Signaling in Legume-Rhizobia Symbiosis.

Legumes represent an important source of food protein for human nutrition and animal feed. Therefore, sustainable production of legume crops is an issue of global importance. It is well-known that legume-rhizobia symbiosis allows an increase in the productivity and resilience of legume crops. The efficiency of this mutualistic association strongly depends on precise regulation of the complex interactions between plant and rhizobia. Their molecular dialogue represents a complex multi-staged process, each step of which is critically important for the overall success of the symbiosis. In particular, understanding the details of the molecular mechanisms behind the nodule formation and functioning might give access to new legume cultivars with improved crop productivity. Therefore, here we provide a comprehensive literature overview on the dynamics of the signaling network underlying the development of the legume-rhizobia symbiosis. Thereby, we pay special attention to the new findings in the field, as well as the principal directions of the current and prospective research. For this, here we comprehensively address the principal signaling events involved in the nodule inception, development, functioning, and senescence.

Effects of Elevated Temperature on Pisum sativum Nodule Development: I-Detailed Characteristic of Unusual Apical Senescence.

Despite global warming, the influence of heat on symbiotic nodules is scarcely studied. In this study, the effects of heat stress on the functioning of nodules formed by Rhizobium leguminosarum bv. viciae strain 3841 on pea (Pisum sativum) line SGE were analyzed. The influence of elevated temperature was analyzed at histological, ultrastructural, and transcriptional levels. As a result, an unusual apical pattern of nodule senescence was revealed. After five days of exposure, a senescence zone with degraded symbiotic structures was formed in place of the distal nitrogen fixation zone. There was downregulation of various genes, including those associated with the assimilation of fixed nitrogen and leghemoglobin. After nine days, the complete destruction of the nodules was demonstrated. It was shown that nodule recovery was possible after exposure to elevated temperature for 3 days but not after 5 days (which coincides with heat wave duration). At the same time, the exposure of plants to optimal temperature during the night leveled the negative effects. Thus, the study of the effects of elevated temperature on symbiotic nodules using a well-studied pea genotype and Rhizobium strain led to the discovery of a novel positional response of the nodule to heat stress.

Detergent-Assisted Protein Digestion-On the Way to Avoid the Key Bottleneck of Shotgun Bottom-Up Proteomics.

Gel-free bottom-up shotgun proteomics is the principal methodological platform for the state-of-the-art proteome research. This methodology assumes quantitative isolation of the total protein fraction from a complex biological sample, its limited proteolysis with site-specific proteases, analysis of the resulted peptides with nanoscaled reversed-phase high-performance liquid chromatography-(tandem) mass spectrometry (nanoRP-HPLC-MS and MS/MS), protein identification by sequence database search and peptide-based quantitative analysis. The most critical steps of this workflow are protein reconstitution and digestion; therefore, detergents and chaotropic agents are strongly mandatory to ensure complete solubilization of complex protein isolates and to achieve accessibility of all protease cleavage sites. However, detergents are incompatible with both RP separation and electrospray ionization (ESI). Therefore, to make LC-MS analysis possible, several strategies were implemented in the shotgun proteomics workflow. These techniques rely either on enzymatic digestion in centrifugal filters with subsequent evacuation of the detergent, or employment of MS-compatible surfactants, which can be degraded upon the digestion. In this review we comprehensively address all currently available strategies for the detergent-assisted proteolysis in respect of their relative efficiency when applied to different biological matrices. We critically discuss the current progress and the further perspectives of these technologies in the context of its advances and gaps.

Dynamics of Reactive Carbonyl Species in Pea Root Nodules in Response to Polyethylene Glycol (PEG)-Induced Osmotic Stress.

