Protein nanofibrils as coformulant

Abstract

The present invention relates to the field of protein nanofibrils originating from Bacillus sp. provided in a composition as additive to a biologically active substance, in particular to a microbial substance such as a pesticide or plant beneficial agent. More specifically, the present invention relates to a composition comprising an agriculturally bioactive substance and Alpha(helical) Endospore appendage (A-ENA) protein nanofibril materials, more precisely wherein said A-ENA protein nanofibrils are recombinantly produced and exogenously added to said bioactive substance of the composition, thereby functioning as coformulant or additive, and preferably increasing the biological activity of said substance. In particular, the composition of the invention comprises B. thuringiensis spores or cells and self-assembled (engineered or functionalized) A-ENA protein nanofibrils as coformulant, providing for an improved efficacy and / or application potential as pesticide or insecticide. The present invention further relates to a method for controlling plant growth and protection of plants or parts thereof from pest damage, in particular by applying said composition of the invention in an effective amount or formulation, wherein the presence of said A-ENA protein nanofibril coformulant or additive results in increased efficacy and / or virulence of the bioactive substance of said composition, in particular for bioactive substances comprising Bacillus sp. strains.

Description

[0001] HaRe / ENAcoformulant / 875-PCT

[0002] PROTEIN NANOFIBRILS AS COFORMULANT

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to the field of protein nanofibrils originating from Bacillus sp. provided in a composition as additive to a biologically active substance, in particular to a microbial substance such as a pesticide or plant beneficial agent. More specifically, the present invention relates to a composition comprising an agriculturally bioactive substance and Alpha(helical) Endospore appendage (A-ENA) protein nanofibril materials, more precisely wherein said A-ENA protein nanofibrils are recombinantly produced and exogenously added to said bioactive substance of the composition, thereby functioning as coformulant or additive, and preferably increasing the biological activity of said substance. In particular, the composition of the invention comprises B. thuringiensis spores or cells and self-assembled (engineered or functionalized) A-ENA protein nanofibrils as coformulant, providing for an improved efficacy and / or application potential as pesticide or insecticide. The present invention further relates to a method for controlling plant growth and protection of plants or parts thereof from pest damage, in particular by applying said composition of the invention in an effective amount or formulation, wherein the presence of said A-ENA protein nanofibril coformulant or additive results in increased efficacy and / or virulence of the bioactive substance of said composition, in particular for bioactive substances comprising Bacillus sp. strains.

[0005] BACKGROUND

[0006] The natural entomopathogenic properties of Bacillus thuringiensis (Bt) strains have led to world-wide adoption of Bt as an industrial biopesticide and vector control agent. Bt was originally registered as a General Use in 1961 but is now found in over 1000 registered American pesticide products (National Pesticide Information Center. NPIC Product Research Online (NPRO): 'Bacillus thuringiensis’. http: / / npic. orst.edu / NPRO / ), positioning Bt-based products dominate the bioinsecticide market, which was globally valued at USD 3.6 billion in 2023 and is projected to reach USD 8.7 billion by 2030, growing at a CAGR of 13.5% (Research and Markets, 2023). Bt's longstanding efficacy and proven safety make it the preferred choice for pest management in both organic and conventional farming. There is a demand to increase the share of biological pesticides within the crop protection market due to increasing regulations on synthetic chemical pesticides, rising consumer demand for organic and 'sustainably' produced food and growing resistance to synthetic chemicals in insect pests.

