote relatives is also identified. These chitin‐related mechanisms are novel approaches to AD whose etiology until now has defied explication.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chitin: A "Hidden Figure" in the Fungal Cell Wall.

Chitin and chitosan are two related polysaccharides that provide important structural stability to fungal cell walls. Often embedded deeply within the cell wall structure, these molecules anchor other components at the cell surface. Chitin-directed organization of the cell wall layers allows the fungal cell to effectively monitor and interact with the external environment. For fungal pathogens, this interaction includes maintaining cellular strategies to avoid excessive detection by the host innate immune system. In turn, mammalian and plant hosts have developed their own strategies to process fungal chitin, resulting in chitin fragments of varying molecular size. The size-dependent differences in the immune activation behaviors of variably sized chitin molecules help to explain how chitin and related chitooligomers can both inhibit and activate host immunity. Moreover, chitin and chitosan have recently been exploited for many biomedical applications, including targeted drug delivery and vaccine development.

Enzymatic treatments as alternative to produce chitin fragments of low molecular weight from Alternaria alternata

ABSTRACTChitin and its oligosaccharides are involved in the plant defense response against fungal pathogens. In most studies, these molecules come from crustacean shell, and there are scarce studies on the use of fungal chitin. Usually the extraction of chitin is by alkaline treatments, which affect the acetylation degree (DA), and the obtaining of oligosaccharides of low molecular weight; so the use of enzymes is proposed as an alternative treatment to obtain chitin oligosaccharides from fungi. The objective of this work was to characterize the chitin fragments of Alternaria alternata extracted by alkaline and enzymatic treatments. The chitin extracted was ultrasonicated and ultrafiltrated to produce chitin fragments. Enzymatic treatments decreased the protein content to 29%, and the bands corresponding to β‐glucans decreased when β‐1,3‐glucanase was applied. Fragments smaller than 1 kDa, DA of 76.7%, and 35.4 μg glucosamine/mg dry weight were obtained by enzymatic treatments. The enzymatic treatments show promising results to extract chitin fragments from A. alternata without severely affect the DA of the molecule. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019, 136, 47339.

Possible Role of Chitin-Like Proteins in the Etiology of Alzheimer's Disease.

Chitin is a β-linked straight chain carbohydrate matrix monopolymer prominent in invertebrates, from fungi to arthropods. Surprisingly, chitin is now documented in vertebrates, including humans, a component of vertebrate physiology that has been neglected until now. Chitin levels are elevated in Alzheimer's disease (AD) patients, not only in the central nervous system but also in the cerebrospinal fluid and plasma. Elevated levels of chitin lectin have been reported in patients with AD. Chitinase activity varies widely in the human population. Chitin levels can increase in individuals with intrinsically low chitinase activity. Elevated amounts of chitin can reflect accumulation of the small chitin fragments that remain wherever rapid hyaluronan synthesis occurs. Another source of chitin may be from remote fungal infections. Chitin can be toxic for neurons, and its accumulation may lead to the development of AD. We present new suggestions for animal models and treatment modalities that could prove useful in future research endeavors. An unexpected connection with Gaucher's disease patients and their heterozygote relatives is also identified. These chitin-related mechanisms are novel approaches to AD whose etiology until now has defied explication.

Suppression of Plant Immunity by Fungal Chitinase-like Effectors

Crop diseases caused by fungi constitute one of themost important problems in agriculture, posing a serious threat to food security [1]. To establish infection, phytopathogens interfere with plant immune responses [2, 3]. However, strategies to promote virulence employed by fungal pathogens, especially non-model organisms, remain elusive [4], mainly because fungi are more complex and difficult to study when compared to the better-characterized bacterial pathogens. Equally incomplete is our understanding of the birth of microbial virulence effectors. Here, we show that the cacao pathogen Moniliophthora perniciosa evolved an enzymatically inactive chitinase (MpChi) that functions as a putative pathogenicity factor. MpChi is among the most highly expressed fungal genes during the biotrophic interaction with cacao and encodes a chitinase with mutations that abolish its enzymatic activity. Despite the lack of chitinolytic activity, MpChi retains substrate binding specificity and prevents chitin-triggered immunity by sequestering immunogenic chitin fragments. Remarkably, its sister species M.roreri encodes a second non-orthologous catalytically impaired chitinase with equivalent function. Thus, a class of conserved enzymes independently evolved as putative virulence factors in these fungi. In addition to unveiling a strategy of host immune suppression by fungal pathogens, our results demonstrate that the neofunctionalization of enzymes may be an evolutionary pathway for the rise of new virulence factors in fungi. We anticipate that analogous strategies are likely employed by other pathogens.

Role of the Nod Factor Hydrolase MtNFH1 in Regulating Nod Factor Levels during Rhizobial Infection and in Mature Nodules of Medicago truncatula.

Establishment of symbiosis between legumes and nitrogen-fixing rhizobia depends on bacterial Nod factors (NFs) that trigger symbiosis-related NF signaling in host plants. NFs are modified oligosaccharides of chitin with a fatty acid moiety. NFs can be cleaved and inactivated by host enzymes, such as MtNFH1 (MEDICAGO TRUNCATULA NOD FACTOR HYDROLASE1). In contrast to related chitinases, MtNFH1 hydrolyzes neither chitin nor chitin fragments, indicating a high cleavage preference for NFs. Here, we provide evidence for a role of MtNFH1 in the symbiosis with Sinorhizobium meliloti Upon rhizobial inoculation, MtNFH1 accumulated at the curled tip of root hairs, in the so-called infection chamber. Mutant analysis revealed that lack of MtNFH1 delayed rhizobial root hair infection, suggesting that excess amounts of NFs negatively affect the initiation of infection threads. MtNFH1 deficiency resulted in nodule hypertrophy and abnormal nodule branching of young nodules. Nodule branching was also stimulated in plants expressing MtNFH1 driven by a tandem CaMV 35S promoter and plants inoculated by a NF-overproducing S. meliloti strain. We suggest that fine-tuning of NF levels by MtNFH1 is necessary for optimal root hair infection as well as for NF-regulated growth of mature nodules.