Drought dramatically affects crop productivity worldwide. For legumes this effect is especially pronounced, as their symbiotic association with rhizobia is highly-sensitive to dehydration. This might be attributed to the oxidative stress, which ultimately accompanies plants' response to water deficit. Indeed, enhanced formation of reactive oxygen species in root nodules might result in up-regulation of lipid peroxidation and overproduction of reactive carbonyl compounds (RCCs), which readily modify biomolecules and disrupt cell functions. Thus, the knowledge of the nodule carbonyl metabolome dynamics is critically important for understanding the drought-related losses of nitrogen fixation efficiency and plant productivity. Therefore, here we provide, to the best of our knowledge, for the first time a comprehensive overview of the pea root nodule carbonyl metabolome and address its alterations in response to polyethylene glycol-induced osmotic stress as the first step to examine the changes of RCC patterns in drought treated plants. RCCs were extracted from the nodules and derivatized with 7-(diethylamino)coumarin-3-carbohydrazide (CHH). The relative quantification of CHH-derivatives by liquid chromatography-high resolution mass spectrometry with a post-run correction for derivative stability revealed in total 194 features with intensities above 1 × 105 counts, 19 of which were down- and three were upregulated. The upregulation of glyceraldehyde could accompany non-enzymatic conversion of glyceraldehyde-3-phosphate to methylglyoxal. The accumulation of 4,5-dioxovaleric acid could be the reason for down-regulation of porphyrin metabolism, suppression of leghemoglobin synthesis, inhibition of nitrogenase and degradation of legume-rhizobial symbiosis in response to polyethylene glycol (PEG)-induced osmotic stress effect. This effect needs to be confirmed with soil-based drought models.

Identification and Expression Analysis of the C-TERMINALLY ENCODED PEPTIDE Family in Pisum sativum L.

The C-TERMINALLY ENCODED PEPTIDE(CEP) peptides play crucial roles in plant growth and response to environmental factors. These peptides were characterized as positive regulators of symbiotic nodule development in legume plants. However, little is known about the CEP peptide family in pea. Here, we discovered in pea genome 21 CEP genes (PsCEPs), among which three genes contained additional conserved motifs corresponding to the PIP (PAMP-induced secreted peptides) consensus sequences. We characterized the expression patterns of pea PsCEP genes based on transcriptomic data, and for six PsCEP genes with high expression levels in the root and symbiotic nodules the detailed expression analysis at different stages of symbiosis and in response to nitrate treatment was performed. We suggest that at least three PsCEP genes, PsCEP1, PsCEP7 and PsCEP2, could play a role in symbiotic nodule development, whereas the PsCEP1 and PsCEP13 genes, downregulated by nitrate addition, could be involved in regulation of nitrate-dependent processes in pea. Further functional studies are required to elucidate the functions of these PsCEP genes.

Bringing New Methods to the Seed Proteomics Platform: Challenges and Perspectives.

For centuries, crop plants have represented the basis of the daily human diet. Among them, cereals and legumes, accumulating oils, proteins, and carbohydrates in their seeds, distinctly dominate modern agriculture, thus play an essential role in food industry and fuel production. Therefore, seeds of crop plants are intensively studied by food chemists, biologists, biochemists, and nutritional physiologists. Accordingly, seed development and germination as well as age- and stress-related alterations in seed vigor, longevity, nutritional value, and safety can be addressed by a broad panel of analytical, biochemical, and physiological methods. Currently, functional genomics is one of the most powerful tools, giving direct access to characteristic metabolic changes accompanying plant development, senescence, and response to biotic or abiotic stress. Among individual post-genomic methodological platforms, proteomics represents one of the most effective ones, giving access to cellular metabolism at the level of proteins. During the recent decades, multiple methodological advances were introduced in different branches of life science, although only some of them were established in seed proteomics so far. Therefore, here we discuss main methodological approaches already employed in seed proteomics, as well as those still waiting for implementation in this field of plant research, with a special emphasis on sample preparation, data acquisition, processing, and post-processing. Thereby, the overall goal of this review is to bring new methodologies emerging in different areas of proteomics research (clinical, food, ecological, microbial, and plant proteomics) to the broad society of seed biologists.

Does Protein Glycation Impact on the Drought-Related Changes in Metabolism and Nutritional Properties of Mature Pea (Pisum sativum L.) Seeds?