[0007] Bt biopesticides act through the combined action of spores and Cry toxin proteins, which are ingested by pests and cause mortality. During the sporulation process, Bt secretes parasporal bodies (PSB) that HaRe / ENAcoformulant / 875-PCT are crystalline agglomerates of different (Cry protein) insecticidal pro-toxins that synergistically target specific insect groups. Typically, PSBs are present in the extracellular milieu, and are therefore subject to environmental dispersal and separation from the infectious body, i.e. the spore. Although some strains such as Bacillus sphaericus (Kalfon et al., 1984) have solved this potential issue by producing PSBs that are embedded within the interstitial space between the spore body and the exosporium, most commonly used commercial strains such as Bt. subsp. Kurstaki (BtK) and Bt. subsp. Israelensis (Btl) produce extrasporal PSBs, raising the question how simultaneous uptake of spore and PSB are ensured during infection. Co-transmission of spores and PSBs is a likely requirement for efficient establishment of the natural Bt lifecycle considering cytotoxicity is predominantly mediated by the PSB, giving ingested spores the opportunity to germinate and proliferate on the nutrients supplied by the insect remains. Insect exposure to PSBs alone will lead to insect death but no subsequent proliferation and dispersion of Bt titers, whereas ingestion of PSB deprived spore suspensions will have minimal cytotoxic effect.

[0008] Btl is a naturally occurring soil bacterium that is used as a larvicide in aquatic and wetland areas where black fly and mosquito-borne disease control is deployed (Charles and De Barjac, 1982). Typical commercial formulations (Aquabac®, Teknar®, Bactimos® , and Vectobac® ) consist of a mixture of spores and extracellular PSBs that are produced through a simple fermentation process that leverages the natural growth cycle of Btl where nutrient starvation and high cell density trigger sporulation. These conditions mimic the natural scenario of Bacillus biofilm formation which consists of a complex network of vegetative cells, spores, PSBs and extracellular matrix (ECM) components. Because most studies on Btl tend to focus on PSB composition, larvicidal activity and specificity, the composition of the extracellular milieu of a spore dominated population remains poorly understood despite obvious academic and industrial interest.

[0009] As first described in Remaut et al. (2025; PCT / EP2024 / 074370) and in Sleutel et al. (2025; submitted), CryoEM analysis resolved authentic protein nanofibrils constituting a Bti spore-based biofilm as a pervasive extrasporal matrix (ESM) constructed of 8 nm diameter protein nanofibers, dubbed A-ENA (see nomenclature below), that function as molecular tethers between spores and PSBs, said A-ENA proteins being extensively covalently cross-linked to via iso-peptide bonds. Beyond the biological role of these A- ENA based nanofibrils in Bti spore-PSB clustering, these insights also form the basis of novel applications due to the nature and unique properties of these A-ENA fibrils, and the capability of recombinantly producing these self-assembling nanofibrils, as described.

[0010] However, in view of today's key challenges to improve the biocidal activity of the Bt-based products, further efforts and insights in the mode of action are of importance to the field, to for instance increase the persistence and longevity of the biocide, since Cry proteins lose their efficacy when exposed to UV HaRe / ENAcoformulant / 875-PCT irradiation; as well as to optimize delivery on plants, crops, and aquatic environments as well as to improve suboptimal concentration, distribution and adhesion of Bt spores and Cry proteins on plant surfaces and near the water surface; to enhance the insecticidal activity for later-stage larvae, further to manage resistance development; and finally maximising the activity window, often affected by diverse environmental conditions (such as intense sun and rainfall), which can impact farmer adoption and trust. So there is a need to improve the biocidal activity, specifically the Bt-activity, in commercial biocidal products.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention is based on the initial observation that the proteinaceous extracellular matrix produced by Bt Israelensis functions as a molecular glue holding spores and PSBs in tight unison within a spore biofilm, and the identification of A-ENA proteins and their self-assembled nanofibrillar structures. Due to the characteristics of these A-ENA nanofibrils, a robust spore-PSB connection is formed and this is particularly logical for Bti given the aquatic lifestyle of its natural hosts, e.g. mosquito larvae. The absence of such a connection is likely to lead to separation of both entities in a liquid environment due to dilution effects. Further investigation of these A-ENA nanofibrils produced upon recombinant expression of the a-ena coding sequence in a bacterial production host, are applicable as additive to Bt spore preparations as to increase the spore clustering and toxin retention, moreover thereby increasing the killing activity of said Bt spore prep as compared to a prep without the A-ENA nanofibril additive. Furthermore, the exogenous addition of purified A-ENA nanofibrils to Bacillus thuringiensis subsp. Kurstaki (Btk) spore suspensions, which natively lack a-ena, led to a similar effect, indicating that A-ENA nanofibrils are widely usable as a spore clustering agent, i.e. as a biofilm for spores, as well as an adjuvant or coformulant to improve the insecticidal activity of any Bt species.

