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Project titleProteomic screening of amyloid proteins in pea (Pisum sativum L.) and root nodule bacterium (Rhizobium leguminosarum)

AffiliationFederal State Budget Scientific Institution All-Russia Research Institute for Agricultural Microbiology,

KeywordsAmyloid, protein fibril, pea, agrobiotechnology, root nodule bacteria, bacteroid, storage proteins, sym31, Pisum sativum, Rhizobium, proteomics, transcriptomics

Annotation
As a result of the Project in 2017-2019, the important data on plant and root nodule bacteria proteins that possess amyloid properties (an ability of proteins to form fibrils with a special ordered spatial structure) have been obtained. For the first time, a plant protein that forms amyloids under physiological conditions has been identified. This protein of the garden pea Pisum sativum L., Vicilin, is one of the main storage proteins in seeds. The amyloid proteins of the nodule bacterium R. leguminosarum, RopA, and RopB, have also been identified for the first time. These proteins form fibrils in the capsule, which is a special extracellular structure formed by this microorganism and playing an important role in its virulence and formation of the initial stages of plant-microbial interactions. Project 2020-2021 implies further studies of functional amyloidogenesis in plants and root nodule bacteria, clarification of its physiological role in the storage of protein in seeds and defense against plant pathogens, as well as plant-microbial interactions. The first area of research will continue the study of the amyloid properties of the Vicilin protein of pea seeds. According to modern concepts, some storage proteins, including cupins, to which Vicilin belongs, are bifunctional, performing not only a storage, but also a defense function. This is due to the presence of carbohydrate-binding lectin domains that have a toxic effect on various pathogenic microorganisms. Within the framework of the Project, the formation of amyloid aggregates in various organs of the garden pea plants (leaves, shoots, roots) by Vicilin will be analyzed, and the toxicity of these aggregates for a number of pathogenic fungi and bacteria will be evaluated. The second area of study will include the investigation of amyloid properties of other storage proteins of pea seeds, Legumin and Convicilin, previously identified as candidates for the role of new amyloids in proteomic and bioinformatic screenings. The ability of these proteins to form fibrils in vitro and their compliance with the basic properties of amyloids including resistance to treatment with ionic detergents and proteases, enrichment with beta sheets, binding with amyloid-specific dyes Thioflavin T and Congo red, will be tested. In the case if Legumin and Convicilin form amyloids in vitro, their amyloid properties will also be investigated in vivo by analyzing their formation of detergent-resistant aggregates in pea seeds and the binding of amyloid-specific dyes to these aggregates. The third focus area will be devoted to an analysis of the role of previously identified amyloid proteins, RopA and RopB, of the nodule bacterium R. leguminosarum in the formation of plant-microbial symbiosis. Within its framework, an analysis of the formation of the RopA and RopB amyloids at the stage of binding of bacterial cells to the roots of the plant, as well as in bacteroids, i.e., a differentiated nitrogen-fixing form of R. leguminosarum located in pea root nodules will be performed. We will also study the effect of deletions of genes encoding RopA and RopB, which entail the impossibility of the formation of amyloid fibrils by these proteins, on the interaction of R. leguminosarum with the host plant, differentiation into bacteroids, and root nodule formation. The fourth area of study within this Project is to analyze the interaction of the plant amyloid proteins identified by us with other amyloid proteins in the in vitro system. In previous studies, we showed that Vicilin amyloids are extremely stable and can withstand treatment with digestive tract enzymes under physiological conditions, and also remain stable in canned peas. In addition, the structure of Vicilin is similar to the structures of the proteins of the outer membrane of gram-negative bacteria, being based on the beta-barrel type domains. On these grounds, it can be assumed that Vicilin amyloids that enter the digestive tract when pea seeds are eaten can potentially induce amyloidogenesis of intestinal bacterial proteins with similar structure, as well as cause aggregation of some human proteins. The potential possibility of such Vicilin amyloids activity will be tested by analyzing their in vitro effect on the efficiency of amyloid formation by proteins of the outer membrane of gram-negative bacteria, as well as by different human amyloidogenic proteins and peptides, including pathological ones (amyloid beta, lysozyme, insulin and alpha-synuclein). In general, the implementation of this Project will allow for the first time to address fundamental questions regarding: the role of amyloidogenesis in plant protection against pathogens; the involvement of various groups of storage proteins in the formation of complexes of amyloid aggregates in plant seeds; the role of amyloidogenesis in the control of plant-microbial interactions; and the possible effect of amyloids of storage proteins of plant seeds on the induction of amyloidogenesis of human and bacterial proteins.

