Synthetic platform, intended to at least partially mimic a cuticle of terrestrial arthropod mouthparts

Abstract

The invention relates to a synthetic platform intended to mimic at least one type of interaction that takes place between a cuticular protein of the CPR family and an agent with cuticular tropism, and that occurs at the natural buccal cuticle of a terrestrial arthropod, said platform comprising a chitosan film in which at least one cuticular protein of the CPR family present in the natural cuticle of said terrestrial arthropod is trapped, while being accessible to interact with at least said agent with cuticular tropism, as well as to the methods for its manufacture and to in vitro evaluation and identification methods which use it.

Description

DESCRIPTION Title of the invention: Synthetic platform, intended to mimic at least partially a cuticle of mouthparts of a terrestrial arthropod technical field

[0001] The present invention relates to the technical field of in vitro evaluations. More specifically, it concerns a synthetic platform capable of mimicking the interaction that can occur in vivo on the surface of the buccal cuticle of a terrestrial arthropod with a so-called cuticulotropic agent, such as pathogens, salivary molecules, or any other substance exhibiting an affinity for these structures (for example, host proteins of a terrestrial arthropod). The study of this type of interaction is particularly relevant in the case of cuticulotropic agents that may be involved in the transmission of phytopathogens or in terrestrial arthropod host interactions. It also relates to a method for manufacturing such a mimetic platform, as well as methods for the in vitro evaluation and identification of cuticular proteins involved in cuticle functionalization using such a synthetic platform. Technological background of the invention

[0002] Crops are constantly threatened by plant pathogen infections, which lead to significant yield losses. Among these plant pathogens, certain bacteria and the majority of plant viruses, like some human and animal pathogens, are transmitted by a vector, most often a terrestrial arthropod. Aphids are vectors of plant pathogenic viruses. They possess piercing-sucking mouthparts. Mouthparts, particularly in terrestrial arthropods, are anatomical structures located around the mouth and adapted for feeding, manipulation, chewing, or sucking. They are composed of several specialized parts that vary in shape and function depending on the diet and lifestyle of the terrestrial arthropod.The aphid's mouthparts consist of a labrum housing a bundle of stylets composed of two mandibular stylets surrounding two maxillary stylets. The maxillary stylets have digitations that allow them to be joined along their length to form a feeding canal through which ingested fluids or compounds pass. The insect has a salivary duct and a salivary canal. These two canals join at the distal end of the stylets to form the common duct. In adult mosquitoes, the mouthparts consist of six segments: the labrum, labium, mandibles, maxillae, and hypopharynx. The mouthparts play an essential role in feeding and can also be involved in the transmission of pathogens in some insect vectors or in host-arthropod interactions. The mouthparts are covered by a cuticle, called the buccal cuticle, the composition of which can vary depending on the terrestrial arthropod. The cuticle of the stylets is made up of an assembly of chitin and cuticular proteins among which, in the aphid, is the styline-01 protein (Deshoux et al., 2020; Webster et al., 2018; Guschinskaya et al., 2020) involved in the transmission of plant viruses (Webster et al. 2018).Indeed, various functions are attributed to cuticular proteins (SO Andersen, 1979), including the recognition of phytopathogens. Cuticular proteins may possess chitin recognition units. These units, also called chitin-binding domains, can differ in their composition of constituent amino acids. However, certain conserved motifs exist that allow for the definition of cuticular protein families, such as the extended Rebers and Riddiford (R&R) consensus (JE Rebers et al. and JH Willis, 1988 and JE Rebers et al. and JH Willis, 2001), found in cuticular proteins of the CPR family, for example (the largest family in the cuticle of terrestrial arthropods), which will be referred to hereafter as the R&R consensus.This R&R consensus states that the chitin-binding domain (CBD) architecture is as follows: Gx(7)-[DEN]-Gx(6)-[FY]-xA-[DGN]-x(2,3)-G-[FY]-x-[AP]-x(6), where x can represent any amino acid, with the number of amino acids x shown in parentheses, where 2,3 means 2 or 3. The amino acids that can be present at that position in the sequence are shown in brackets (one from those listed). Three distinct variants of the R&R consensus exist and classify CPR proteins according to the RR-1, RR-2, or RR-3 type CBD (Andersen, 2000). In 2018, CG Webster et al. identified for the first time the cuticular proteins styline-01 and styline-02 containing an RR-1 type CBD at the tips of aphid mouthparts. At the time of the proteomic study by Deshoux et al. (2020), there were 150 CPRs in the pea aphid.The stylines are deeply embedded in a matrix of chitin fibers and possess an accessible domain on the surface of the cuticle. They co-localize with the sites of. Cauliflower mosaic virus (CaMV) retention. Thus, the original function of certain cuticular proteins (which may be to interact with a salivary molecule before being delivered to the host, for example, as is the case with the salivary effector Mp10 in aphids, which interacts with the cuticular protein stylin-03 (Deshoux et al. 2022); or which may be to interact with the salivary protein LIPS2 in the case of the cuticular protein Cp19 of the CPR family with RR-2-type CBD in mosquitoes (Arnoldi et al. 2022)) can be hijacked by phytopathogens by acting as receptors for certain pathogen surface proteins. More specifically, surface proteins of certain pathogens, particularly viruses, bind to certain cuticular proteins, which are considered receptors for such pathogens.

[0003] Furthermore, when aphids settle on the plant to feed, the introduction of their stylets triggers the plant's immune response to the aggressor. The effector Mp10 is present in aphid saliva. It is described as being able to suppress the production of ROS (Reactive Oxygen Species) and reduce the plant's immune responses (Bos et al., 2010). Mp10 also interferes with hormonal signaling pathways (jasmonic acid, salicylic acid) involved in plant defenses (Rodriguez et al., 2014). Mp10 interacts with the acrostyle and is able to bind to the cuticular protein stylin-03 of the aphid stylets (Deshoux et al., 2022). It is then injected into the plant with the saliva (Mugford et al., 2016). The interactions between a cuticular protein and an effector would therefore allow the modulation of host (plant, human, animal) - terrestrial arthropod interactions.

[0004] The study of cuticular protein / protein interactions involved in pathogen propagation or host (plant / human / vertebrate / animal) - terrestrial arthropod interactions, or the search for inhibitors of such interactions, is difficult to perform in vivo. Therefore, the development of solutions enabling such studies to be carried out in vitro is of great interest. In this context, the present invention aims to provide a synthetic platform for mimicking the interactions that one or more cuticular proteins, particularly those of the CPR family, present in the buccal cuticle of a terrestrial arthropod, may have. Summary of the invention

[0005] In this context, the present invention relates to a synthetic platform, intended to mimic at least one type of interaction occurring between a cuticular protein, in particular of the CPR family, and a cuticulotropic agent occurring at the level of the natural buccal cuticle of a terrestrial arthropod, comprising a chitosan film in which at least one cuticular protein present in the natural cuticle of said terrestrial arthropod is trapped, while being accessible to interact with at least said cuticulotropic agent.

[0006] It was by no means obvious that it would be possible, using chitosan rather than chitin, to immobilize a cuticular protein, particularly one from the CPR family, in an architecture that would leave at least one binding domain for a cuticulotropic agent accessible on the surface of the formed chitosan film. A cuticulotropic agent is understood to be an entity that has an affinity for the cuticle of a terrestrial arthropod in its natural environment, or even interacts specifically with it. Therefore, the synthetic platform proposed within the framework of the invention will allow the study of a number of interactions occurring in nature at the level of the cuticle of a terrestrial arthropod, but artificially in the laboratory.The synthetic platform proposed within the framework of the invention, i.e. prepared by a chemical process and not existing in the natural state, will allow the study of interactions, which will vary according to the cuticular protein(s) immobilized in the chitosan film, in particular chosen from among the interactions with a surface protein of a pathogen, a salivary molecule such as an effector, or any other binding entity having an affinity with said immobilized cuticular protein, and in particular involved in the transmission of a pathogen and / or in host / terrestrial arthropod interactions, but this without using the living terrestrial arthropod, which is why this type of study is called an in vitro study.Without being linked by any mechanism of action, it is suggested that the orientation of the cuticular protein, in particular of the CPR family, according to a conformation that mimics the natural state, is due to an assembly with chitosan determined by very strong CBD interactions, which leads to a conformation close to that which it has in the chitin of natural oral cuticles.

[0007] The synthetic platforms according to the invention can also be defined as follows: they comprise a chitosan film in which at least one cuticular protein, particularly from the CPR family, present in the natural cuticle of a terrestrial arthropod, is encapsulated. This cuticular protein remains accessible on the surface of the chitosan film by presenting a binding domain, notably for a cuticulotropic agent. This binding domain, accessible on the surface of the chitosan film, is also accessible on the surface of a natural buccal cuticle of said terrestrial arthropod. In other words, the cuticular protein, particularly from the CPR family, immobilized on the mimetic platform according to the invention possesses, in its natural state, a domain accessible on the surface of the cuticle, allowing its interaction with at least one cuticulotropic agent.Similarly, in the synthetic platform according to the invention, the immobilized cuticular protein, in particular of the CPR family, also has such an accessible domain on the surface of the cuticle allowing its interaction with at least one cuticulotropic agent.