Protein glycation is usually referred to as an array of non-enzymatic post-translational modifications formed by reducing sugars and carbonyl products of their degradation. The resulting advanced glycation end products (AGEs) represent a heterogeneous group of covalent adducts, known for their pro-inflammatory effects in mammals, and impacting on pathogenesis of metabolic diseases and ageing. In plants, AGEs are the markers of tissue ageing and response to environmental stressors, the most prominent of which is drought. Although water deficit enhances protein glycation in leaves, its effect on seed glycation profiles is still unknown. Moreover, the effect of drought on biological activities of seed protein in mammalian systems is still unstudied with respect to glycation. Therefore, here we address the effects of a short-term drought on the patterns of seed protein-bound AGEs and accompanying alterations in pro-inflammatory properties of seed protein in the context of seed metabolome dynamics. A short-term drought, simulated as polyethylene glycol-induced osmotic stress and applied at the stage of seed filling, resulted in the dramatic suppression of primary seed metabolism, although the secondary metabolome was minimally affected. This was accompanied with significant suppression of NF-kB activation in human SH-SY5Y neuroblastoma cells after a treatment with protein hydrolyzates, isolated from the mature seeds of drought-treated plants. This effect could not be attributed to formation of known AGEs. Most likely, the prospective anti-inflammatory effect of short-term drought is related to antioxidant effect of unknown secondary metabolite protein adducts, or down-regulation of unknown plant-specific AGEs due to suppression of energy metabolism during seed filling.

s-dePooler: determination of polymorphism carriers from overlapping DNA pools.

BACKGROUND: Samples pooling is a method widely used in studies to reduce costs and labour. DNA sample pooling combined with massive parallel sequencing is a powerful tool for discovering DNA variants (polymorphisms) in large analysing populations, which is the base of such research fields as Genome-Wide Association Studies, evolutionary and population studies, etc. Usage of overlapping pools where each sample is present in multiple pools can enhance the accuracy of polymorphism detection and allow identifying carriers of rare-variants. Surprisingly there is a lack of tools for result interpretation and carrier identification, i.e. for "depooling". RESULTS: Here we present s-dePooler, the application for analysis of pooling experiments data. s-dePooler uses the variants information (VCF-file) and the pooling scheme to produce a list of candidate carriers for each polymorphism. We incorporated s-dePooler into a pipeline (dePoP) for automation of pooling analysis. The performance of the pipeline was tested on a synthetic dataset built using the 1000 Genomes Project data, resulting in the successful identification 97% of carriers of polymorphisms present in fewer than ~ 10% of carriers. CONCLUSIONS: s-dePooler along with dePoP can be used to identify carriers of polymorphisms in overlapping pools, and is compatible with any pooling scheme with equivalent molar ratios of pooled samples. s-dePooler and dePoP with usage instructions and test data are freely available at https://github.com/lab9arriam/depop .

Thin Film Chemical Deposition Techniques as a Tool for Fingerprinting of Free Fatty Acids by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Metabolic fingerprinting is a powerful analytical technique, giving access to high-throughput identification and relative quantification of multiple metabolites. Because of short analysis times, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is the preferred instrumental platform for fingerprinting, although its power in analysis of free fatty acids (FFAs) is limited. However, these metabolites are the biomarkers of human pathologies and indicators of food quality. Hence, a high-throughput method for their fingerprinting is required. Therefore, here we propose a MALDI-TOF-MS method for identification and relative quantification of FFAs in biological samples of different origins. Our approach relies on formation of monomolecular Langmuir films (LFs) at the interphase of aqueous barium acetate solution, supplemented with low amounts of 2,5-dihydroxybenzoic acid, and hexane extracts of biological samples. This resulted in detection limits of 10-13-10-14 mol and overall method linear dynamic range of at least 4 orders of magnitude with accuracy and precision within 2 and 17%, respectively. The method precision was verified with eight sample series of different taxonomies, which indicates a universal applicability of our approach. Thereby, 31 and 22 FFA signals were annotated by exact mass and identified by tandem MS, respectively. Among 20 FFAs identified in Fucus algae, 14 could be confirmed by gas chromatography-mass spectrometry.

MtNODULE ROOT1 and MtNODULE ROOT2 Are Essential for Indeterminate Nodule Identity.