Expected results
Expected results of the Project 2020-2021 will have high novelty and significance. The garden pea Pisum sativum L. and root nodule bacterium Rhizobium leguminosarum will be used as main objects of research. The characterization of amyloids identified within the Project 2017-2019 will continue primarily from the perspective of a detailed analysis of the physiological role of amyloidogenesis in plants and root nodule bacteria, as well as the identification of new amyloids. The bifunctionality of storage proteins of plant seeds is the subject of discussions, since it has recently become clear that many of those proteins perform not only a storage but also a protective function acting as carbohydrate-binding lectin proteins that cause agglutination of pathogenic microorganisms. Within the framework of the Project, the study of amyloid properties of the Vicilin storage protein of pea seeds will be continued. Novel results will be obtained on the analysis of the formation of amyloid aggregates by this protein in various plant organs (leaves, stems, roots), as well as the toxicity of these aggregates for a number of pathogenic fungi and bacteria. These data will allow us to assess more broadly the role of amyloidogenesis of storage proteins, not only in the context of the formation of amyloids as stable structures for long-term storage in seeds, but also as protective structures that are toxic to pathogens. One of the most important questions of this study is which groups of seed storage proteins form amyloids. Previous proteomic screening revealed not only Vicilin, the amyloid properties of which were fully characterized by us in vivo and in vitro, but also representatives of two other key groups of storage proteins: Legumin and Convicilin. For these proteins, the results of a complete characterization of the in vitro amyloid properties will be obtained (the formation of fibrils, verification of their resistance to detergents and proteases, binding the amyloid-specific dye Thioflavin T, green birefringence upon binding of Congo red dye, enrichment with beta sheets). If Legumin and Convicilin form amyloids in vitro, the results of characterization of their amyloid properties in vivo will be obtained, including analysis of the detergent- and protease resistance of their aggregates in seed lysates, as well as the colocalization of antibodies against these proteins with amyloid-specific dyes on histological sections. The importance of studying the role of amyloidogenesis in the control of plant-microbial interactions can not be overestimated, since, in fact, this phenomenon could represent a fundamentally new mechanism of interaction in the symbiont-host system. Within the framework of this Project, the role of previously identified by us amyloid proteins, RopA and RopB, of the root nodule bacterium R. leguminosarum in the interaction with the host plant P. sativum L. will be continued. Using the western blot hybridization, optical and electron microscopy, we will obtain comparative data on the presence and quantity of amyloids of these proteins at various phases of the bacterial life cycle (free-living culture; cells interacting with the roots of the host plants; bacteroids). The effects of deletions of these genes (if they have no lethal consequences) on the ability of bacteria to interact with plant roots and form bacteroids will also be studied. The results obtained will allow us to make conclusions on the physiological role of amyloidogenesis of RopA and RopB in the control of plant-microbial interactions. The cross-interaction of amyloids is a factor contributing to the development of several incurable human diseases. It is well known that amyloids of some proteins can specifically induce amyloidogenesis of others. The amyloids of the plant protein Vicilin that we identified are resistant to the action of digestive tract enzymes, therefore, they can potentially interact in the digestive tract with the structurally similar proteins of bacteria of the intestinal microbiome as well as with human proteins. In this regard, an important goal is to check the possible effect of Vicilin amyloids on the induction of amyloidogenesis of bacterial and human proteins, including those forming pathogenic amyloids. Within the framework of the Project, results assessing the effect of Vicilin amyloids on the induction of bacterial proteins amyloidogenesis (primarily, porins of the outer membrane of gram-negative bacteria involved in the pathogenesis of a number of diseases), as well as human amyloidogenic proteins and peptides (insulin, lysozyme, amyloid beta, alpha synuclein) will be obtained. In the case if Vicilin amyloids induce amyloidogenesis of these proteins, this can be considered as the first evidence of the influence of plant-derived food on the amyloidoses and other diseases related with amyloid proteins aggregation. In general, it is expected that during the Project 2020-2021 we will obtain globally novel data on the diversity of the biological roles of amyloidogenesis of plant storage proteins in the context of its possible impact not only on the formation and maintenance of the reservoir of nutrients, but also on the protection of plants from pathogens; on a diversity of seed storage proteins forming amyloids in vitro and in vivo; on the role of amyloidogenesis of proteins of the root nodule bacteria in the control of plant-microbial interactions, as well as on the effect of plant amyloid proteins on the amyloidogenesis of bacterial and human proteins.