[0008] In certain embodiments, the terrestrial arthropod is an insect, specifically an aphid. Therefore, in this case, the synthetic platform is designed to mimic at least one type of interaction between a cuticular protein and a cuticulotropic agent occurring in the natural buccal cuticle of an insect, particularly an aphid. In this instance, the immobilized cuticular protein is located in the natural buccal cuticle of the insect, specifically aphids, for example, at the acrostyle.

[0009] Advantageously, in the synthetic platforms according to the invention, chitosan has a degree of acetylation (DA) in the range of 0.5% to 85%, particularly in the range of 0.5% to 76%, and preferably in the range of 54% to 76%. With such a DA, the binding domains of the cuticular protein within the chitosan film are oriented and presented on the surface of the chitosan film in a manner even closer to the conformation of the cuticular protein found in the natural cuticle.

[0010] Advantageously in the synthetic platforms according to the invention, the chitosan film comprises a cuticular protein / chitosan mass ratio of 0.09% to 9%, in particular 0.5% to 1.3%.

[0011] Particularly advantageously, in the synthetic platforms according to the invention, chitosan has a degree of DA acetylation in the range of 0.5% to 85%, especially in the range of 0.5% to 76%, preferably in the range of 54% to 76% and a cuticular protein mass ratio of the CPR family / chitosan of 0.5% to 1.3%.

[0012] Preferably in the synthetic platforms according to the invention, chitosan has a weight-average molar mass (Mw) of 15 to 800 kg / mol, and preferably of 100 to 300 kg / mol. Such molecular weights promote satisfactory stability for the chitosan film.

[0013] According to particular embodiments of the invention, said cuticular protein is in its natural state a binding protein for a cuticulotropic agent, said cuticulotropic agent being selected from the surface proteins of a pathogen, salivary effectors, and any other molecules, in particular proteins, involved in the transmission of a pathogen or in host-arthropod interactions. In particular, said cuticular protein belongs to the CPR family, and is, in particular, styline-01.

[0014] According to one embodiment of the synthetic platforms of the invention, the chitosan film is itself immobilized by covalent grafting onto a support. This embodiment makes the synthetic platform easier to handle and less fragile during manipulation. In particular, the support may consist, at least in part, of a sheet of glass, silicon, silicone, titanium, polymer, gold, or mica.

[0015] In particular, in such a variant, the covalent coupling of the chitosan film to the support can be achieved via the amine functions of the chitosan.

[0016] The invention also relates to a method for preparing a synthetic platform, intended to mimic at least one type of interaction occurring between a cuticular protein, in particular of the CPR family, and a cuticulotropic agent occurring at the level of the natural buccal cuticle of a terrestrial arthropod, as defined within the scope of the invention, said method comprising the following successive steps: i) having an acidic aqueous solution comprising a mixture of chitosan and of at least one cuticular protein present in the natural buccal cuticle of said terrestrial arthropod, ii) deposit said solution on a support base, in sufficient quantity to form a continuous layer on said support base, iii) carry out a drying process leading to the formation of a film of said chitosan trapping said cuticular protein, with at least one binding domain of said cuticular protein to a cuticulotropic agent which is accessible on the surface of the chitosan film.

[0017] Such a process may, in particular, include the following successive steps: a) having a support functionalized with reactive functions, said support acting as a base support, b) having an acidic aqueous solution comprising a mixture of chitosan and at least one cuticular protein present in the natural buccal cuticle of said terrestrial arthropod, c) depositing said solution on said support, in sufficient quantity to form a continuous layer on the support, d) applying conditions suitable for forming covalent bonds between the reactive functions and amine functions of chitosan or functions derived from amine functions of chitosan, and then carrying out a drying process leading to the formation of a film of said chitosan trapping said cuticular protein, with at least one binding domain of said cuticular protein to a cuticulotropic agent which is accessible on the surface of the chitosan film.

[0018] Advantageously, the reactive functions of the functionalized support are chosen from acid, anhydride, aldehyde, halogenated derivatives, acyl chlorides and epoxy functions, with epoxy functions being preferred.

[0019] According to preferred embodiments of the process according to the invention, step iii) or step d) is carried out at a temperature not exceeding 150°C, in particular in the range of 60 to 80°C, preferably at a temperature of the order of

[0020] In general, the acidic aqueous solution comprises 0.1 to 3% by weight, including 0.5 to 1.5% by weight of chitosan, these % being given in relation to the total mass of the solution.

[0021] In the processes according to the invention, the acidic aqueous solution comprising a mixture of chitosan and at least one cuticular protein will preferably comprise a cuticular protein / chitosan mass ratio of 0.09% to 9%, in particular 0.5% to 1.3%.

[0022] Advantageously, in the process according to the invention, the acidic aqueous solution comprising a mixture of chitosan and at least one cuticular protein is an acidic aqueous solution, comprising at least one acid, in particular acetic acid, in which the ratio H+ of the acid(s) present: NH2 functions of chitosan is between 1.4:1 and 0.8:1, and is preferably 1:1.

[0023] The invention also relates to a method for evaluating the interaction of a cuticular protein, in particular of the CPR family, present in the natural buccal cuticle of a terrestrial arthropod, with a cuticulotropic agent, wherein the interaction is studied in vitro by making said cuticulotropic agent interact with a synthetic platform according to the invention.

[0024] Another object of the invention relates to a method for identifying a cuticulotropic agent of a natural buccal cuticle of a terrestrial arthropod, characterized in that the identification is carried out in vitro by making a candidate cuticulotropic agent interact with a synthetic platform according to the invention.

[0025] Another object of the invention relates to a method for identifying an inhibitor of the interaction of a cuticular protein present in the natural buccal cuticle occurring in the natural state with a given cuticulotropic agent, of a terrestrial arthropod, wherein the identification is carried out in vitro by making an inhibitor candidate interact with a synthetic platform according to the invention. Detailed description of the invention

[0026] Other features, details and advantages of the invention will become apparent from the description provided with reference to the accompanying figures given for illustrative purposes, which represent:

[0027] Figure 1 shows the evolution of the amount of styline-01 retained on the surface of a chitosan film, as a function of the amount deposited, in a study carried out in the experimental part of the description.

[0028] Figure 2 presents the results obtained in the immunolabeling examples allowing the detection of the C-terminal (Cter, 1), N-terminal (Nter, 2) or CBD (3) domains of styline-01 on the surface of the different platforms developed using specific antibodies directed against these three domains.

[0029] Figure 3 presents the results obtained in the examples of fluorescence detection of the retention of the CaMV P2 viral protein fused to a fluorescent-GFP protein (P2-GFP) on the surface of the different platforms developed.

[0030] Before going into the details of the invention, the meaning of certain terms used in its context will be reviewed. Unless otherwise specified, the terms used to describe the invention have the general meanings that a person skilled in the art would normally attribute to them.

[0031] The notion of "in the order of" or "approximately" means that a variation of at most 5%, 4%, 3%, 2%, and preferably 1%, around the given value is tolerated. However, an exact match with the given value is preferred.

[0032] The term "chitin-binding domain" or "CBD" refers to the specific region of a protein that allows it to bind to chitin. This specific region is characterized by the conservation of certain amino acids at key positions, forming a recognizable motif (chain of amino acids) that enables the formation of a sufficient number of interactions (in particular, of the hydrogen or hydrophobic type) to bind to chitin.

[0033] The term "cuticular protein-cuticulotropic agent binding domain" refers to the region of the cuticular protein that allows it to bind, specifically or non-specifically, to a cuticulotropic agent. The types of binding that can occur between a cuticular protein and a cuticulotropic agent include non-covalent bonds such as hydrogen bonds, electrostatic bonds, Van der Waals forces, and / or hydrophobic bonds.

[0034] An "effector," as classically understood in biology, is a molecule, often a protein, produced by an organism, that is secreted or injected into the tissues or cells of the host, and that acts directly on the host to modulate its responses. Effectors can inhibit immune responses, disrupt signaling pathways, alter metabolism, or exploit various cellular functions to promote the survival, colonization, or replication of the organism that produces them. They play a crucial role in host-pathogen or host-arthropod interactions, particularly in defense and parasitism strategies.

[0035] The term "host" refers to the plant or animal organism on which the terrestrial arthropod will land or inhabit, feed, develop, and reproduce. This can be a plant, a fungus, an animal, or a human being.

[0036] The acrostyle is the cuticular micro-territory identified in the mouthparts of aphids, located within the common canal, at the end of the two maxillary stylets.