[0013] Furthermore, the observations that rather non-specific interactions drive the binding of A-ENA fibrils to the surface of the Btk crystals, points towards a general promiscuity of A-ENA mediated interactions with a diverse range of surfaces (spore, PSB) amplified through avidity (display density of patchy, sticky surfaces on A-ENA fibrils combined with fibril super bundling).

[0014] In addition, A-ENA protein nanofibrils were used as induced temporary biofilms herein, confirming their application potential as a generic platform for the agglutination or clustering of particulates such as microbial spores, specifically exemplified herein by different Bt spores. Moreover, also particulates such as bacterial cells, toxins, protein crystals and / or combinations thereof, may be bound together by a matrix of A-ENA nanofibrils acting as 'clustering, aggregation or biofilm formation' formulant.

[0015] Ultimately, the presence of A-ENA nanofibril coformulants may thus be applied as a marked gain of virulence, enabling the rational, non-GMO functionalization of a biological pest control agent. HaRe / ENAcoformulant / 875-PCT

[0016] Next to the biological implications, A-ENA nanofibrils have remarkable material properties. Although the chemical and physical robustness of spore derived self-assembly structures has been demonstrated for numerous systems (Pradhan et al., 2021) (Henriques and Moran, 2007; Sleutel et al., 2024; Terry et al., 2017), those structures are either susceptible to depolymerization under reducing conditions or unfold under extreme conditions (e.g. 100% (w / v) formic acid for L-ENA). For A-ENA, an unfolding or depolymerization transition point could not be identified, making it one of the most resilient (natural) protein structures studied to date, which is attributed to the covalent crosslinks combined with the compact size of the protomers. Spores are non-motile dormant structures, subject to a wide range of conditions as a result of environmental dispersion, that may include desiccation, UV radiation, high temperatures, proteolytic attack, and shear stress. By producing a durable net that guarantees comigration of the PSBs together with the spore right up until the moment of ingestion by the host, Bti has developed a way to optimize for virulence and with it, the opportunity for further propagation. So by the breakthrough of identifying the nature of this net, i.e. the structural units of the A-ENA protein nanofibrils, and through developing processes for isolation and recombinant production and purification of these self-assembling A-ENA nanofibrils, a novel type of bionanomaterial is provided, with surprisingly an application as coformulant for pesticides due to its biological function as a virulence factor.

[0017] Further aspects of the invention involve the use of modified or engineered A-ENA protein nanofibrils, which lead to functionalized fibrils that are applicable for further advantageous properties in agricultural formulations, such as but not limited to plant retention and UV-damage, as described herein.

[0018] A first aspect of the invention thus relates to a composition comprising an agriculturally bioactive substance and a protein nanofibril, which comprises a self-assembled endospore appendage (ENA) protein nanofibril classified as an A-ENA protein, according to the definition provided herein and in Remaut et al. (2025; PCT / EP2024 / 074370). More specifically, the invention relates to said composition comprising an agriculturally bioactive substance and one ore more, or a plurality of protein nanofibril(s), wherein said protein nanofibril(s) comprise one ore more A-ENA proteins, and the nanofibril(s) are exogenously added to said agriculturally bioactive substance. In particular, said A-ENA protein nanofibril may be made recombinantly and / or heterogeneously, and added as an A-ENA protein nanofibril component that was isolated from its production host, and / or purified from a host cell. In a specific embodiment, the composition described herein comprises an agriculturally bioactive substance comprising or consisting of a microbial spore suspension, microbial cells, microbial toxins, microbial proteins or microbial metabolites, or a formulation of any one or of a combination thereof. In a further specific embodiment, the composition described herein comprises an agriculturally bioactive substance which contains or is any one of said structural components (microbial spore suspension, microbial cells, microbial toxins, microbial proteins or microbial metabolites, or a formulation of any one or of a HaRe / ENAcoformulant / 875-PCT combination thereof) originating from a Bacillus sp., preferably a B. thuringiensis sp., more preferably a Bt israelensis or Bt kurstaki sp., and said composition comprises an A-ENA protein nanofibril, wherein said nanofibril is heterogenous to the Bacillus sp. and / or is made recombinantly in a difference host for (exogenous) addition to said composition. So said composition of the invention comprises an agriculturally bioactive substance and an A-ENA protein nanofibril which is recombinantly made and / or exogenously added to the composition. It is thus specified herein that said A-ENA protein nanofibril of the composition is defined herein as an A-ENA protein containing fibrous assembly that is not endogenously produced by said agriculturally bioactive substance, in particular said Bt strain, of said composition.