Symbiotic interactions between legume plants and rhizobia result in the formation of nitrogen-fixing nodules, but the molecular actors and the mechanisms allowing for the maintenance of nodule identity are poorly understood. Medicago truncatula NODULE ROOT1 (MtNOOT1), Pisum sativum COCHLEATA1 (PsCOCH1), and Lotus japonicus NOOT-BOP-COCH-LIKE1 (LjNBCL1) are orthologs of Arabidopsis (Arabidopsis thaliana) AtBLADE-ON-PETIOLE1/2 and are members of the NBCL gene family, which has conserved roles in plant development and is essential for indeterminate and determinate nodule identity in legumes. The loss of function of MtNOOT1, PsCOCH1, and LjNBCL1 triggers a partial loss of nodule identity characterized by the development of ectopic roots arising from nodule vascular meristems. Here, we report the identification and characterization of a second gene involved in regulating indeterminate nodule identity in M. truncatula, MtNOOT2MtNOOT2 is the paralog of MtNOOT1 and belongs to a second legume-specific NBCL subclade, the NBCL2 clade. MtNOOT2 expression was induced during early nodule formation, and it was expressed primarily in the nodule central meristem. Mtnoot2 mutants did not present any particular symbiotic phenotype; however, the loss of function of both MtNOOT1 and MtNOOT2 resulted in the complete loss of nodule identity and was accompanied by drastic changes in the expression of symbiotic, defense, and root apical meristem marker genes. Mtnoot1 noot2 double mutants developed only nonfixing root-like structures that were no longer able to host symbiotic rhizobia. This study provides original insights into the molecular basis underlying nodule identity in legumes forming indeterminate nodules.

Profiling of Seed Proteome in Pea (Pisum sativum L.) Lines Characterized with High and Low Responsivity to Combined Inoculation with Nodule Bacteria and Arbuscular Mycorrhizal Fungi.

Legume crops represent the major source of food protein and contribute to human nutrition and animal feeding. An essential improvement of their productivity can be achieved by symbiosis with beneficial soil microorganisms-rhizobia (Rh) and arbuscular mycorrhizal (AM) fungi. The efficiency of these interactions depends on plant genotype. Recently, we have shown that, after simultaneous inoculation with Rh and AM, the productivity gain of pea (Pisum sativum L) line K-8274, characterized by high efficiency of interaction with soil microorganisms (EIBSM), was higher in comparison to a low-EIBSM line K-3358. However, the molecular mechanisms behind this effect are still uncharacterized. Therefore, here, we address the alterations in pea seed proteome, underlying the symbiosis-related productivity gain, and identify 111 differentially expressed proteins in the two lines. The high-EIBSM line K-8274 responded to inoculation by prolongation of seed maturation, manifested by up-regulation of proteins involved in cellular respiration, protein biosynthesis, and down-regulation of late-embryogenesis abundant (LEA) proteins. In contrast, the low-EIBSM line K-3358 demonstrated lower levels of the proteins, related to cell metabolism. Thus, we propose that the EIBSM trait is linked to prolongation of seed filling that needs to be taken into account in pulse crop breeding programs. The raw data have been deposited to the ProteomeXchange with identifier PXD013479.

Methodology of Drought Stress Research: Experimental Setup and Physiological Characterization.

Drought is one of the major stress factors affecting the growth and development of plants. In this context, drought-related losses of crop plant productivity impede sustainable agriculture all over the world. In general, plants respond to water deficits by multiple physiological and metabolic adaptations at the molecular, cellular, and organism levels. To understand the underlying mechanisms of drought tolerance, adequate stress models and arrays of reliable stress markers are required. Therefore, in this review we comprehensively address currently available models of drought stress, based on culturing plants in soil, hydroponically, or in agar culture, and critically discuss advantages and limitations of each design. We also address the methodology of drought stress characterization and discuss it in the context of real experimental approaches. Further, we highlight the trends of methodological developments in drought stress research, i.e., complementing conventional tests with quantification of phytohormones and reactive oxygen species (ROS), measuring antioxidant enzyme activities, and comprehensively profiling transcriptome, proteome, and metabolome.

NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes.

During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ.

Volumetric Modification of Transparent Materials with Two-Color Laser Irradiation: Insight from Numerical Modeling.

