Lipo-chitooligosaccharide Signals Research Articles

The Medicago truncatula hydrolase MtCHIT5b degrades Nod factors of Sinorhizobium meliloti and cooperates with MtNFH1 to regulate the nodule symbiosis.

Nod factors secreted by nitrogen-fixing rhizobia are lipo-chitooligosaccharidic signals required for establishment of the nodule symbiosis with legumes. In Medicago truncatula, the Nod factor hydrolase 1 (MtNFH1) was found to cleave Nod factors of Sinorhizobium meliloti. Here, we report that the class V chitinase MtCHIT5b of M. truncatula expressed in Escherichia coli can release lipodisaccharides from Nod factors. Analysis of M. truncatula mutant plants indicated that MtCHIT5b, together with MtNFH1, degrades S. meliloti Nod factors in the rhizosphere. MtCHIT5b expression was induced by treatment of roots with purified Nod factors or inoculation with rhizobia. MtCHIT5b with a fluorescent tag was detected in the infection pocket of root hairs. Nodulation of a MtCHIT5b knockout mutant was not significantly altered whereas overexpression of MtCHIT5b resulted in fewer nodules. Reduced nodulation was observed when MtCHIT5b and MtNFH1 were simultaneously silenced in RNA interference experiments. Overall, this study shows that nodule formation of M. truncatula is regulated by a second Nod factor cleaving hydrolase in addition to MtNFH1.

Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors

Plants and animals use cell surface receptors to sense and interpret environmental signals. In legume symbiosis with nitrogen-fixing bacteria, the specific recognition of bacterial lipochitooligosaccharide (LCO) signals by single-pass transmembrane receptor kinases determines compatibility. Here, we determine the structural basis for LCO perception from the crystal structures of two lysin motif receptor ectodomains and identify a hydrophobic patch in the binding site essential for LCO recognition and symbiotic function. We show that the receptor monitors the composition of the amphiphilic LCO molecules and uses kinetic proofreading to control receptor activation and signaling specificity. We demonstrate engineering of the LCO binding site to fine-tune ligand selectivity and correct binding kinetics required for activation of symbiotic signaling in plants. Finally, the hydrophobic patch is found to be a conserved structural signature in this class of LCO receptors across legumes that can be used for in silico predictions. Our results provide insights into the mechanism of cell-surface receptor activation by kinetic proofreading of ligands and highlight the potential in receptor engineering to capture benefits in plant-microbe interactions.

Distinct genetic basis for root responses to lipo-chitooligosaccharide signal molecules from different microbial origins.

Lipo-chitooligosaccharides (LCOs) were originally found as symbiotic signals called Nod Factors (Nod-LCOs) controlling the nodulation of legumes by rhizobia. More recently, LCOs were also found in symbiotic fungi and, more surprisingly, very widely in the kingdom Fungi, including in saprophytic and pathogenic fungi. The LCO-V(C18:1, fucosylated/methyl fucosylated), hereafter called Fung-LCOs, are the LCO structures most commonly found in fungi. This raises the question of how legume plants such as Medicago truncatula can discriminate between Nod-LCOs and Fung-LCOs. To address this question, we performed a genome-wide association study on 173 natural accessions of M. truncatula, using a root branching phenotype and a newly developed local score approach. Both Nod-LCOs and Fung-LCOs stimulated root branching in most accessions, but the root responses to these two types of LCO molecules were not correlated. In addition, the heritability of the root response was higher for Nod-LCOs than for Fung-LCOs. We identified 123 loci for Nod-LCO and 71 for Fung-LCO responses, of which only one was common. This suggests that Nod-LCOs and Fung-LCOs both control root branching but use different molecular mechanisms. The tighter genetic constraint of the root response to Fung-LCOs possibly reflects the ancestral origin of the biological activity of these molecules.

Phytomicrobiome Coordination Signals Hold Potential for Climate Change-Resilient Agriculture.