[0019] Further embodiments relates to said composition described herein, wherein said agriculturally bioactive substance comprises a biocidal active substance, such as an insecticide, nematicide, pesticide, fungicide, herbicide, herbicide safener, or comprises a plant growth stimulating substance such as a plant growth regulator, biostimulant, biofertilizer, and / or combinations of any of said substances. In a specific embodiment, said composition described herein comprises an agriculturally bioactive substance comprising a Bacillus sp. strain or a Pasteuria sp. strain., preferably a B. thuringiensis strain, or a biocidal mutant variant thereof.

[0020] A further specific embodiment relates to said composition described herein, wherein the agriculturally bioactive substance comprises a spore-containing suspension, preferably selected from Bacillus sp., Paenibacillus sp., Pasteuria sp., Brevibacillus laterosporus, Clostridium bifermentans, Lysinibacillus sphaericus, or a biocidal mutant variant thereof.

[0021] More specific embodiments relate to the composition described herein, wherein the Bacillus sp. is selected from any one of the strains of the list of: Bacillus thuringiensis, in particular any one of Bt. Israelensis, Bt.Kurstaki, Bt. Aizawai, Bt. Kenyae, or Bt. Tenebrionis, or an insecticidal mutant variant of any one thereof.

[0022] Specific embodiments described herein also refer to said composition described herein, wherein the protein nanofibril comprises one or more engineered or functionalized A-ENA proteins. In a specific embodiment, said engineering or functionalization relates to an A-ENA protein nanofibril modification to obtain a nanofibril that is a plant-binding agent, or plant-part-specific binder (e.g. providing for seed or leaf retention), and / or provides protection to hostile conditions (e.g. UV, by engineering A-ENA to include aromatic amino acids on its surface), and / or that improves uptake or ingestion by larvae.

[0023] Further embodiments relate to a composition comprising an agriculturally bioactive substance and an A- ENA protein nanofibril wherein due to clustering of the substance, which may be a spore, cell, and / or protein toxin or metabolite, within the A-ENA protein nanofibrils, results in an 'induced temporary HaRe / ENAcoformulant / 875-PCT biofilm'. So a specific embodiment relates to a biofilm comprises exogenous A-ENA protein nanofibrils and an agriculturally active substance, which is preferably a microbial spore or cell, more preferably a Bt spore or cell.

[0024] Further embodiments relate to said composition described herein formulated to contain a preservative, a structuring agent or surfactant, a buffer, a dye, an anti-foam agent, a wetting agent, or a freeze-thaw stabilizing agent. Alternatively, a formulation comprising the composition described herein is provided, which may be a tank mix formulation, a wettable granule, a spray, or floatable briquettes.

[0025] A second aspect relates to a method for protecting plants or plant parts, and / or for biocidal control of plant development, the method comprising to apply an effective dose or formulation of the composition to an area containing said plant or plant parts. More specifically said area or plant part may comprise or consist of leaves, shoots, seeds, fruits, full plants, or roots. An alternative embodiment relates to a method for control of mosquito pests to prevent disease, wherein an effective amount of the composition or formulation is applied to an area in need thereof.