Annotation of the results obtained in 2021
In the scope of the Project in 2021 we analyzed the amyloid properties of plant proteins and root nodule bacteria in vivo, as well as their interactions and effects on the aggregation of each other and the amyloidogenesis of different proteins. We analyzed the amyloid properties of RopA and RopB proteins in bacteroids and undifferentiated cells extracted from pea root nodules of the Sprint-2 and Sprint-2Fix- lines, respectively. The ability of these proteins to form amyloid fibrils in a free-living culture of R. leguminosarum in the stationary growth phase was shown by us earlier. In the present work, we demonstrated that similar fibrils are also formed by R. leguminosarum bacteroids located in root nodules that may indicate the potential functional significance of RopA and RopB amyloidogenesis. It is known that the expression of the ropA and ropB genes increases at the early stages of nodulation, and the amount of RopA amyloids in a free-living culture increases upon stimulation of cells with the luteolin flavonoid, which mimics the initial stages of the formation of symbiotic relationships. These data also support the involvement of RopA and RopB amyloidogenesis in supraorganismal interactions. The amyloid properties of RopA and RopB in bacteroids and undifferentiated cells isolated from pea nodules of lines Sprint-2 and Sprint-2Fix-, respectively, are shown in this study using a range of methods, including analysis of resistance to treatment with ionic detergents, transmission electron and polarization microscopy. Also, RopA and RopB were identified by proteomic analysis in detergent-resistant protein fractions of root nodules of the Sprint-2 and Sprint-2Fix- pea lines inoculated with R. leguminosarum. Proteomic analysis also revealed the formation of detergent-resistant aggregates by different storage proteins (vicilin, legumins, and convicillin) in the root nodules of Sprint-2 garden pea line. It is noteworthy that the nodules of the Sprint-2Fix- pea line, in which the differentiation of R. leguminosarum cells into bacteroids does not occur, do not contain aggregates of storage proteins. These data support the specificity of the formation of amyloid aggregates of pea storage proteins in nodules containing differentiated bacteroids of R. leguminosarum, which is possibly associated with specific mechanisms of the supraorganismal interactions control; however, this assumption requires further analysis. The phenomenon of amyloid aggregation of pea storage proteins in root nodules during the formation of effective symbiotic relationships which we have described has significant novelty. It should be noted that as we demonstrated in 2021 different storage proteins of pea seeds have amyloid properties in native conditions in vivo, and their aggregates interact. Thus, we completely demonstrated the amyloid properties of legumin A2 in vivo (detergent resistance, formation of fibrils, green birefringence upon binding of Congo red). Also, using co-immunoprecipitation we revealed co-aggregation of vicilin and legumin amyloids. Thus, amyloidogenesis seems to involve various storage proteins of seeds, and this process is accompanied by the formation of complex amyloid structures including aggregates of various proteins. Another important result obtained in 2021 was the identification of the inhibitory effect of “seeds” prepared from fibrils of plant amyloid proteins on the amyloidogenesis of pathological human amyloid proteins. This effect was established when evaluating the effect of “seeds” from plant amyloids on the fibrillogenesis of lysozyme, insulin, beta-2-microglobulin, and human amyloid peptide-beta. This effect, in our opinion, is very important and unexpected since it shows that amyloid fibrils of plant storage proteins that enter the human body with food do not induce amyloidogenesis of human proteins which could be assumed based on the known facts about the cross-induction of amyloidogenesis during the interactions of various amyloid proteins. On the contrary, we have demonstrated plant amyloid fibrils suppress pathological amyloidogenesis of human proteins, which can potentially represent a fundamentally new mechanism of protection against amyloidosis. Overall, within the framework of 2021 stage of the Project we have shown the formation of amyloids RopA and RopB by bacteroids R. leguminosarum, have shown for the first time the amyloid properties of the legumin A2 protein in pea seeds in vivo and have demonstrated the interaction of amyloids of legumin and vicilin. The formation of amyloids of plant storage proteins in effective nodules of P. sativum L. is described. For the first time, the inhibitory effect of “seeds” prepared from amyloid fibrils of plants on the amyloidogenesis of human proteins has been established. These results have high novelty and demonstrate that the system of interactions between amyloids of various organisms is selective and participates in the control of the performance of important biological functions. Publications of the Project in 2021: 1. Sulatskaya A.I., Kosolapova A.O., Bobylev A.G., Belousov M.V., Antonets K.S., Sulatsky M.I., Kuznetsova I.M., Turoverov K.K., Stepanenko O.V., Nizhnikov A.A. β-Barrels and Amyloids: Structural Transitions, Biological Functions, and Pathogenesis // International Journal of Molecular Sciences, 2021, V.22, e11316. https://doi.org/10.3390/ijms222111316 2. Belousov M., Zykin P., Andreeva E., Kosolapova A., Shtark O., Vasileva E., Antonets K., Nizhnikov A. The Aggregates of Vicilin Protein Are Found in the Roots and Nodules of Pisum sativum L. // The FASEB Journal V.35, S1, 02280. https://doi.org/10.1096/fasebj.2021.35.S1.02280 In addition, one of the publications on this Project was published at the end of 2020 and was not included in the report for the last year: 3. Malovichko Y.V., Shtark O.Y., Vasileva E.N., Nizhnikov A.A., Antonets K.S. Transcriptomic signatures of seed maturation heterochrony in garden pea (Pisum sativum L) accessions // BMC Bioinformatics, 2020, V.21, Suppl 20, e567, P10. https://doi.org/10.1186/s12859-020-03838-2