A plant growing under natural conditions is always associated with a substantial, diverse, and well-orchestrated community of microbes—the phytomicrobiome. The phytomicrobiome genome is larger and more fluid than that of the plant. The microbes of the phytomicrobiome assist the plant in nutrient uptake, pathogen control, stress management, and overall growth and development. At least some of this is facilitated by the production of signal compounds, both plant-to-microbe and microbe back to the plant. This is best characterized in the legume nitrogen fixing and mycorrhizal symbioses. More recently lipo-chitooligosaccharide (LCO) and thuricin 17, two microbe-to-plant signals, have been shown to regulate stress responses in a wide range of plant species. While thuricin 17 production is constitutive, LCO signals are only produced in response to a signal from the plant. We discuss how some signal compounds will only be discovered when root-associated microbes are exposed to appropriate plant-to-microbe signals (positive regulation), and this might only happen under specific conditions, such as abiotic stress, while others may only be produced in the absence of a particular plant-to-microbe signal molecule (negative regulation). Some phytomicrobiome members only elicit effects in a specific crop species (specialists), while other phytomicrobiome members elicit effects in a wide range of crop species (generalists). We propose that some specialists could exhibit generalist activity when exposed to signals from the correct plant species. The use of microbe-to-plant signals can enhance crop stress tolerance and could result in more climate change resilient agricultural systems.

The NMN Module Conducts Nodule Number Orchestra.

Legumes control nodule number through nodulation and autoregulation of nodulation (AON) pathways. Nodule Inception (NIN) is essential for rhizobial infection and nodule organogenesis in legumes. The GmNINa-miR172c-NNC1 (NMN) module, which consists of two positive regulators, GmNINa and miR172c, and a suppressor, NNC1, integrates both pathways. GmNINa activates miR172c to downregulate NNC1, leading to nodulation, while NNC1 inhibits miR172c expression, forming a negative feedback loop. GmNINa and NNC1 interact with each other and antagonistically fine-tune GmRIC1/RIC2 expression, turning AON on and off. Conversely, activation of AON inhibits GmNINa and miR172c expression, thereby reducing their inhibitory effects on NNC1 to attenuate both nodulation signaling and AON. The NMN module functions not only as an “accelerator” of the nodulation signal to promote nodulation but also as a “brake” on the signal by activating AON to orchestrate nodule number.

Mini-Review: Nod Factor Regulation of Phytohormone Signaling and Homeostasis During Rhizobia-Legume Symbiosis.

The rhizobia-legume symbiosis is a mutualistic association in which bacteria provide plants with nitrogen compounds and the plant provides bacteria with carbon sources. A successful symbiotic interaction relies on a molecular dialog between the plant and the bacteria, and generally involves rhizobial lipo-chitooligosaccharide signals called Nod factors (NFs). In most cases, specific NF perception is required for rhizobia to enter root cells through newly formed intracellular structures called infection threads (ITs). Concomitantly to IT formation in root hairs, root cortical cells start to divide to create a new root organ called the nodule, which will provide the bacteria with a specific micro-environment required for symbiotic nitrogen fixation. During all these steps of plant–bacteria interaction, new plant cellular compartments and developmental programs are activated. This interaction is costly for the plant that tightly controls symbiosis establishment and functioning. Phytohormones are key regulators of cellular and developmental plasticity in plants, and they are influential endogenous signals that rapidly control plant responses. Although early symbiotic responses were known for decades to be linked to phytohormone-related responses, new data reveal the molecular mechanisms involved and links between phytohormones and the control of early symbiotic events. Reciprocally, NF signaling also targets phytohormone signaling pathways. In this review, we will focus on the emerging notion of NF and phytohormone signaling crosstalk, and how it could contribute to the tight control of symbiosis establishment in legume host plants.

Down-regulation of a Phaseolus vulgaris annexin impairs rhizobial infection and nodulation

Legumes establish a symbiotic relationship with N2-fixing soil bacteria known as rhizobia. This process is initiated by a molecular dialog between roots and bacteria. Rhizobia secrete lipochitooligosaccharide signals, known as Nod factors, into the rhizosphere that are perceived by root hairs and induce an increase in Ca2+ levels, ROS production, and transcriptional activation of nodulin genes. Plant annexins are a family of calcium- and membrane-binding proteins involved in growth and development and in several abiotic and biotic responses. In this study, we identified thirteen annexin genes in the Phaseolus vulgaris (common bean) genome. We show that PvAnn1 transcript abundance was higher in root hairs than in root apices and stripped roots, and that during symbiosis, PvAnn1 transcript abundance was substantially higher in nodules than in nodule-stripped roots. RNAi knock-down of PvAnn1 resulted in reduced root hair lengths and ROS production. Furthermore, PvAnn1-RNAi transgenic roots that were inoculated with rhizobia showed a decrease in the progression and number of infection threads, possibly due to changes in Ca2+-ROS homeostasis and nodulin signaling. Furthermore, a reduced number of nodules and less nitrogen fixation was observed in the transgenic plants at 21 days post infection. Together, these data suggest that PvAnn1 is involved in rhizobial infection and nodule development and function.