[0026] A further aspect relates to a method for producing the composition described herein, comprising the steps of a) generating A-ENA or engineered A-ENA protein nanofibrils in a host cell, b) extracting the self-assembled nanofibrils from said host cell, and c) bringing the agriculturally bioactive substance and the extracted self-assembled nanofibrils together as to obtain the composition. In a specific embodiment, said A-ENA or engineered A-ENA protein nanofibril is functionalized after extraction from the host cells in step b. prior to step c. as to obtain an A-ENA-based protein nanofibril with particular properties (e.g. plant-binding specificity).

[0027] The invention further relates to uses of the composition described herein, such as used for pest controlling, for crop protection, or disease control, and / or as a biocidal product, or alternatively as plant growth stimulating substance, such as fertilization.

[0028] Finally, also the use of the A-ENA protein nanofibril described herein, to enhance pesticidal activity of a bacterial endospore, preferably a Bacillus endospore, wherein said pesticidal activity is preferably insecticidal activity of a B. thuringiensis endospore is provided herein. Specific uses of said A-ENA protein nanofibril as described herein relate to the application as coformulant for enhancing virulence or pesticidal activity of a bacterial endospore, preferably insecticidal activity of a B. thuringiensis endospore.

[0029] DESCRIPTION OF THE FIGURES

[0030] The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. HaRe / ENAcoformulant / 875-PCT

[0031] Figure 1. A-ENA clusters spores and PSBs and acts as a virulence factor, (a) Phase-contrast microscopy of the different Histodenz fractions (supernatant (SN), 40% and 60%) comparing Bti WT and Aa-ena and different centrifugal forces (100 and 10,000 g). PSBs are false colored blue, scale bar = 5 pm. (b) SDS- PAGE of high-pH dissolved PSBs in the different density fractions. Input (before fractionation) shows same starting toxin load. Bands correspond to CryllAa (♦),Crylla fragments (O) and CytlAa ( ▼ ). (c) Normalized OD600 (i.e. relative spore content) of the different density fractions of Bti WT and Aa-ena.

[0032] (d) nsTEM images from Bti WT and Aa-ena. PSBs and spores are labelled with blue and white arrowheads, respectively. Scale bar is 3 pM. (e) Phase contrast images of Btk WT (left), or Btk recombinantly expressing (Btk p-A-ENA; middle) or with exogenously added A-ENA fibrils (Btk + A-ENA at 4.18 mg / ml; right). PSBs are false colored blue, scale bar = 5 pm.

[0033] Figure 2. Recombinant expression or exogenous addition of A-ENA results in endospore - PSB clustering in Bacillus thuringiensis Kurstaki. (a) SDS-PAGE analysis of dissolved crystal proteins from Btk (left) and Bti (right). For Btk, samples include WT, Btk pAS_A-ENA (pA, expressing a-ena from a plasmid under its native promoter), and recombinant A-ENA (4.18 mg / ml final concentration) mixed with Btk WT ('+A'), all normalized by ODgoo- For Bti, WT and Aa-ena (AA) were similarly normalized. Bands were analyzed by mass spectrometry (MS) fingerprinting, with identified proteins indicated next to each band, (b) SDS-PAGE analysis of different fractions after Histodenz cushion separation (supernatant (SN), 40% and 60%). The input (before fractionation) confirms equal toxin loading across conditions. The fraction type, strain, and centrifugation speed are indicated above the gel. (c) Light microscopy images of the indicated strains reveal clustering induced by the presence of A-ENA. scale bar = 10 pm. (d) nsTEM images of the analyzed strains show that A-ENA, whether expressed from a plasmid (pA-ENA) or recombinantly added to WT cells, induces phenotypes similar to those observed in Bti. Scale bar = 3 pm.

[0034] (e) Normalized ODgoo values of different fractions and strains after fractionation, (f) nsTEM of purified Btk spores obtained using a sucrose gradient. In the 80% sucrose fraction, pure spores from WT remain unclustered, whereas pA-ENA spores are associated with PSCs, forming clusters. White arrowheads indicate spores, while blue arrowheads indicate PSCs. Scale bar = 3 pm.