[0037] The term "cuticular protein" refers to a structural protein found in the cuticle of arthropods. These proteins interact with chitin to form a rigid and resistant matrix that provides protection, support, and impermeability in natural cuticles. Cuticular proteins in terrestrial arthropods, particularly insects, encompass all proteins with a structural function located within the cuticle. These proteins contain conserved motifs, such as CBD. Different CBDs can be identified using pattern recognition tools, such as hidden Markov models (HMMs, for example, the R&R-Rebers and Riddiford type CBD, the most prevalent in the acrostyle of aphids and in the cuticles of other terrestrial arthropods).Depending on the protein's orientation within the cuticle, the N-terminal or C-terminal end of the protein may be located on the cuticle surface (as is the case with stylins or the Cp19 protein in mosquitoes) or possibly internally and therefore not exposed to the surface. Thus, CPR cuticular proteins (for "Cuticular Proteins with an R&R Consensus") constitute an important family of cuticular proteins characterized by the presence of a conserved Rebers & Riddiford consensus motif (R&R consensus). Stylins are cuticular proteins identified in aphid stylets that possess a motif or peptide accessible on the cuticle surface. Since all proteins in the CPR family adhere to the consensus principle... R&R, they all exhibit the central CBD chitin-binding domain described previously in paragraph

[0002] whose structure is predicted to be in the form of flat beta sheets. On either side of this central CBD domain, N- and C-terminal regions, more or less structured according to the proteins, are present.

[0038] A "vector" refers to any organism that actively transmits an infectious agent (pathogenic or not) from one host to another (whether animal or plant). The concept of active transmission requires that the vector, through its behavior, enables the transmission of an infectious agent by acquiring it from one host and transferring it to another. The infectious agent may or may not multiply within the vector.

[0039] The term "pathogen" refers to viruses, bacteria, and infectious agents that can affect plants, animals, or humans. In particular, examples include viruses of the genera Caulimovirus (including cauliflower mosaic virus (CaMV)), Cucumovirus (including cucumber mosaic virus (CMV)), and Potyvirus (including turnip mosaic virus (TuMV) and potato virus Y (PVY)), which are phytopathogenic viruses transmitted by aphids. Other examples include Closteroviruses (including citrus tristeza virus (CTV), transmitted by aphids) and Criniviruses (including lettuce yellows virus (LiYV), transmitted by whiteflies). Regarding bacteria, Xylella fastidosa, a xylem-dwelling bacterium transmitted by various hemipterans in Europe and the United States, is a notable example. It lodges in the vector notably via the cuticular chitin (Labroussaa et al., 2017, Almeida et al., 2003).

[0040] By "surface protein" of a pathogen, in the case of a viral protein, we mean a protein that is located on the surface of the capsid or viral envelope and plays a crucial role in receptor recognition or infection of host cells.

[0041] A "binding entity" generally refers to a molecule, a protein, or more generally a structure capable of associating with another entity through specific binding forces, whether chemical or physical, in order to form a complex or facilitate a functional interaction. In the context of this invention, a cuticulotropic agent constitutes a binding entity for a cuticular protein. Some examples of cuticular protein / agent pairs are given. cuticulotropes are listed in Table 1 below, for a given vertebrate arthropod. Table 1

[0042] "Receptor" refers to a biological molecule, usually a protein, that binds to a specific molecule (often called a ligand or here a binding entity) and can either trigger a biological response or activation of a particular cellular mechanism, or promote entry into a cell, infection, or transmission of a pathogen when it is a receptor-pathogen interaction.

[0043] Terrestrial arthropods are invertebrates belonging to the phylum Arthropoda, characterized by a segmented body, a chitinous exoskeleton, and jointed appendages, which have evolved to live in terrestrial ecosystems. Terrestrial arthropods include members of the classes Insecta, Arachnida, Myriapoda, and some terrestrial crustaceans.

[0044] Chitosan is a linear copolysaccharide composed of D-glucosamine (GIcN) and N-acetyl-D-glucosamine (GIcNAc) units linked by [3(1->4)] glycosidic bonds. Chitosan can therefore be represented by the following formula:

[0045] in which DA is the degree of acetylation, which can be defined as the percentage of the number of acetylated units, i.e., N-acetyl-D-glucosamine units, relative to the total number of units constituting the polymer of chitosan. In other words, DA is the % of N-acetyl-D-glucosamine units, relative to the total number of units constituting the chitosan polymer (i.e., the N-acetyl-D-glucosamine and D-glucosamine units).

[0046] Within the framework of the invention, the chitosan used may be a statistical chitosan, i.e. the N-acetyl-D-glucosamine and glucosamine units are distributed statistically (randomly), which is not reflected in the formula shown above.

[0047] Chitosan is generally obtained by alkaline deacetylation of chitin, which is found in nature, notably in insect cuticles or crustacean shells. However, there is no official nomenclature defining the alkaline deacetylation (AD) threshold between chitin and chitosan. Classically, the term chitosan refers to the family of such copolysaccharides that are sufficiently N-deacetylated and soluble in acidic aqueous media (acidic aqueous media with a stoichiometric amount of acidic function relative to the NH2 groups of chitosan, with the exception of sulfuric acid). The solubility of a chitosan can, in particular, be evaluated in water acidified with a stoichiometric amount of acetic acid (Vàrum et al., 1994). As an illustration, in insect cuticles, chitin, which is not soluble in aqueous media, has a DA of 80 to 90% (KY Zhu et al., 2016).Chitosan thus represents a family of deacetylated chitin derivatives soluble in weakly acidic aqueous media, which can exhibit different degrees of acetylation (DA) and different weight-average molar masses (Mw). The advantage of chitosan is therefore its ability to be soluble in acidic aqueous media, unlike chitin, which facilitates its use in the fabrication of synthetic platforms within the scope of the invention. Advantageously, within the scope of the invention, the chitosan forming the film of the synthetic platform has a degree of acetylation (DA) in the range of 0.5% to 85%, particularly in the range of 0.5% to 76%, and preferably in the range of 54% to 76%. The degree of acetylation can be determined experimentally by proton nuclear magnetic resonance (¹H NMR) using the method described by Hirai et al.Mw can be determined by size exclusion chromatography (SEC), as described by M. Dumont et al. Dispersion, which is a measure of the distribution of molecular weights, can. can also be determined by this technique. The dispersity of chitosan present in the films of the invention is preferably from 1 to 3, in particular from 1 to 2.

[0048] The weight-average molar mass (Mw) of chitosan can depend on the source of chitin used to prepare the chitosan and its degree of acetylation. For example, chitosan with a low degree of acetylation (DA < 5%) typically has an Mw of 150 to 210 kg / mol when obtained from shrimp chitin, and 530 to 570 kg / mol when obtained from squid chitin. Chitosan with a low Mw can be obtained from chitosan with a higher Mw by applying any method known in the prior art, notably described by G.G. Allan.

[0049] Various types of chitin and chitosan are commercially available. The chitosan used in the context of the invention can also be obtained by chemical or enzymatic deacetylation of chitin. Depending on the desired DA, chitosan can also be obtained by reacetylation of chitosan with a lower DA. Deacetylation can be carried out with an enzyme, generally chitin deacetylase, or with a chemical agent, generally a base, such as sodium or potassium hydroxide. Acetylation or reacetylation is controlled to obtain the desired DA and can be carried out using adjusted amounts of acetic anhydride, for example, in a hydroalcoholic solution of chitosan (containing a polyalcohol such as, for example, 1,2-propanediol) containing acetic acid. Such procedures are described by L. Vachoud et al.

[0050] The DA and Mw of the chitosan forming the film of the synthetic mimetic platform are controlled upstream of film formation. In other words, a chitosan with the desired DA and Mw is dissolved in the acidic aqueous solution that will be used to form the film.

[0051] In particular embodiments of the synthetic mimetic platform, the chitosan film incorporating the cuticular protein(s) has a thickness between 5 and 500 nm, preferably between 40 and 200 nm. The thickness can be determined, in particular, by atomic force microscopy, ellipsometry or spectral reflectance.

[0052] In the said film, chitosan may present a semi-crystalline structure, that is to say that X-ray diffraction analysis highlights the presence of the alpha allomorph of chitin for high DA or hydrated or anhydrous allomorph for low DA.

[0053] In the context of the invention, at least one cuticular protein found in the natural cuticle of a terrestrial arthropod, particularly one of the CPR family, is trapped within the chitosan film present in the synthetic platform. In its natural state, as well as in the synthetic platform of the invention, said cuticular protein has one or more binding domains for a cuticulotropic agent that is accessible on the surface of said cuticle (in its natural state), and also on the surface of the chitosan film (in the synthetic platform according to the invention), and which is therefore capable of interacting with a cuticulotropic agent, in particular such as surface proteins of a pathogen, salivary effectors, or other molecules involved in pathogen transmission or terrestrial host-arthropod interactions. This cuticular protein is specific to the cuticle that is to be mimicked.The invention relates to a platform designed to mimic at least one type of interaction between a cuticular protein and a cuticulotropic agent occurring at the level of the natural buccal cuticle of a terrestrial arthropod. This means that the platform will mimic the presentation of at least one cuticular protein, particularly from the CPR family, as it occurs on the buccal cuticle of a terrestrial arthropod, and in particular on the buccal cuticle of an aphid or mosquito.Thus, the synthetic platform according to the invention will make it possible to mimic the interactions that occur in the natural state between the cuticular protein and a cuticulotropic agent which may be a protein of a pathogen (in particular a surface protein of a virus), a salivary effector or any other molecule which is a binding entity for said cuticular protein and which may be involved in the transmission of pathogens and / or in terrestrial host / arthropod interactions.