The strigolactone biosynthesis gene DWARF27 is co-opted in rhizobium symbiosis

BackgroundStrigolactones are a class of plant hormones whose biosynthesis is activated in response to phosphate starvation. This involves several enzymes, including the carotenoid cleavage dioxygenases 7 (CCD7) and CCD8 and the carotenoid isomerase DWARF27 (D27). D27 expression is known to be responsive to phosphate starvation. In Medicago truncatula and rice (Oryza sativa) this transcriptional response requires the GRAS-type proteins NSP1 and NSP2; both proteins are essential for rhizobium induced root nodule formation in legumes. In line with this, we questioned whether MtNSP1-MtNSP2 dependent MtD27 regulation is co-opted in rhizobium symbiosis.ResultsWe provide evidence that MtD27 is involved in strigolactone biosynthesis in M. truncatula roots upon phosphate stress. Spatiotemporal expression studies revealed that this gene is also highly expressed in nodule primordia and subsequently becomes restricted to the meristem and distal infection zone of a mature nodules. A similar expression pattern was found for MtCCD7 and MtCCD8. Rhizobium lipo-chitooligosaccharide (LCO) application experiments revealed that of these genes MtD27 is most responsive in an MtNSP1 and MtNSP2 dependent manner. Symbiotic expression of MtD27 requires components of the symbiosis signaling pathway; including MtDMI1, MtDMI2, MtDMI3/MtCCaMK and in part MtERN1. This in contrast to MtD27 expression upon phosphate starvation, which only requires MtNSP1 and MtNSP2.ConclusionOur data show that the phosphate-starvation responsive strigolactone biosynthesis gene MtD27 is also rapidly induced by rhizobium LCO signals in an MtNSP1 and MtNSP2-dependent manner. Additionally, we show that MtD27 is co-expressed with MtCCD7 and MtCCD8 in nodule primordia and in the infection zone of mature nodules.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0651-x) contains supplementary material, which is available to authorized users.

Lipochitooligosaccharides modulate plant host immunity to enable endosymbioses.

Symbiotic nitrogen-fixing rhizobium bacteria and arbuscular mycorrhizal fungi use lipochitooligosaccharide (LCO) signals to communicate with potential host plants. Upon a compatible match, an intimate relation is established during which the microsymbiont is allowed to enter root (-derived) cells. Plants perceive microbial LCO molecules by specific LysM-domain-containing receptor-like kinases. These do not only activate a common symbiosis signaling pathway that is shared in both symbioses but also modulate innate immune responses. Recent studies revealed that symbiotic LCO receptors are closely related to chitin innate immune receptors, and some of these receptors even function in symbiosis as well as immunity. This raises questions about how plants manage to translate structurally very similar microbial signals into different outputs. Here, we describe the current view on chitin and LCO perception in innate immunity and endosymbiosis and question how LCOs might modulate the immune system. Furthermore, we discuss what it takes to become an endosymbiont.

Aphanomyces euteiches Cell Wall Fractions Containing Novel Glucan-Chitosaccharides Induce Defense Genes and Nuclear Calcium Oscillations in the Plant Host Medicago truncatula