[0035] Figure 3. Bacillus thuringiensis Sv. Israelensis (Bti) spores produce Endospore Appendages (ENA), (a) Schematic drawing of the sporulation cycle of B. thuringiensis. (i) Asymmetric cell division results in the formation of a forespore and a larger mother cell, (ii-iii) The mother cell engulfs the forespore and produces the parasporal body (PSB) with insecticidal proteins, (iv) Consecutive protective layers develop around the maturing spore, while the mother cell prepares for lysis, (v) Upon mother cell lysis, the mature spore and PSB are released into the environment, (vi) The cycle is completed as spores germinate under favorable conditions, returning to the vegetative state. Created using BioRender.com. (b) Phase contrast microscopy image of a sporulating Bti cells. M: mother cell, S: spore, P: PSB. (c) Wide-field view HaRe / ENAcoformulant / 875-PCT of sporulating cells, showing clusters of varying sizes, (d) Inset of the dotted square in panel (c) showing a small cluster composed of spores and PSBs. (e) TEM micrograph of a resuspended Bti spore biofilm, composed of endospores (S) and PSBs (P).

[0036] Figure 4. Enhanced entomopathogenic activity of Bacillus thuringiensis on Trichoplusia ni (cabbage looper) larvae with purified recombinant A-ENA fibrils, a. Representative ns-EM images of wildtype Btk spore suspensions incubated with purified recombinant A-ENA fibrils (4.18 mg / mL, produced in E. coli) show that the exogenous addition of fibrils to spore preparations results in the clustering of spores and Cry toxin crystals in a network of A-ENA fibrils, b. representative picture of dead and alive Trichoplusia ni (cabbage looper) larvae, following feeding on medium supplemented with Btk spore suspensions, c. Survival curve of Trichoplusia ni larvae feeding on media supplemented with different formulations: spore suspensions of wild-type B. thuringiensis Sv. Kurstaki ('BTK WT'), recombinant B. thuringiensis Sv. Kurstaki expressing A-ENA ('BTK + A-ENA'), or wild-type B. thuringiensis Sv. Israelensis ('BTI'), and phosphate buffered saline as a negative control ('PBS'), d. Survival curves of T. ni larvae feeding on media supplemented with spore suspensions of wild-type B. thuringiensis Sv. Kurstaki ('BTK WT'); spore suspensions of wild-type B. thuringiensis Sv. Kurstaki ('BTK WT + A-ENA fibrils') incubated with purified recombinant A-ENA (4.18 mg / ml, produced in E. coli); and as a negative control, purified recombinant A- ENA fibrils (4.18 mg / ml), to rule out any toxic effects.

[0037] Figure 5. Genetic organisation of A-ENA and ruffle genes in Bacillus and Pasteuria. (a) Genetic organization of ruffle and A-ENA genes in the genome of Bacillus cereus SIBC65; (b) multiple sequence alignment of A-ENA from Bti (A-ENA / l-101=SEQ ID NO:1) and ORFs RS29610 and RS29620 from B.cereus SIBC65 (SEQ ID NOs:2-3): catalytic residues marked with +, IPB residues marked with *; (c) AF3 multimer prediction of a putative B.cereus ruffle / A-ENA-like complex: N-terminal A-ENA-like domains of the ruffle protein interlock with the A-ENA protomers; (d) Zoom-in of the type 4 IPB site in the protomer-protomer and protomer-ruffle interfaces; (e) Schematic representation of two putative modes of A-ENA ruffle decoration: Terminated ruffle: a single trimeric ruffle caps the A-ENA fibril terminus, Distributed ruffles: multiple trimeric ruffles are integrated in random positions into the A-ENA fibril. (f,h) Genetic organization of ruffle and A-ENA genes in the genome of Pasteuria ramosa; (g) Multiple sequence alignment of A-ENA from Bti (SEQ ID NO:1 without last residue as shown in SEQ ID NO:4) and ORFs 00694, 00695, 00696, and AKNPHEEL_01218 (SEQ ID NOs: 5-8) from Pasteuria ramosa: catalytic residues marked with +, IPB residues marked with *; (i) AF3 multimer prediction of a putative P. ramose ruffle / A-ENA-like complex: N-terminal A-ENA-like domains of the ruffle protein interlock with the A-ENA protomers.