Traditionally, single-color laser beams are used for material processing and modifications of optical, mechanical, conductive, and thermal properties of different materials. So far, there are a limited number of studies about the dual-wavelength laser irradiation of materials, which, however, indicate a strong enhancement in laser energy coupling to solid targets. Here, a theoretical study is reported that aimed at exploring the volumetric excitation of fused silica with dual-wavelength (800 nm and 400 nm) ultrashort laser pulses focused on the material's bulk. Numerical simulations are based on Maxwell's equations, accounting for the generation of conduction electrons, their hydrodynamic motion in the laser field, and trapping into an excitonic state. It is shown that, by properly choosing the energies of the two laser harmonics successively coupling with the material, it is possible to strongly enhance the laser energy absorption as compared to the pulses of a single wavelength with the same total energy. Laser energy absorption strongly depends on the sequence of applied wavelengths, so that the shorter wavelength pre-irradiation can yield a dramatic effect on laser excitation by the following longer-wavelength pulse. The predictions of this study can open a new route for enhancing and controlling the highly localized absorption of laser energy inside transparent materials for optoelectronic and photonic applications.

Genomic and Transcriptomic Analysis of Pea (Pisum sativum L.) Breeding Line 'Triumph' with High Symbiotic Responsivity.

Pea (Pisum sativum L.), like most legumes, forms mutualistic symbioses with nodule bacteria and arbuscular mycorrhizal (AM) fungi. The positive effect of inoculation is partially determined by the plant genotype; thus, pea varieties with high and low symbiotic responsivity have been described, but the molecular genetic basis of this trait remains unknown. Here, we compare the symbiotically responsive breeding line 'Triumph' of grain pea with its parental cultivars 'Vendevil' (a donor of high symbiotic responsivity) and 'Classic' (a donor of agriculturally valuable traits) using genome and transcriptome sequencing. We show that 'Triumph' inherited one-fourth of its genome from 'Vendevil', including the genes related to AM and nodule formation, and reveal that under combined inoculation with nodule bacteria and AM fungi, 'Triumph' and 'Vendevil', in contrast to 'Classic', demonstrate similar up-regulation of the genes related to solute transport, hormonal regulation and flavonoid biosynthesis in their roots. We also identify the gene PsGLP2, whose expression pattern distinguishing 'Triumph' and 'Vendevil' from 'Classic' correlates with difference within the promoter region sequence, making it a promising marker for the symbiotic responsivity trait. The results of this study may be helpful for future molecular breeding programs aimed at creation of symbiotically responsive cultivars of pea.

The Effects of Rhizophagus irregularis Inoculation on Transcriptome of Medicago lupulina Leaves at Early Vegetative and Flowering Stages of Plant Development.

The study is aimed at revealing the effects of Rhizophagus irregularis inoculation on the transcriptome of Medicago lupulina leaves at the early (second leaf formation) and later (flowering) stages of plant development. A pot experiment was conducted under conditions of low phosphorus (P) level in the substrate. M. lupulina plants were characterized by high mycorrhizal growth response and mycorrhization parameters. Library sequencing was performed on the Illumina HiseqXTen platform. Significant changes in the expression of 4863 (padj < 0.01) genes from 34049 functionally annotated genes were shown by Massive Analysis of cDNA Ends (MACE-Seq). GO enrichment analysis using the Kolmogorov-Smirnov test was performed, and 244 functional GO groups were identified, including genes contributing to the development of effective AM symbiosis. The Mercator online tool was used to assign functional classes of differentially expressed genes (DEGs). The early stage was characterized by the presence of six functional classes that included only upregulated GO groups, such as genes of carbohydrate metabolism, cellular respiration, nutrient uptake, photosynthesis, protein biosynthesis, and solute transport. At the later stage (flowering), the number of stimulated GO groups was reduced to photosynthesis and protein biosynthesis. All DEGs of the GO:0016036 group were downregulated because AM plants had higher resistance to phosphate starvation. For the first time, the upregulation of genes encoding thioredoxin in AM plant leaves was shown. It was supposed to reduce ROS level and thus, consequently, enhance the mechanisms of antioxidant protection in M. lupulina plants under conditions of low phosphorus level. Taken together, the obtained results indicate genes that are the most important for the effective symbiosis with M. lupulina and might be engaged in other plant species.