N-acetylglucosamine-based saccharides (chitosaccharides) are components of microbial cell walls and act as molecular signals during host-microbe interactions. In the legume plant Medicago truncatula, the perception of lipochitooligosaccharide signals produced by symbiotic rhizobia and arbuscular mycorrhizal fungi involves the Nod Factor Perception (NFP) lysin motif receptor-like protein and leads to the activation of the so-called common symbiotic pathway. In rice and Arabidopsis, lysin motif receptors are involved in the perception of chitooligosaccharides released by pathogenic fungi, resulting in the activation of plant immunity. Here we report the structural characterization of atypical chitosaccharides from the oomycete pathogen Aphanomyces euteiches, and their biological activity on the host Medicago truncatula. Using a combination of biochemical and biophysical approaches, we show that these chitosaccharides are linked to β-1,6-glucans, and contain a β-(1,3;1,4)-glucan backbone whose β-1,3-linked glucose units are substituted on their C-6 carbon by either glucose or N-acetylglucosamine residues. This is the first description of this type of structural motif in eukaryotic cell walls. Glucan-chitosaccharide fractions of A. euteiches induced the expression of defense marker genes in Medicago truncatula seedlings independently from the presence of a functional Nod Factor Perception protein. Furthermore, one of the glucan-chitosaccharide fractions elicited calcium oscillations in the nucleus of root cells. In contrast to the asymmetric oscillatory calcium spiking induced by symbiotic lipochitooligosaccharides, this response depends neither on the Nod Factor Perception protein nor on the common symbiotic pathway. These findings open new perspectives in oomycete cell wall biology and elicitor recognition and signaling in legumes.

LysM domains of Medicago truncatula NFP protein involved in Nod factor perception. Glycosylation state, molecular modeling and docking of chitooligosaccharides and Nod factors

The establishment of the symbiosis between legume plants and rhizobial bacteria depends on the production of rhizobial lipo-chitooligosaccharidic signals (the Nod factors) that are specifically recognized by roots of the host plant. In Medicago truncatula, specific recognition of Sinorhizobium meliloti and its Nod factors requires the NFP (Nod factor perception) gene, which encodes a putative serine/threonine receptor-like kinase (RLK). The extracellular region of this protein contains three tandem lysin motifs (LysMs), a short peptide domain that is implicated in peptidoglycan or chitin binding in various bacterial or eukaryotic proteins, respectively. We report here the homology modeling of the three LysM domains of M. truncatula NFP based on the structure of a LysM domain of the Escherichia coli membrane-bound lytic murein transglycosidase D (MltD). Expression of NFP in a homologous system (M. truncatula roots) revealed that the protein is highly N-glycosylated, probably with both high-mannose and complex glycans. Surface analysis and docking calculations performed on the models of the three domains were used to predict the most favored binding modes for chitooligosaccharides and Nod factors. A convergent model can be proposed where the sulfated, O-acetylated lipo-chitooligosaccharidic Nod factor of S. meliloti binds in similar orientation to the three LysM domains of M. truncatula NFP. N-glycosylation is not expected to interfere with Nod factor binding in this orientation.

Pharmacological evidence that multiple phospholipid signaling pathways link Rhizobium nodulation factor perception in Medicago truncatula root hairs to intracellular responses, including Ca2+ spiking and specific ENOD gene expression.

Rhizobium nodulation (Nod) factors are specific lipochito-oligosaccharide signals essential for initiating in root hairs of the host legume developmental responses that are required for controlled entry of the microsymbiont. In this article, we focus on the Nod factor signal transduction pathway leading to specific and cell autonomous gene activation in Medicago truncatula cv Jemalong in a study making use of the Nod factor-inducible MtENOD11 gene. First, we show that pharmacological antagonists that interfere with intracellular ion channel and Ca2+ pump activities are efficient blockers of Nod factor-elicited pMtENOD11-beta-glucuronidase (GUS) expression in root hairs of transgenic M. truncatula. These results indicate that intracellular Ca2+ release and recycling activities, essential for Ca2+ spiking, are also required for specific gene activation. Second, pharmacological effectors that inhibit phospholipase D and phosphoinositide-dependent phospholipase C activities are also able to block pMtENOD11-GUS activation, thus underlining a central role for multiple phospholipid signaling pathways in Nod factor signal transduction. Finally, pMtENOD11-GUS was introduced into all three Nod-/Myc- dmi M. truncatula mutant backgrounds, and gene expression was evaluated in response to the mastoparan peptide agonist Mas7. We found that Mas7 elicits root hair MtENOD11 expression in dmi1 and dmi2 mutants, but not in the dmi3 mutant, suggesting that the agonist acts downstream of DMI1/DMI2 and upstream of DMI3. In light of these results and the recently discovered identities of the DMI gene products, we propose an integrated cellular model for Nod factor signaling in legume root hairs in which phospholipids play a key role in linking the Nod factor perception apparatus to downstream components such as Ca2+ spiking and ENOD gene expression.