[0054] The present invention is applicable for mimicking the cuticular protein / binding entity interactions that occur in the buccal cuticle of a large number of terrestrial arthropods. It is possible to immobilize in the chitosan film one or more cuticular proteins present in the cuticle of a terrestrial arthropod, particularly of the CPR family, the choice of the one or more Cuticular proteins are defined as those specific to the terrestrial arthropod cuticle being mimicked, and therefore to the specific terrestrial arthropod. When several cuticular proteins are immobilized in the chitosan film, they will generally be cuticular proteins from the same terrestrial arthropod, for example, styline-01 and styline-02, styline-03, or styline-04, which are found in the oral cuticles of aphids. The terrestrial arthropod can be an insect, for example, chosen from among the Hemiptera and Thysanoptera (aphids, leafhoppers, scale insects, whiteflies, psyllids, thrips), which are known to be vectors of plant pathogens, from among the Diptera (mosquitoes), or from among other terrestrial arthropods, such as ticks, which are vectors of pathogens to human or animal vertebrates.In the context of the invention, according to a particular embodiment, the synthetic platform produced is intended and / or suitable for mimicking at least in part the buccal cuticle of aphids, and in particular is suitable and / or intended to mimic at least in part the cuticle of the acrostyle of the stylet of aphids, and in particular of pea aphids or green peach aphids.

[0055] Various cuticular proteins are found in the different cuticles of a terrestrial arthropod. A description of the different types of cuticular proteins (14 families of cuticular proteins have been described to date) that may be present in the synthetic platforms according to the invention is available in Loannidou et al., 2014, and Guschinskaya et al., 2020, which may be consulted for further details. The majority of stylins are cuticular proteins of the CPR family, which have an RR-1 chitin-binding domain located in the cuticle of the acrostyle of the aphid stylet (a cuticular micro-territory at the stylet apex) and are involved in biotic interactions. These stylines are styline-01, styline-02, styline-03, styline-04 and styline-04bis (Deshoux et al. 2020 er Webster et al. 2018).The acrostyle of the aphid stylet also contains other CPR proteins that have an RR-2 type chitin-binding domain and CPAP3 proteins that have three ChtBD2 type chitin-binding domains (Uzest et al. 2010 and Deshoux et al. 2022). Such proteins are known to interact with at least one pathogen and thus promote its transmission by the terrestrial arthropod vector. By "interacting with at least one pathogen," we mean that it is the receptor for a binding entity involved in the transmission of the pathogen, for example, by being the receptor for a... A viral protein, specifically a surface protein of a pathogen (a pathogen-encoded protein that can be a "structural" protein, a component of the viral capsid, or a non-structural protein that forms a molecular link between the pathogen and the cuticular substrate by interacting with both). Stylins can also bind other molecules, such as effectors contained in aphid saliva. These molecules are then injected into the host and are capable of modulating the plant's defenses (Wang et al. 2023). If the synthetic platform aims to mimic the interactions occurring at the level of the aphid's buccal cuticle, one or more cuticular proteins of the CPR family, such as stylins, will be immobilized in the chitosan film. In particular, stylin-01 with the RR-1 motif, the sequence of which is described in CG Webster et al., 2018 and Deshoux et al., 2020.In the case of another terrestrial arthropod, the immobilized cuticular protein(s) will be different. For example, in the case of the mosquito, the Cp19 protein, which is a cuticular protein of the CPR family with an RR-2 type binding domain (whose sequence and interaction with the salivary effector LIPS-2 is described, Arnoldi et al., 2022), may be immobilized in the chitosan film.

[0056] In the synthetic platforms according to the invention, the cuticular protein(s) present is / are accessible for interaction with at least one binding entity, called a cuticulotropic agent, which may be involved in the transmission of a pathogen, or be a salivary effector or another molecule, particularly a protein, involved in terrestrial host-arthropod interactions that can modulate host defenses. That is to say, the binding domain of the cuticular protein to the binding entity involved in pathogen transmission or interaction with the terrestrial host-arthropod is accessible on the surface of the chitosan film. The binding entity involved in the transmission of said pathogen may be a surface protein of the pathogen.The linking entity that is involved in host-arthropod interactions may be a salivary effector that can impact the biting mechanism in a biting terrestrial arthropod such as the mosquito (Arnoldi et al., 2022).

[0057] In the context of the invention, the chitosan film of the proposed synthetic platform traps one or more cuticular proteins present in the cuticle of a terrestrial arthropod, by presenting its binding domain to a The binding entity (called the cuticulotropic agent) is notably involved in pathogen transmission or terrestrial host-arthropod interactions. The surface-accessible binding domain of the chitosan film can be either the C-terminal or N-terminal end of the protein. The surface-accessible C-terminal or N-terminal end of the protein specifically comprises the last 10 to 20 amino acids of that protein end, and typically the last 15 amino acids. For example, the surface-accessible protein binding domain of the chitosan film will be the C-terminal end of the cuticular protein when the latter is styline-01, present in aphids, or the Cp19 protein, present in mosquitoes.The pathogen that is transmitted by the terrestrial arthropod may be directed against plants, as is the case with viruses of the genera Caulimovirus [including cauliflower mosaic virus (CaMV)], Cucumovirus [including cucumber mosaic virus (CMV)], or of the genus Potyvirus [including turnip mosaic virus (TuMV), potato virus Y (PVY) and many other potyviruses], or potentially directed against humans or animals for other terrestrial arthropods.

[0058] In the case of styline-01 in aphids, it is the C-terminal portion of the protein that interacts with the non-structural viral protein Helper P2 of cauliflower mosaic virus. This non-structural viral protein establishes a molecular link between the viral proteins on the virus's surface and styline (the binding entity involved in pathogen transmission). Therefore, it is the C-terminal portion of styline-01, specifically the last fifteen amino acids of the sequence, that constitutes the binding domain for the cuticulotropic agent (in particular, the non-structural viral protein Helper P2 of cauliflower mosaic virus). Consequently, it is the C-terminal portion of styline-01 that will be accessible on the surface of the chitosan film of the synthetic platform according to the invention, when the latter comprises styline-01 immobilized within the chitosan film.In this case, this platform will be designed and capable of mimicking at least one type of interaction occurring between styline-01 and a cuticulotropic agent, specifically the non-structural viral protein Helper P2, occurring at the level of the natural buccal cuticle of the aphid. In the case of the Cp19 protein in the mosquito, it is also the C-terminal part of the protein that interacts with the salivary effector LIPS-2 (a binding entity involved in the insect's feeding behavior and foraging). blood vessels during a mosquito bite (Arnoldi et al., 2022). Therefore, here again, it is the C-terminal portion of the Cp19 protein that is the binding domain for the cuticulotropic agent (specifically the salivary effector LIPS-2). Thus, it is the C-terminal portion of the Cp19 protein that will be accessible on the surface of the chitosan film of the synthetic platform according to the invention, when the latter comprises the Cp19 protein immobilized within the chitosan film. In this case, this platform will be designed and capable of mimicking at least one type of interaction occurring between the Cp19 protein and a cuticulotropic agent, specifically the salivary effector LIPS-2, at the level of the mosquito's natural oral cuticle.

[0059] The following examples of a synthetic platform according to the invention can therefore be given: - a platform comprising a chitosan film in which styline-01 is encapsulated, with styline-01 remaining accessible on the surface of the chitosan by presenting a styline-01 binding domain to a cuticulotropic agent (in particular the non-structural viral protein Helper P2 of cauliflower mosaic virus, CaMV), said binding domain also being accessible in a natural aphid buccal cuticle. In particular, said platform comprises a chitosan film in which styline-01 is encapsulated, with styline-01 presenting its C-terminal portion on the surface of the chitosan film. - a platform comprising a chitosan film in which Cp19 protein is encapsulated, with the Cp19 protein remaining accessible on the surface of the chitosan by presenting a Cp19 protein binding domain to a cuticulotropic agent (in particular the salivary effector LIPS-2), said binding domain also being accessible in a natural mosquito buccal cuticle. In particular, said platform comprises a chitosan film in which Cp19 protein is encapsulated, and presents its C-terminal portion on the surface of the chitosan film.

[0060] When the mimetic platform includes a support on which the chitosan is immobilized, it can have a variety of shapes and dimensions. Preferably, it will be a flat support, such as a plate, slab, blade, or strip, these terms being used according to the nature and thickness of the support. The support is generally flat and can vary in shape and thickness. The thickness is generally between 0.1 and 2 mm, typically from 0.13 mm to 1.5 mm. In particular, the support consists, at least in part, of a blade. of glass, a silicon surface (silicon sheet or silicon layer on another type of substrate, for example), silicone, titanium, polymer, gold or mica.

[0061] When a support is present, the chitosan layer, which traps at least one cuticular protein, is covalently attached to the support, specifically to one of its large faces. To achieve this, the support is functionalized with reactive groups capable of forming covalent bonds directly with the chitosan's NH2 groups (more precisely, GIcN units) or with derived groups obtained from the chitosan's NH2 groups (more precisely, GIcN units). Any technique well-known to those skilled in the art for coupling molecules bearing NH2 groups to functionalized supports, particularly those with -OH, -COOH, aldehyde, anhydride, acyl chloride, halogenated derivative, or epoxy groups, can be used.