Publications

1. Sulatskaya A.I., Kosolapova A.O., Bobylev A.G., Belousov M.V., Antonets K.S., Sulatsky M.I., Kuznetsova I.M., Turoverov K.K., Stepanenko O.V., Nizhnikov A.A. β-Barrels and Amyloids: Structural Transitions, Biological Functions, and Pathogenesis International Journal of Molecular Sciences, V.22, e11316 (year - 2021) https://doi.org/10.3390/ijms222111316

2. Belousov M., Zykin P., Andreeva E., Kosolapova A., Shtark O., Vasileva E., Antonets K., Nizhnikov A. The Aggregates of Vicilin Protein Are Found in the Roots and Nodules of Pisum sativum L. FASEB JOURNAL, V.35, S1, 02280. (year - 2021) https://doi.org/10.1096/fasebj.2021.35.S1.02280

3. Yury V. Malovichko, Oksana Y. Shtark, Ekaterina N. Vasileva, Anton A. Nizhnikov, Kirill A. Antonets Transcriptomic signatures of seed maturation heterochrony in garden pea (Pisum sativum L) accessions BMC BIOINFORMATICS, V.21, Suppl 20, e567, P10 (year - 2020) https://doi.org/10.1186/s12859-020-03838-2

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5. - Петербургские ученые открыли новую форму белков у растений и бактерий Санкт-Петербургские ведомости, https://spbvedomosti.ru/news/country_and_world/peterburgskie-uchenye-otkryli-novuyu-formu-belkov-u-rasteniy-i-bakteriy/ (year - )

6. - Благодаря нацпроекту «Наука и университеты» поддержку получают самые прогрессивные исследования Первый канал, - (year - )