[0038] Figure 6. Exogenous addition of recombinantly produced A-ENA nanofibrils results in induced temporary biofilm (ITB) formation in spore and toxin preparations of Bacillus thuringiensis Aizawai HaRe / ENAcoformulant / 875-PCT

[0039] (Bta) and Bacillus thuringiensis Tenebrionis (Btt). (a, b) Phase contrast microscopy of Btt (a) and Bta (b) spore preparations with and without the addition of exogenous A-ENA nanofibrils. Spores are seen as bright phase white particles, toxin crystals as phase dark particles, (c) SDS-PAGE of high-pH dissolved parasporal bodies (PSBs) in different Histodenz fractions (supernatant (SN), 40% and 60%) of Btt WT and Btt with exogenous added A-ENA (at 1 mg / ml). “IN” (before fractionation) shows the input material at 1 :10 dilution, displaying the same starting toxin load.

[0040] Figure 7. Coformulation of microbial particulates with A-ENA nanofibrils results in "Induced Temporary Biofilm" (ITB) formation. Schematic representation of the generalizing concept of "Induced Temporary Biofilm" formation by the exogenous addition of purified A-ENA nanofibrils to micrometer scale particulates. Said particulates can be single or combinations of bacterial or fungal spores and cells, protein crystals or other particulates of micrometer scale diameter. In the formed ITBs, particulates are bound together in a fibre matrix composed of A-ENA nanofibrils, non-specifically entrapping the particulate materials. As an exogenously added matrix, the ITB biofilm nature is temporary, and independent of any endogenous biofilm inducing pathways or components in the encapsulated microbial cells or spores.

[0041] Figure 8. A-ENA ITBs increase retention of Btt or Bta on plant leaf surfaces. (a,b) A-ENA increases retention of the spore - PSB mixture on leaf tissue. SDS-PAGE of Btt (a) and Bta (b) spore preparations applied on tomato leaf disks, and subsequently subjected to consecutive washes with washed with X mL. ‘IN’ refers to the input material prior to leaf application.

[0042] Figure 9. Bio assays to score the pupation of Spodoptera upon treatment with Btk or Btk+A-ENA. Pupation success of Spodoptera exigua LI larvae (12 DAT) feeding on artificial diet treated with increasing concentrations of Bt kurstaki, with (1 mg / ml) or without A-ENA supplementation (20 larvae per treatment, 1 replicate).

[0043] Figure 10. Bio assays to score the mortality of Plutella xylostella upon treatment with Btk or Btk+A- ENA. Mortality of Plutella xylostella L3 larvae (4 DAT) feeding on artificial diet supplemented with increasing concentrations of Bt kurstaki strain ABTS-351 (spore suspension, panel a), or Dipel (Bt kurstaki strain ABTS-351)(liquid commercial formulation, panel b), with or without A-ENA supplementation (1 mg / ml) (10 larvae per treatment, 4 replicates each).

[0044] Figure 11. Bio assays to score the mortality of Plutella xylostella upon treatment with Bta or Bta +A- ENA. Mortality of Plutella xylostella L3 larvae (4 DAT) feeding on artificial diet supplemented with increasing concentrations of Btaizawai, with (1 mg / ml) or without A-ENA supplementation. The bioassay was performed twice (a and b), focusing on higher concentrations in the second assay (5 larvae per treatment, 2 replicates each). HaRe / ENAcoformulant / 875-PCT

[0045] Figure 12. Testing UV damage on Btt spore suspension. Left panel, controlled UV-dosing is performed using a Dymax Redicure UV-light at 2 cm height and 100 % power on 5 pl of dried in preparations of spore-PSB mixtures. Afterwards, the mixtures are redissolved using 10 pl H2O and 5 pl DX buffer (for SDS- PAGE analysis) or using 50 pl H2O (for spore viability analysis). Detection of UV-induced damage on Cry toxins is followed by SDS-PAGE (right panel) or Western blot analysis. Here, upon extended UV treatment, degradation of the toxins is witnessed by a decrease in intensity of the Cry toxin main band (70kDa), upon UV induced degradation (appearing as a band with slightly lower molecular weight on SDS-PAGE) compared to the input material, as well as the appearance of a high MW band (indicative of the aggregated Cry toxins).