[0062] In particular, in a classical manner, a glass or silica support presents on the surface hydroxy functions which can be converted into epoxy functions (reactive functions capable of forming covalent bonds with the NH2 functions of chitosan), by coupling with a trimethoxysilane carrying an epoxy function, such as 3-glycidyloxypropyltrimethoxysilane.

[0063] Many other covalent coupling techniques are available in the literature and will be adapted depending on the selected support. Examples include: - dopamine coupling to a titanium substrate, leading to reactive -aldehyde functions (Shi et al., 2008; Shi et al., 2009), - coupling of a succinic triethoxysilylpropyl anhydride to a titanium substrate, leading to reactive anhydride functions (Campos et al., 2015), - dopamine coupling to silicone, leading to reactive OH functions (Wang et al., 2012), - a coupling of 3-aminopropyldimethylmono-ethoxysilane on a glass substrate, followed by a deposition of PEMAh (Poly(ethylene-alt-maleic anhydride) - acrylic acid, leading to reactive anhydride functions (Bratskaya et al., 2007), - a grafting of acrylic acid onto polypropylene, corresponding to reactive -COOH functions (Morais et al., 2020).

[0064] Coupling to reactive substrate functions, whether of the -OH, -COOH, aldehyde, anhydride, epoxy, or other types, can occur directly to the chitosan's -NH2 amine group or to derived functions. These derived functions are obtained by reacting the -NH2 amine group with a reagent, leading, for example, to quaternary ammonium or -COOH groups (by coupling p-benzoic acid to the chitosan's -NH2 groups, as described by Zhu et al., 2002). The reactive function pair on the substrate / function pair on the chitosan (-NH2 or a derivative of the -NH2 group) will be chosen to induce a covalent bond under appropriate conditions.

[0065] In the processes according to the invention, the film formation conditions, which can incorporate this covalent coupling when a support is present, also lead to the desired orientation of the cuticular protein(s) present in the deposited solution during the final functionalization of the resulting chitosan film. This is because the cuticular protein(s) will be trapped within the chitosan film during its formation. By desired orientation, it is understood that the binding domain of said cuticular protein to a cuticulotropic agent is located on the surface of the chitosan film and is therefore accessible for studying the interaction with said binding entity. The resulting functionalized chitosan film is thus suitable for use as an interaction surface with a cuticulotropic agent, particularly one involved in pathogen transmission or terrestrial host-arthropod interactions.

[0066] In the processes according to the invention, for producing the film, an acidic aqueous solution comprising chitosan and at least one cuticular protein is used. Generally, said acidic aqueous solution comprises from 0.1 to 3% by weight, in particular from 0.5 to 1.5% by weight, of chitosan, these percentages being given relative to the total mass of the solution. Said solution also comprises the cuticular protein(s) that will be trapped in the chitosan film during its formation. In particular, said solution comprises a mixture of chitosan having selected DA and Mw values ​​and at least one cuticular protein with a cuticular protein / chitosan mass ratio of 0.09% to 9%, in particular from 0.5 to 1.3%. When several cuticular proteins are to be trapped in the chitosan film, a cuticular protein / chitosan mass ratio of 0.09% to 9%, specifically 0.5% to 1.3%, will be used for each of these cuticular proteins. According to a method of In a particular embodiment of the invention, a single cuticular protein, present in the desired quantity, is trapped in the chitosan film.

[0067] The manufacturing process for the synthetic platform according to the invention is implemented with selected chitosan and selected cuticular protein(s) to obtain the desired cuticular protein concentration and film thickness. Such a process comprises the following successive steps: i) preparing an acidic aqueous solution comprising a mixture of chitosan and at least one cuticular protein present in the natural buccal cuticle of said terrestrial arthropod, in particular of the CPR family; ii) depositing said solution onto a support base in sufficient quantity to form a continuous layer on said support base; and iii) carrying out drying, ultimately resulting in the formation of a film.

[0068] When manufacturing a synthetic platform corresponding to a self-supporting film, the deposition is carried out on a support base, and the chitosan film, once formed, can then be peeled off. In this case, the support base has no reactive surface functions.

[0069] For practical reasons, particularly in terms of ease of handling and implementation, it is preferable to have a synthetic platform in which the chitosan film is immobilized by covalent grafting onto a support.Typically, the process according to the invention comprises the following successive steps: a) having a support functionalized with reactive functions, said support acting as a base support, b) having an acidic aqueous solution comprising a mixture of chitosan and at least one cuticular protein present in the natural buccal cuticle of a terrestrial arthropod, in particular of the CPR family, c) depositing said solution on said support, in sufficient quantity to form a continuous layer on the support, d) applying conditions suitable for forming covalent bonds between the reactive functions and amine NH2 functions of chitosan or derived functions, and then carrying out drying leading to the final formation of a film.

[0070] In this case, the supporting base has reactive functionalities on its surface that are capable of reacting with the NH2 groups of chitosan or with functionalities derived from these NH2 groups. Preferably, the covalent coupling occurs directly with the NH2 groups of chitosan. The reactive functionalities present on the surface of the support that are capable of reacting with the NH2 groups of chitosan or with functionalities derived from these NH2 groups are, in particular, -COOH, anhydride, aldehyde, acyl chloride, halogenated derivative, or epoxy groups, and preferably epoxy. Preferably, the covalent coupling occurs directly with the NH2 groups of chitosan. When the grafting reaction occurs with functionalities derived from these NH2 groups, the deposited solution directly comprises chitosan containing these derived functionalities.

[0071] Apart from this possible difference, whether a functionalized support or a simple base is used, the acidic aqueous solution that is deposited can be the same. The acidic aqueous solution typically has a pH of around 5.5. Such an acidic pH is obtained through the presence of at least one acid. This acid in the acidic aqueous solution can be an organic acid, an inorganic acid, or a mixture of both. Hydrochloric acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, nitric acid, sulfuric acid, hexafluorophosphoric acid, tetrafluoroboric acid, trifluoroacetic acid, acetic acid, sulfonic acid such as methanesulfonic acid, and mono- or polycarboxylic acids are typical examples of acids that can be used. Preferably, the acid present in the deposited acidic solution is acetic acid or hydrochloric acid.The concentration of the acid in the acidic aqueous solution can be adjusted so that the ratio (H+ of the acid or of all the acids present: NH2 groups of chitosan) is between 1.4:1 and 0.8:1, and preferably 1:1 (stoichiometric amount relative to the amine NH2 groups of chitosan). A larger quantity of acid may also be used.

[0072] Chitosan, the cuticular protein(s) present, particularly those of the CPR family, and their quantity have been described previously. These are preferably placed in water acidified by the addition of acetic acid, advantageously with an amount of acid stoichiometric with respect to the free amine groups of the dissolved chitosan. It is also possible to add a surfactant to the solution to promote surface orientation. Chitosan from a binding domain (for at least one cuticulotropic agent) of the cuticular protein to be trapped is hydrophilic or hydrophobic. For example, it is possible to create chemically modified chito-oligosaccharide "surfactants" (Berthalon et al., 2024). Their presence would modulate the hydrophobicity of the film, thus trapping the most hydrophobic part of the protein within the film and exposing the hydrophilic part of the protein on the surface. In the case of styline-01, for example, the C-terminus of the protein is more hydrophilic and would therefore be more readily exposed on the surface. In any case, due to the presence of the R&R consensus within the CPR protein family, the presence of the common CBD motif promotes the desired immobilization under conditions that can be adapted by those skilled in the art, depending on whether the C-terminus or N-terminus is variable.In cases where covalent coupling is desired between chitosan and a support, one or more coupling-enhancing additives may, if necessary, be added to the solution.

[0073] The deposition can be carried out using any suitable method that allows for film formation, and in the case of a film deposited on a support that allows for film formation and covalent chitosan / support bonds, a deposition technique under suitable conditions will be used to obtain a film of the desired thickness. In particular, spin coating (also known as centrifugal coating) or a solvent evaporation deposition technique may be used.

[0074] The conditions necessary for forming covalent bonds between the reactive functional groups present on the surface of the substrate and the amine groups of chitosan or derived functional groups will be adapted by a person skilled in the art, depending on the covalent coupling that occurs during film formation and its grafting to the substrate. In the case of an epoxy / amine covalent coupling, the coupling occurs by simple hydrolysis, with heating to a temperature in the range of 60 to 80°C, preferably 70°C, under a humid atmosphere (particularly with a humidity level of 100%). In the case of an anhydride / amine covalent coupling, the deposited solution will preferably contain a polyalcohol, such as polyethylene glycol or glycerol. In all cases, the coupling reaction is carried out over a sufficiently long period to achieve the desired grafting.

[0075] These coupling conditions, as well as the drying process, will be carried out under gentle conditions, meaning conditions that do not cause denaturation or alteration of the cuticular proteins present. When there is no covalent coupling, drying can be performed at room temperature (around 22°C), or more generally at a temperature of 20 to 30°C. Drying can also be carried out simultaneously or almost simultaneously with the deposition of the solution, particularly in the spin coating technique. In the case of covalent coupling, heating will most often be used for drying. In particular, during step d) mentioned above, heating at a temperature not exceeding 150°C, specifically in the range of 60 to 80°C, preferably 70°C when heating is necessary to achieve covalent coupling, will be used.Drying is carried out at such a temperature, typically for 24 to 72 hours, for example for 24 hours.