Genetic and genomic analysis in model legumes bring Nod-factor signaling to center stage.

The control of host-specificity in the Rhizobium-legume symbiosis has been a topic of long-standing interest to plant biologists. By the early 1990s, biologists had deciphered the chemical signals that trigger early symbiotic responses. Flavonoids from the plant root trigger bacterial gene expression and the production of lipo-chitooligosaccharide signals (called Nod factors) that are recognized by the plant host. Genetic differences between bacterial strains modify the oligosaccharide backbone, for example by the addition of sulfate, acetate or fucose, and simultaneously alter the host-specificity of the purified Nod factor and the bacterium. Recent studies have begun to reveal the genetic and molecular basis of Nod-factor perception in legumes, a signaling system that also controls plant interactions with mycorrhizal fungi.

Dissection of Nod factor signalling in legumes: cell biology, mutants and pharmacological approaches.

Nodulation factors (NFs) are lipochito-oligosaccharide signal molecules excreted by soil-living rhizobia. These molecules elicit a range of responses in the legume roots, with which the bacteria can live in symbiosis. In this review we focus on the genetic, pharmacological and cell biological approaches that have been, and are being, undertaken to decipher the signalling pathways that lead to the symbiotic responses in the plant.

Structural determination of the lipo-chitin oligosaccharide nodulation signals produced by Rhizobium giardinii bv. giardinii H152

Rhizobium giardinii bv. giardinii is a microsymbiont of plants of the genus Phaseolus and produces extracellular signal molecules that are able to induce deformation of root hairs and nodule organogenesis. We report here the structures of seven lipochitooligosaccharide (LCO) signal molecules secreted by R. giardinii bv. giardinii H152. Six of them are pentamers of GlcNAc carrying C 16:0, C 18:0, C 20:0 and C 18:1 fatty acyl chains on the non-reducing terminal residue. Four are sulfated at C-6 of the reducing terminal residue and one is acetylated in the same position. Six of them are N-methylated on the non-reducing GlcN residue and all the nodulation factors are carbamoylated on C-6 of the non-reducing terminal residue. The structures were determined using monosaccharide composition and methylation analyses, 1D- and 2D-NMR experiments and a range of mass spectrometric techniques. The position of the carbamoyl substituent on the non-reducing glucosamine residue was determined using a CID-MSMS experiment and an HMBC experiment.

A receptor kinase gene regulating symbiotic nodule development.

Leguminous plants are able to establish a nitrogen-fixing symbiosis with soil bacteria generally known as rhizobia. Metabolites exuded by the plant root activate the production of a rhizobial signal molecule, the Nod factor, which is essential for symbiotic nodule development. This lipo-chitooligosaccharide signal is active at femtomolar concentrations, and its structure is correlated with host specificity of symbiosis, suggesting the involvement of a cognate perception system in the plant host. Here we describe the cloning of a gene from Medicago sativa that is essential for Nod-factor perception in alfalfa, and by genetic analogy, in the related legumes Medicago truncatula and Pisum sativum. The identified 'nodulation receptor kinase', NORK, is predicted to function in the Nod-factor perception/transduction system (the NORK system) that initiates a signal cascade leading to nodulation. The family of 'NORK extracellular-sequence-like' (NSL) genes is broadly distributed in the plant kingdom, although their biological function has not been previously ascribed. We suggest that during the evolution of symbiosis an ancestral NSL system was co-opted for transduction of an external ligand, the rhizobial Nod factor, leading to development of the symbiotic root nodule.

Nod factor induction of reactive oxygen species production is correlated with expression of the early nodulin gene rip1 in Medicago truncatula.

Plant genes that are specifically activated by the rhizobial lipochitooligosaccharide signal molecule (Nod factor) in legume hosts are collectively referred to as nodulins. Although nodulin gene expression is both spatially and temporally correlated with symbiosis, the function of these genes and the molecular events underlying their expression remain unknown. Sequence analysis of rip1, an early nodulin gene encoding a putative peroxidase protein, revealed the existence of sequence motifs with homology to reactive oxygen species (ROS) responsive cis elements. Here we report that recognition of compatible Nod factor rapidly stimulates a spatially localized production of reactive oxygen species in legume roots. Sinorhizobium meliloti mutants that produce an altered Nod factor structure and a nonnodulating plant mutant, dmi1-1, that is implicated in Nod factor signal transduction are equally impaired in the ability to elicit ROS production and rip1 expression. Interestingly, both rip1 transcription and ROS production exhibit the same tissue-specific pattern of localization. Moreover, exogenous hydrogen peroxide is sufficient to activate rip1 transcription. Taken together, these results suggest that ROS production is a consequence of specific Nod factor perception and implicate H2O2 produced during this response as a mediator of Nod factor-induced rip1 expression.