War and Peas: Molecular Bases of Resistance to Powdery Mildew in Pea (Pisum sativum L.) and Other Legumes.

Grain legumes, or pulses, have many beneficial properties that make them potentially attractive to agriculture. However, the large-scale cultivation of legumes faces a number of difficulties, in particular the vulnerability of the currently available cultivars to various diseases that significantly impair yields and seed quality. One of the most dangerous legume pathogens is powdery mildew (a common name for parasitic fungi of the order Erisyphales). This review examines the methods of controlling powdery mildew that are used in modern practice, including fungicides and biological agents. Special attention is paid to the plant genetic mechanisms of resistance, which are the most durable, universal and environmentally friendly. The most studied legume plant in this regard is the garden pea (Pisum sativum L.), which possesses naturally occurring resistance conferred by mutations in the gene MLO1 (Er1), for which we list here all the known resistant alleles, including er1-12 discovered by the authors of this review. Recent achievements in the genetics of resistance to powdery mildew in other legumes and prospects for the introduction of this resistance into other agriculturally important legume species are also discussed.

Draft Genome Sequence of Rhizobium ruizarguesonis (Rhizobium leguminosarum) Strain 1TK341.

Rhizobium ruizarguesonis (Rhizobium leguminosarum) strain 1TK341 was isolated from pink nodules of fixation-negative mutant line P61 of pea (Pisum sativum L.) grown in soil. Here, we report the draft genome sequence of the strain.

Intestinal Microbiota Correction in the Treatment and Prevention of Urinary Tract Infection.

Intestinal microbiota is a topical subject of modern research. The maintenance of a healthy intestinal micro biota is an important component of homeostasis, and violations of its composition and functions, called dysbiosis, are associated with a number of diseases, including urinary tract infections. Antimicrobial therapy leads to significant changes in the intestinal microbiota and causes the possibility of urinary tract infection recurrence. In this regard, it is important to study methods of microbiota correction in order to restore its structural and functional integrity.

RopB protein of Rhizobium leguminosarum bv. viciae adopts amyloid state during symbiotic interactions with pea (Pisum sativum L.).

Amyloids represent protein aggregates with highly ordered fibrillar structure associated with the development of various disorders in humans and animals and involved in implementation of different vital functions in all three domains of life. In prokaryotes, amyloids perform a wide repertoire of functions mostly attributed to their interactions with other organisms including interspecies interactions within bacterial communities and host-pathogen interactions. Recently, we demonstrated that free-living cells of Rhizobium leguminosarum, a nitrogen-fixing symbiont of legumes, produce RopA and RopB which form amyloid fibrils at cell surface during the stationary growth phase thus connecting amyloid formation and host-symbiont interactions. Here we focused on a more detailed analysis of the RopB amyloid state in vitro and in vivo, during the symbiotic interaction between R. leguminosarum bv. viciae with its macrosymbiont, garden pea (Pisum sativum L.). We confirmed that RopB is the bona fide amyloid protein since its fibrils exhibit circular x-ray reflections indicating its cross-ß structure specific for amyloids. We found that fibrils containing RopB and exhibiting amyloid properties are formed in vivo at the surface of bacteroids of R. leguminosarum extracted from pea nodules. Moreover, using pea sym31 mutant we demonstrated that formation of extracellular RopB amyloid state occurs at different stages of bacteroid development but is enhanced in juvenile symbiosomes. Proteomic screening of potentially amyloidogenic proteins in the nodules revealed the presence of detergent-resistant aggregates of different plant and bacterial proteins including pea amyloid vicilin. We demonstrated that preformed vicilin amyloids can cross-seed RopB amyloid formation suggesting for probable interaction between bacterial and plant amyloidogenic proteins in the nodules. Taken together, we demonstrate that R. leguminosarum bacteroids produce extracellular RopB amyloids in pea nodules in vivo and these nodules also contain aggregates of pea vicilin amyloid protein, which is able to cross-seed RopB fibrillogenesis in vitro. Thus, we hypothesize that plant nodules contain a complex amyloid network consisting of plant and bacterial amyloids and probably modulating host-symbiont interactions.