[0076] Ultimately, in the synthetic platforms of the invention, a chitosan film of selected DA and Mw is formed, trapping one or more cuticular proteins within it. The cuticular protein(s) (which are naturally present on the cuticle of a terrestrial arthropod) is / are accessible on the surface of the chitosan film to interact with at least one cuticulotropic agent. That is to say, the binding domain between the cuticular protein, particularly one from the CPR family, and said binding entity is present on the surface of the chitosan film.Thus, the synthetic platform can be described as a biomimetic platform for the cuticle of this terrestrial arthropod, since it will mimic the interactions that occur naturally at the cuticle level between the cuticular protein and a cuticulotropic agent. This agent could be a surface protein of a pathogen, a viral protein, a salivary effector, or any other molecule involved in pathogen transmission or host-arthropod interactions. These interactions can be of various types: hydrophobic, electrostatic, hydrogen bonding, IT interactions, or Van der Waals forces, as in antigen / antibody interactions.Thus, the synthetic platforms according to the invention can be used for various in vitro applications, allowing the study of the interactions of said cuticular protein with a known or potential binding entity whose interactions with the immobilized cuticular protein are to be studied. The interaction conditions will be... These techniques are adapted by experts in the field, depending on the cuticular protein / binding entity pair being studied, and specifically to mimic as closely as possible the interaction that can occur in nature. Incubation conditions for detecting antigen / antibody interactions are well-established and can be adjusted.

[0077] Advantageously, the synthetic platform will be used, particularly in in vitro studies, within one month of its preparation. In the meantime, it should preferably be stored at a temperature below 30°C, specifically at room temperature (20 to 25°C) in an airtight container to avoid storage in a humid environment, but rather in a dry atmosphere (humidity level below 35%).

[0078] Any method for in vitro evaluating the interaction of a cuticular protein, particularly one from the CPR family, present in the natural cuticle of a terrestrial arthropod and interacting with at least one cuticulotropic agent using a synthetic platform according to the invention, forms an integral part of the invention. The invention also relates to any method for identifying a cuticulotropic agent in the natural buccal cuticle of a terrestrial arthropod, wherein the identification is performed in vitro by interacting a candidate cuticulotropic agent with a synthetic platform according to the invention.The invention also relates to any method for identifying an inhibitor of the interaction between a cuticular protein present in the natural buccal cuticle of a naturally occurring terrestrial arthropod and a cuticulotropic agent, wherein the identification is performed in vitro by interacting a candidate inhibitor with a synthetic platform according to the invention. Such a method for identifying an inhibitor of the interaction between a cuticular protein present in the natural buccal cuticle of a naturally occurring terrestrial arthropod and a cuticulotropic agent may be performed by interacting a candidate inhibitor with a synthetic platform according to the invention, with or without said cuticulotropic agent.

[0079] In these different methods, the cuticulotropic agent is a cuticular protein binding entity immobilized and accessible on the surface of the chitosan film, as described in the present description.

[0080] The descriptions given in the description of the synthetic platforms according to the invention apply mutatis mutandis to the preparation processes, as well as to the evaluation and identification methods according to the invention.

[0081] Experimental section

[0082] Abbreviations

[0083] AcOH, acetic acid; GPS, (3-glycidyloxypropyl)trimethoxysilane; SEC, size-exclusion chromatography; NMR, nuclear magnetic resonance; CBD, chitin-binding domain; Cter, C-terminal region of the protein; Nter, N-terminal region of the protein; ROI, region of interest; RFU, relative fluorescence unit; ChNF, chitin nanofibrils

[0084] Materials and Methods

[0085] Shrimp-derived chitosan was obtained from Mahtani Chitosan Ltd. The degree of acetylation (DA) was determined by 1H NMR and was 0.5%. The DA of the chitosan was determined using a Bruker Advance III proton magnetic resonance (1H NMR) spectrometer, 300 MHz, 64 scans, according to the method described by Hirai et al. The weight-average molar mass of the chitosan was determined by size-exclusion chromatography coupled with SEC-MALLS multi-angle laser light scattering (Mw = 190 kg / mol, dispersity D = 2.3). Acetic acid (AcOH, > 99% w / w), ammonium hydroxide (NH4OH, 28% w / w), hydrogen peroxide (H2O2, 40% w / w), sulfuric acid (H2SO4, 96% w / w), deuterium oxide (99.9% D), HEPES buffer (as a powder), Tween®20 and Imidazole (C3H4N2, 99%) were purchased from Sigma Aldrich.Toluene, anhydrous toluene, 1,2-propanediol, acetic anhydride (>99% w / w), sodium chloride (NaCl), potassium chloride (KCl), disodium phosphate (Na₂HPO₄), and potassium phosphate dihydrogen (KH₂PO₄) were purchased from Carlo Erba. (3-Glycycloxypropyl)trimethoxysilane (GPS >98%) was purchased from TCI. Ni-NTA His*Bind® Superflow™ resin, AcTev protease, DTT (C₄H₁₀O₂S₂), and Laemmli SDS 6X (ref: J61337.AC) were purchased from Thermo Fisher. All pre-made kD™ Mini-PROTEAN® TGX™ protein gels, Precision Plus Protein Dual Color standards, 10x Tris / Glycine / SDS, and Coomassie Bio-Safe™ stain were purchased from BioRad. Microscope glass slides (rectified to 90°) were purchased from [source missing]. Carl Roth. Chitin nanofibrils (2 wt%) were obtained from Sugino (Namerikawa, Japan) and used diluted in Milli-Q water at a concentration of 0.02 wt%. The ultrapure Milli-Q water was obtained using an ultrapure water system (Milli-Q Simplicity® Ultrapure Water System, resistivity of 18.2 MQ.cm).

[0086] Methods used

[0087] 1. Chitosan preparation

[0088] The chitosan used for film formation was purified and reacetylated as described by L. Vachoud et al., to DAs of 15%, 35%, 54%, 67%, and 76%. The weight-average molar mass was checked by SAC-MALLS analysis, and the DA was estimated by 1H NMR as described by Hirai et al.

[0089] 2. Preparation of recombinant Styline-01

[0090] The styline-01 gene, encoding the mature styline-01 protein lacking a signal peptide, was cloned into the pETtrxl b plasmid (EMBL, Heidelberg, Germany) fused with a histidine tag and thoredoxine for bacterial production (E. coli strain BL21). Recombinant styline-01 was produced and purified according to the protocol described for the production and purification of the effector Mp10 in Deshoux et al. 2022. Styline-01 was then dialyzed with HEPES buffer (0.01 M, pH 7). The purity of the obtained styline-01 was verified by electrophoresis, and its concentration was assessed using a Nanodrop One instrument at 280 nm. The protein was frozen as aliquots at -20°C until further use.

[0091] 3. Fluorescein (FITC) marking of styline-01

[0092] Styline-01 was coupled to fluorescein (FITC) to allow for fluorescence quantification. The FITC labeling kit was purchased from Thermo Fisher (FluoReporter™ FITC Protein Labeling Kit, ref: F6434) and used according to the protocol described in the kit. Briefly, styline-01 was mixed with a 1 M reactive FITC stock solution in sodium bicarbonate buffer (pH=9) for 1 h. The solution was then dialyzed against 0.01 M HEPES buffer until all unbound FITC molecules were removed. The presence of unbound FITC was verified by gel electrophoresis using the BioRad GelDoc™ Go imaging system. The concentration styline-01-FITC was determined with a Nanodrop one device at 280nm and 494nm and showed a concentration of 1.5 mg / mL with an F / P factor of 2 (i.e., number of FITCs per protein).

[0093] 4. Preparation of P2-GFP and P2Rev5-GFP proteins

[0094] The CaMV virus encodes the non-structural helper protein P2, which is essential for transmission by aphids. The mutated version P2Rev5 (Q-to-Y substitution at position 6) impairs CaMV transmission (A. Moreno et al. 2005), and the mutated protein is no longer able to interact with receptors on the cuticle surface of mouthparts (Uzest et al. 2007). Both the P2 and P2Rev5 proteins were fused to a green fluorescent protein (GFP) at their C-terminus to allow their observation in the mouthparts of insect vectors and were expressed in insect / Sf9 baculovirus cells as previously described by M. Uzest et al. After production, the P2-GFP and P2Rev5-GFP fusion viral proteins were sedimented, resuspended in DB5-0 buffer, and then frozen as aliquots at -20°C until use.The concentration of P2-GFP or P2Rev5-GFP protein is difficult to assess precisely because the proteins are not purified and they aggregate even at low concentrations. The production protocol has been standardized to ensure reproducibility in the production of viral fusion proteins.

[0095] 5. Formation of films grafted onto glass slides

[0096] Glass slides (75 x 25 mm²) were immersed in a piranha solution (H₂SO₄ / H₂O₂, 7 / 3 v / v) at 150 °C for 15 min and then grafted with a 2% GPS solution in anhydrous toluene for 5 h. Surface-functionalized glass slides with epoxy groups were thus obtained. A 1% aqueous solution (1% w / v) was prepared from each of the different chitosans obtained by adding a stoichiometric amount of acetic acid relative to the GIcN units.