Annotation of the results obtained in 2020
In the scope of the Project in 2020 we studied diversity of plant amyloid proteins and their functions. We for the first time studied the amyloid properties of the legumin A2 and convicilin storage proteins of the seeds of garden pea P. sativum L. These proteins were previously identified in the proteomic screening of proteins that form potentially amyloidogenic detergent-resistant aggregates in pea seeds. The data obtained during this stage of the work showed that, under certain conditions, both of these proteins form fibrils that are resistant to treatment with cold ionic detergents, bind the amyloid-specific dye Thioflavin-T and exhibit birefringence upon binding of the Congo red dye. These results support amyloidogenic properties of legumin A2 and convicilin in vitro. The binding of Congo red and the formation of birefringence by bacterial cells secreting legumin A2 and convicilin in the C-DAG system shows that these proteins can form amyloid-like aggregates even under conditions close to in vivo. The amyloid properties of these proteins in vivo are planned to be tested at the next stage of work. These results are of considerable interest, since they indicate that different storage proteins could be present in the amyloid form in seeds and, potentially, form complex aggregates consisting of proteins of different groups. The interaction of various plant amyloids with each other and with human and bacterial amyloids is planned to be studied in 2021, for which, within the framework of this stage of the project, fibrils of the required proteins were obtained in preparative quantities. It should be noted that legumins, convicilin and vicilin, whose amyloid properties were shown by us [Antonets et al., PLOS Biology, 2020], belong to seed globulins that have domains of the ancient Cupin superfamily, presented in proteins of both prokaryotes and eukaryotes, and having a beta barrel structure. Based on our data, it can be assumed that these domains are the key determinants of the amyloid properties of seed globulins, and could also potentially determine their interaction in the amyloid form. Another aspect of the functional properties of amyloids of plant seed storage proteins, which was studied in the framework of this study, was the possible involvement of these proteins in defense of plants against pathogens. It is well known that cupin proteins, which include seed globulins, play an important role in plant defense against pathogens, primarily due to their lectin activity. We studied the characteristics of the production of the vicillin protein in various organs of the pea plant and found that despite a rather low production of this protein is observed in the leaves and stem, in the root vicilin is produced at a much higher level and forms aggregates clearly detected by fluorescence microscopy. Moreover, a particularly high level of vicilin production was observed in the root nodules of pea, where both diffuse fluorescence, which may indicate the presence of vicilin in a soluble or oligomeric form, and large aggregates were detected. The data on natural overproduction of vicilin in the roots and nodules of peas correspond to its possible involvement in supra-organismal plant-microbial interactions. Despite the fact that the interactions of plants with root nodule bacteria are mutualistic, the molecular systems that underlay pathogen-host and symbiont-host interactions have significant similarities [see, for example, the review by Kosolapova et al., Int. J. Mol. Sci., 2020]; therefore, the interaction of vicilin with bacteria in nodules could also suggest that it interacts with pathogens. We analyzed the effect of vicillin amyloid fibrils on the growth of pathogenic bacteria Rhodococcus fascians and the fungus Fusarium solani, as a result of which it was found that vicilin amyloids inhibit the growth of these species, although the effects against F. solani were weaker, which may be due to the specific features of the used environments and modes of cultivation. Moreover, using the yeast S. cerevisiae, we have shown that disruption of vicilin fibrils by ultrasound leads to the elimination of toxicity; thus, it is the fibrillar amyloid state of vicilin that inhibits fungal growth. Thus, the data obtained by us in the framework of this stage of the Project show that vicilin fibrils have a significant inhibitory effect on the growth of pathogenic microorganisms, which indicates the possible participation of this protein in the amyloid form in defense against pathogens. Overall, within the framework of this stage of the Project, novel data were obtained on the in vitro amyloid properties of legumin A2 and convicilin storage proteins of pea P. sativum L. seeds, and it was also shown that vicilin is produced and aggregated in various organs of this plant, and the higher production levels of this protein is achieved in the root and root nodules, and its amyloids are toxic to fungi and bacteria pathogenic for the plant. Publications of the Project in 2020: 1. Kosolapova A.O., Antonets K.S., Belousov M.V., Nizhnikov A.A. Biological functions of prokaryotic amyloids in the interspecies interactions: facts and assumptions // International Journal of Molecular Sciences, 2020, V.21, e7240. 2. Antonets K.S., Belousov M.V., Sulatskaya A.I., Belousova M.E., Kosolapova A.O., Sulatsky M.I., Andreeva E.A., Zykin P.A., Malovichko Y.V., Shtark O.Y., Lykholay A.N., Volkov K.V., Kuznetsova I.M., Turoverov K.K., Kochetkova E.Y., Bobylev A.G., Usachev K.S., Demidov O.N., Tikhonovich I.A., Nizhnikov A.A. Accumulation of storage proteins in plant seeds is mediated by amyloid formation // PLOS Biology, 2020, V.18(7), e3000564.

Publications

1. Antonets K.S., Belousov M.V., Sulatskaya A.I., Belousova M.E., Kosolapova A.O., Sulatsky M.I., Andreeva E.A., Zykin P.A., Malovichko Y.V., et al., Nizhnikov A.A. Accumulation of storage proteins in plant seeds is mediated by amyloid formation PLOS Biology, V.18(7), e3000564 (year - 2020) https://doi.org/10.1371/journal.pbio.3000564

2. Kosolapova A.O., Antonets K.S., Belousov M.V., Nizhnikov A.A. Biological functions of prokaryotic amyloids in the interspecies interactions: facts and assumptions International Journal of Molecular Sciences, V.21, e7240 (year - 2020) https://doi.org/10.3390/ijms21197240

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