Structure-function analysis of nod factor-induced root hair calcium spiking in Rhizobium-legume symbiosis.

In the Rhizobium-legume symbiosis, compatible bacteria and host plants interact through an exchange of signals: Host compounds promote the expression of bacterial biosynthetic nod (nodulation) genes leading to the production of a lipochito-oligosaccharide signal, the Nod factor (NF). The particular array of nod genes carried by a given species of Rhizobium determines the NF structure synthesized and defines the range of legume hosts by which the bacterium is recognized. Purified NF can induce early host responses even in the absence of live Rhizobium One of the earliest known host responses to NF is an oscillatory behavior of cytoplasmic calcium, or calcium spiking, in root hair cells, initially observed in Medicago spp. and subsequently characterized in four other genera (D.W. Ehrhardt, R. Wais, S.R. Long [1996] Cell 85: 673-681; S.A. Walker, V. Viprey, J.A. Downie [2000] Proc Natl Acad Sci USA 97: 13413-13418; D.W. Ehrhardt, J.A. Downie, J. Harris, R.J. Wais, and S.R. Long, unpublished data). We sought to determine whether live Rhizobium trigger a rapid calcium spiking response and whether this response is NF dependent. We show that, in the Sinorhizobium meliloti-Medicago truncatula interaction, bacteria elicit a calcium spiking response that is indistinguishable from the response to purified NF. We determine that calcium spiking is a nod gene-dependent host response. Studies of calcium spiking in M. truncatula and alfalfa (Medicago sativa) also uncovered the possibility of differences in early NF signal transduction. We further demonstrate the sufficiency of the nod genes for inducing calcium spiking by using Escherichia coli BL21 (DE3) engineered to express 11 S. meliloti nod genes.

Rhizobium sp. BR816 produces a complex mixture of known and novel lipochitooligosaccharide molecules.

Rhizobial lipochitooligosaccharide (LCO) signal molecules induce various plant responses, leading to nodule development. We report here the LCO structures of the broadhost range strain Rhizobium sp. BR816. The LCOs produced are all pentamers, carrying common C18:1 or C18:0 fatty acyl chains, N-methylated and C-6 carbamoylated on the nonreducing terminal N-acetylglucosamine and sulfated on the reducing/terminal residue. A second acetyl group can be present on the penultimate N-acetylglucosamine from the nonreducing terminus. Two novel characteristics were observed: the reducing/terminal residue can be a glucosaminitol (open structure) and the degree of acetylation of this glucosaminitol or of the reducing residue can vary.

Nod factors of Rhizobium leguminosarum bv. viciae and their fucosylated derivatives stimulate a nod factor cleaving activity in pea roots and are hydrolyzed in vitro by plant chitinases at different rates.

Nod factors (NFs) are rhizobial lipo-chitooligosaccharide signals that trigger root nodule development in legumes. Modifications of NF structures influence their biological activity and affect their degradation by plant chitinases. Nodulation of certain pea cultivars by Rhizobium leguminosarum bv. viciae requires modification of NFs at the reducing end by either an O-acetyl or a fucosyl group. Fucosylated NFs were produced by an in vitro reaction with NodZ fucosyltransferase and purified. Their biological activity on pea was tested by measuring their capacity to stimulate the activity of a hydrolase that cleaves NFs. Nonmodified and fucosylated NFs displayed this activity at nano- to picomolar concentrations, while a sulfated NF from Sinorhizobium meliloti was inactive. In an additional series of experiments, the stability of non-modified and fucosylated NFs in the presence of purified tobacco chitinases was compared. The presence of the fucosyl group affected the degradation rates and the accessibility of specific cleavage sites on the chitooligosaccharide backbone. These results suggest that the fucosyl group in NFs also weakens the interaction of NFs with certain chitinases or chitinase-related proteins in pea roots.