[0097] A first series of tests was carried out for comparison, first creating a chitosan film, grafting it onto the glass slide, and then depositing Styl Ine-01 onto the previously formed chitosan film. In this case, chitosans with DA concentrations of 0.5%, 15%, 35%, 54%, 67%, and 76% were used.

[0098] The prepared chitosan solutions were deposited onto the functionalized glass slides by centrifugal coating (3000 rpm, 5 min), and covalent grafting was achieved by heat treatment in the presence of water (100% relative humidity, 70 °C, 24 h). The resulting chitosan film was then washed first with acetic acid solution (0.01 M, pH 5.5, 500 mL, 15 h), then with Milli-Q water (30 min, 3 times). The slides bearing the chitosan film were then stored in a glass container at room temperature (T = 23 °C, relative humidity = 35%). Next, styline-01 was deposited as follows: 1.5pL drops of different concentrations of styline-01-FITC were deposited onto the chitosan film grafted onto the glass slide, using an IBIDI® device, a bottomless removable network to facilitate the physical separation of the different deposited drops.After 30 minutes, the resulting spots were washed twice with 250 pL of HEPES buffer (0.01 M, pH 7), then twice with 250 pL of Tween20®-HEPES buffer (0.01 M HEPES, 0.05% w / v in Tween20®), and finally twice with 250 pL of HEPES buffer. A final wash was performed with 250 pL of Milli-Q water (wash protocol A) to remove any proteins that had not interacted with the surface. The surfaces were then dried under a fume hood with a slightly perforated aluminum foil to prevent photobleaching.

[0099] Fluorescence was measured using a Leica THUNDER microscope (475 nm, 50 ms exposure) by defining the region of interest (ROI) around the spots. A calibration curve was used to convert the measured fluorescence into the equivalent concentration of bound styline-01 per mm² of chitosan film, according to the following equation: P x MxA

[0100] C = NaxV d

[0101] with M (mg / mol) being the molar mass of styline-01-FITC (13.18 kDa), A being the area of ​​the spot formed by the deposited droplet in mm², P being the number of proteins deposited per mm², Na being Avogadro's number (6.022 x 10 23 mol⁻¹) and Vd, which is the volume of the deposited droplet (0.0015 mL). The contact area of ​​a 1.5 pL droplet was considered to vary depending on the chitosan film: it represented 3.60, 3.41, 4.11, 4.46, 4.43, and 3.99 mm². 2 for a DA of 0.5%, 15%, 35%, 54%, 67%, and 76%, respectively. The tests were carried out in triplicate.

[0102] Figure 1 shows the change in the number of proteins attached to the film after washing, as a function of the number of styline-01 proteins deposited before washing. It appears that regardless of the DA of chitosan, as the concentration of styline-01 increases, the amount of styline-01 also increases until it reaches a plateau, indicating saturation of the interaction sites present on the chitosan. Contrary to what one might expect given the DA of the chitin in natural cuticles, it is the chitosan with a DA of 67% (9 x 10⁻¹¹) that exhibits the highest concentration of styline-01. 11 proteins per mm 2 ), and not the DA 76% chitosan, which has the highest affinity for styline-01. The DA 0.5% chitosan has the lowest affinity, which is still very high (2 x 00). 11 proteins per mm 2 ).

[0103] To fabricate the platforms according to the invention, chitosans with DA concentrations of 0.5% and 67% were selected. In this case, before performing the centrifugal coating, these chitosan solutions were used as is (platform A) or mixed for 30 minutes with styline-01 (C=0.0816 mg / mL in the resulting mixture, platform B) or with styline-01 and ChNF (C=0.0816 mg / mL of styline-01 and 0.002 wt% of ChNF in the resulting mixture, platform C). Once the film had formed, 1.5 pL drops of styline-01 (C=0.5 mg / mL) were deposited onto platforms A1-1 and A2-1, and drops of a mixture of styline-01 and chitin nanofibrils were deposited on the surface of platforms A1-2 and A2-2. Tests with chitin fibers have been carried out to approximate the natural structure of terrestrial arthropod cuticles where chitin is present in the form of fibers linked to cuticular proteins accessible on the surface of the cuticle (CG Webster, et al).The concentration of styline-01 corresponded to the concentration required to saturate the interaction sites of chitosan having a DA of 67%, when styline-01 was deposited on an already formed chitosan film.

[0104] The prepared solutions were then deposited onto glass slides functionalized by centrifugal coating (3000 rpm, 5 min), and covalent grafting was achieved by heat treatment in the presence of water (100% relative humidity, 70 °C, 24 h). In this case, rinsing with acetic acid was omitted to avoid the risk of protein degradation. The slides bearing a chitosan and protein film were then stored in a glass container at room temperature (T = 23 °C, humidity = 35%). Chitosan / ChNF / styline-01 hybrid films were also grafted onto silicon surfaces and used for [various applications]. SEM observations were performed using an FEI Quanta 250 FEG microscope. As a negative control, a chitosan DA 67% / WGA (wheat germ agglutinin protein) film was also produced with the same amount of protein as in the styline-01 case. Table 2 summarizes all the platforms produced.

[0105] Table 2

[0106] 6. Study of the orientation of the protein trapped in chitosan films

[0107] In aphids, certain viral retention sites are located on the acrostyle, a specific area of ​​the cuticle. The Cter portion of styline-01 is accessible on the surface of the acrostyle and can be recognized by a specific antibody directed against this end. There is some competition for interaction between the antibody directed against the Cter end of styline-01 and the CaMV P2 viral protein in the interaction with styline-01 on the cuticle surface. This result suggests that styline-01 can act as a receptor for the CaMV virus (CG Webster, et al.). The mimetic platform according to the invention therefore had to present styline-01 in the same way to be biomimetic to the aphid cuticle.

[0108] In such a case, when styline-01 interacts with the chitosan film, its binding is achieved through an interaction of its CBD with the carbohydrate. The Cter and Nter ends of the protein can then be trapped within the chitosan film or accessible on its surface. Immunostaining was used to detect the Cter, Nter, or CBD portion of the protein on the different films. Antibodies were produced against specific parts of the styline-01 sequence: anti-Nter (1.01), anti-Cter (1.11), and anti-CBD (1.09) antibodies, as described in Webster et al., 2018. The peptides of the Cter, Nter, or CBD portion of styline-01 were synthesized by Eurogentec (Kanaka Eurogentec SA, Seraing, Belgium) and used to immunize rabbits. The antibodies produced were collected and purified against the peptides used for immunization.The secondary antibody used was an anti-rabbit IGG antibody coupled with Alexa Fluor 488 (A11070, Thermo Fisher Scientific). All antibodies were used diluted in HEPES-Tween20® buffer (0.05 wt% in Tween20®) at a 1 / 1600 ratio from a purified or commercial solution. A bottomless removable IBIDI® device was placed on the different candidate platforms to physically separate the various immunostaining samples. First, 50 µL of a 5 wt% skimmed milk powder solution was loaded into the wells formed by the IBIDI® device. After 30 min of incubation, the milk was removed and the wells were washed according to wash protocol A. Milk is commonly used as a blocking agent for non-specific interactions in Western blot techniques, which allowed for the specific detection of styline-O1-antibody interactions.The second step involved the use of a labeled primary antibody. For type A platforms (A 1-1, 1-2, 2-1, and 2-2), 50 pL of antibody 1.11, 1.01, or 1.09 was loaded into wells 1, 2, and 3 of each platform, respectively. The antibody was loaded throughout the well, while styline-01 was present as a droplet only in the center of each well. Thus, positive labeling of the antibody would result in a fluorescent circle. For type B and C platforms, 1.5 pL of antibody 1.11, 1.01, or 1.09 was loaded into wells 1, 2, and 3 of each platform, respectively. For these platforms, the antibody was loaded as a droplet only in the center of each well, while styline-01 was present throughout the well. Thus, here again, positive antibody labeling would lead to a fluorescent circle. After 1 hour of incubation, the... The contents of the wells were removed and the antibodies were washed. The third step consisted of binding with a second fluorescent antibody, followed by washing. For this, 50 pL of the second labeled fluorescent antibody was loaded into wells 1, 2, and 3 of each platform. After 1 h of incubation with planar shaking (300 rpm), the contents of the wells were removed, and the wells were washed and dried according to the washing and drying protocol A described previously. The surfaces were then observed with a Leica Thunder microscope at 475 nm with an exposure of 500 ms. Fluorescence was quantified from the ROI corresponding to the area of ​​the labeled antibody (central circle in the well). RFUs were corrected by subtracting the fluorescence from the background noise of the antibody loading in the IBIDI® well, knowing that each of these fluorescences was also corrected by subtracting the autofluorescence of the grafted glass slide.

[0109] First, visually, no Cter, Nter, or CBD labeling was observed for the A platforms (Figure 2). The relative fluorescence units (RFUs) for Cter, Nter, and CBD detection on the A platforms showed that Cter was poorly accessible at the surface. For the B2 and C2 platforms, the fluorescence in well 1 was significantly higher than in wells 2 and 3 (RFUs of 1457 and 1526 for Cter compared to -57 and 16 for Nter, and 259 and 74 for CBD, respectively). This reflects the accessibility of the Cter in styline-01 and the lower accessibility of Nter and CBD, which must therefore be trapped within the film. CBD appears more accessible and more strongly labeled with fluorescence than Nter. This indicates that Nter is trapped deeper within the film than CBD. For the Cter, the RFUs of well 1 are similar for platforms B2 and C2 and around 1500 (1457 and 1526 respectively).This shows that the presence of the ChNF has no impact on the orientation of the styline-01 which has its Cter part accessible on the surface.

[0110] For platforms B1 and C1, the fluorescence obtained for well 1 is higher than that of wells 2 and 3. The RFUs were 239 and 158 for Cter, 185 and 131 for Nter, and 121 and 102 for CBD for platforms C1 and B1, respectively. Therefore, DA influences the fluorescence obtained and thus the accessibility of styline-01. But, again, the platform B1, the fluorescence of the Cter is greater than that of the Nter, which is itself greater than that of the CBD, which clearly reflects the orientation of the Cter part of the protein towards the outside and on the surface of the chitosan film.

[0111] 7. Study of binding to viral proteins P2 and P2Rev5

[0112] CaMV transmission in aphids is non-circulative. This means that the virus is transiently retained on the cuticle of the aphid mouthparts without being internalized into the aphid body and hemolymph. CaMV receptors are located on the acrostyle, a specific cuticular zone at the tip of the maxillary stylets of aphids. The non-structural protein P2 of CaMV is responsible for viral retention on the acrostyle due to its attachment to the surface of the acrostyle of dissected stylets (Uzest et al. 2007).

[0113] Competition between the antibody directed against the C-terminus of styline-01 and the CaMV P2 viral protein shows that the CaMV receptor and the C-terminus of styline-01 co-localize on the cuticle surface (CG Webster, et al., 2018). When the N-terminus of the P2 viral protein is mutated, this results in a lack of retention at the tips of aphid stylets.

[0114] To evaluate the biological function of styline-01, as present in the natural cuticle of aphids, found on the platforms, its binding to the helper protein P2 was investigated, as well as its binding to its mutated version, P2Rev5. As in section 6, an IBIDI® device was applied to the surface of the chitosan film, and milk blocking was performed. The interaction with the P2 protein was assessed by depositing either 50 pL of a P2-GFP solution (1 / 10 of the aliquoted supernatant) for platforms A or a 1.5 pL drop of the same P2-GFP solution for platforms B, C, and D. P2 was deposited over the entire surface of the well for platforms A, while styline-01 was only present as a circle in the center of the well. Thus, positive retention of P2-GFP corresponds to a ring of fluorescence.For platforms B and C, styline-01 was present over the entire surface of the well, and P2 was only present as a circle in the center of the well; therefore, again, positive retention of P2-GFP corresponds to a fluorescence circle. A 0.5 pL drop of a P2Rev5-GFP solution (1 / 10 of the... aliquoted supernatant) was also deposited for platform B2. After 30 minutes of interaction, the contents of the wells were removed, the wells were washed and dried and the fluorescence measured, as described in paragraph 6.

[0115] In the case of platforms A, no fluorescence corresponding to the retention of the P2-GFP protein was detected (Figure 3). Weak fluorescence corresponding to P2-GFP protein retention was detected for platforms B1 and C1, while in the case of platforms B2 and C2, the fluorescence was intense. This shows that with a DA of 0.5%, there is indeed retention of the viral P2 protein on the surface of the chitosan film, but in a smaller quantity than in the case of DA 67%. No fluorescence was observed for platform D (negative control), which confirms the reliability of the test. Finally, the deposition of P2Rev5-GFP on platform B2 also resulted in no fluorescence, which confirms the biomimicry of the platforms according to the invention. References

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Claims

DEMANDS 1. Synthetic platform, intended to mimic at least one type of interaction occurring between a cuticular protein and a cuticulotropic agent occurring at the level of the natural buccal cuticle of a terrestrial arthropod, comprising a chitosan film in which at least one cuticular protein of the CPR family present in the natural cuticle of said terrestrial arthropod is trapped, with at least one binding domain of said cuticular protein to a cuticulotropic agent which is accessible on the surface of the chitosan film.

2. Synthetic platform according to claim 1, characterized in that the terrestrial arthropod is an insect, and in particular an aphid.

3. Synthetic platform according to claim 1 or 2, characterized in that the chitosan has a degree of acetylation DA in the range of 0.5% to 85%, in particular in the range of 0.5% to 76%, preferably in the range of 54% to 76%.

4. Synthetic platform according to any one of claims 1 to 3, characterized in that chitosan has a weight average molar mass Mw of 15 to 800 kg / mol, and preferably of 100 to 300 kg / mol.

5. Synthetic platform according to any one of claims 1 to 4, characterized in that said cuticular protein is in its natural state a binding protein for a cuticulotropic agent selected from among the surface proteins of a pathogen, salivary effectors and any other molecules involved in the transmission of a pathogen or in host-arthropod interactions.

6. Synthetic platform according to any one of claims 1 to 5, characterized in that said cuticular protein is styline-01, styline-02, styline-03, styline-04, styline-04bis or Cp19 and, in particular, is styline-01.

7. Synthetic platform according to any one of claims 1 to 6, characterized in that the chitosan film is itself immobilized by covalent grafting onto a support.

8. Synthetic platform according to claim 7, characterized in that the support is made at least in part of a sheet of glass, silicon, silicone, titanium, polymer, gold or mica.

9. Synthetic platform according to claim 7 or 8, characterized in that the covalent coupling of the chitosan film on the support is obtained via the amine functions of the chitosan.

10. A method for preparing a synthetic platform, intended to mimic at least one type of interaction occurring between a cuticular protein of the CPR family and a cuticulotropic agent occurring at the level of the natural buccal cuticle of a terrestrial arthropod, as defined in any one of claims 1 to 9, characterized in that it comprises the following successive steps: i) having an acidic aqueous solution comprising a mixture of chitosan and at least one cuticular protein of the CPR family present in the natural buccal cuticle of said terrestrial arthropod, ii) depositing said solution onto a support base, in sufficient quantity to form a continuous layer on said support base, iii) carrying out a drying process leading ultimately to the formation of a film of said chitosan trapping said cuticular protein,with at least one binding domain of said cuticular protein to a cuticulotropic agent that is accessible on the surface of the chitosan film.

11. A preparation method according to claim 10, characterized in that it comprises the following successive steps: a) having a support functionalized with reactive functions, said support acting as a base support, b) having an acidic aqueous solution comprising a mixture of chitosan and at least one cuticular protein of the CPR family present in the natural buccal cuticle of said terrestrial arthropod, c) depositing said solution onto said support, in sufficient quantity to form a continuous layer on the support, d) apply conditions suitable for forming covalent bonds between reactive functions and amine functions of chitosan or functions derived from amine functions of chitosan, then carry out a drying process leading to the formation of a film of said chitosan trapping said cuticular protein, with at least one binding domain of said cuticular protein to a cuticulotropic agent which is accessible on the surface of the chitosan film.

12. A process according to claim 11, characterized in that the reactive functions of the functionalized support are chosen from acid, anhydride, aldehyde, acyl chloride, halogenated derivatives and epoxy functions, epoxy functions being preferred.

13. A method according to any one of claims 10 to 12, characterized in that step iii) or step d) is carried out at a temperature not exceeding 150°C, in particular in the range of 60 to 80°C, preferably at a temperature of around 70°C.

14. A process according to any one of claims 10 to 13, characterized in that the acidic aqueous solution comprises from 0.1 to 3% by weight, in particular from 0.5 to 1.5% by weight of chitosan, these % being given in relation to the total mass of the solution.

15. A process according to any one of claims 10 to 13, characterized in that the acidic aqueous solution comprising a mixture of chitosan and at least one cuticular protein of the CPR family comprises a CPR family cuticular protein / chitosan mass ratio of 0.09% to 9%, in particular 0.5% to 1.3%.

16. A method according to any one of claims 10 to 14, characterized in that the acidic aqueous solution comprising a mixture of chitosan and at least one cuticular protein of the CPR family is an acidic aqueous solution, comprising at least one acid, in particular acetic acid, in which the ratio of H+ of the acid(s) present to the NH2 functions of chitosan is between 1.4:1 and 0.8:1, and is preferably 1:

1.

17. Method for evaluating the interaction of a cuticular protein of the CPR family present in the natural buccal cuticle of a terrestrial arthropod, with a cuticulotropic agent, characterized in that the interaction is studied in vitro by interacting said cuticulotropic agent with a synthetic platform according to any one of claims 1 to 9.

18. Method for identifying a cuticulotropic agent of a natural buccal cuticle of a terrestrial arthropod, characterized in that the identification is carried out in vitro by interacting a candidate cuticulotropic agent with a synthetic platform according to any one of claims 1 to 9.

19. Method for identifying an inhibitor of the interaction of a cuticular protein of the CPR family present in the natural buccal cuticle occurring in the natural state with a given cuticulotropic agent, of a terrestrial arthropod, characterized in that the identification is carried out in vitro by making an inhibitor candidate interact with a synthetic platform according to any one of claims 1 to 9.

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