[0046] DESCRIPTION

[0047] The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter.

[0048] Definitions

[0049] Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where the term "essentially consisting of" or "consisting essentially of" or "comprising substantially" is used herein for chemical matter or compounds such as proteins, this means that specific further components or matter can be present, namely those not materially affecting the essential characteristics of the chemical matter. Where specifically mentioned herein, "comprising substantially" or "essentially consisting of" or "consisting essentially of" refer to the majority or bulk of the chemical matter, such as a polypeptide, wherein the functional outcome is defined thereby, but may contain further matter, such as further amino acids. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments, of the invention described herein are HaRe / ENAcoformulant / 875-PCT capable of operation in other sequences than described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in molecular biology, biochemistry, structural biology, and / or computational biology).

[0050] The terms "protein", "polypeptide", and "protein domain" are interchangeably used further herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. A monomeric or protomer is defined as a single polypeptide chain from amino-terminal end (also referred to herein as N-term or N-terminus or N-terminal end) to carboxy-terminal end (also referred to herein as C-term or C-terminus or C-terminal end). A "protein subunit" as used herein refers to a monomer or protomer, which may form part of a multimeric protein complex or assembly. A "protein domain" as used herein refers to a folded protein, or folded part of a protein as a distinct functional and / or structural unit in a protein. Usually a protein domain is responsible for a particular function or interaction, contributing to the overall role of a protein. Domains may exist in a variety of biological contexts, where similar domains can be found in proteins with different functions. Protein secondary structure elements typically spontaneously form as an intermediate before the protein folds into its three dimensional tertiary structure. The two most common secondary structural elements of proteins are alpha helices and beta (P) sheets, though -turns and omega loops occur as well. Beta sheets consist of beta strands (also p-strand) connected laterally by at least two or three back-bone hydrogen bonds, forming a generally twisted, pleated sheet. A p-strand is a stretch of poly-peptide chain typically 3 to 10 amino acids long with backbone in an extended conformation. A p-turn is a type of non-regular secondary structure in proteins that causes a change in direction of the polypeptide chain. Beta turns (P turns, p- turns, p-bends, tight turns, reverse turns) are very common motifs in proteins and polypeptides, which mainly serve to connect p-strands.

[0051] In the context of the present invention, 'self-assembly' refers to the spontaneous organization of molecules in ordered supramolecular structures thanks to their mutual non-covalent interactions without external control or template. The chemical and conformational structures of individual molecules carry the instructions of how these are assembled. The same or different molecules may constitute the building blocks of a molecular self-assembling system. Generally, interactions are established in a less ordered state, such as a solution, random coil, or disordered aggregate leading to HaRe / ENAcoformulant / 875-PCT an ordered final state, which can be a crystal or folded macromolecule, or a further assembly of macromolecules. The association of small molecules or proteins into well-ordered structures is driven by thermodynamic principles, thus, based on energy minimization. The interactions involved in the molecular assembly process are electrostatic, hydrophobic, hydrogen bonding, van der Waals interactions, aromatic stacking, and / or metal coordination. Although non-covalent and individually weak, these forces can generate highly stable assemblies and govern the shape and function of the final assembly (Lombardi et al., 2019; Pharmaceutics, 11, 166). Said self-assembling protein subunits described herein, and called A-ENA monomeric proteins herein, are capable of self-assembling into monomers, further multimerize and fold and engage into protein fibrils as described herein. The fibrous assemblies can be obtained from the pre-existing components termed building blocks, or subunits, more specifically the isolated self-assembling A-ENA monomeric proteins as described herein.

[0052] The terms "chimeric polypeptide", "chimeric protein", "chimer", "fusion polypeptide", "fusion protein", or "heterologous fusion", are used interchangeably herein and refer to a protein that comprises at least two separate and distinct polypeptide components that may or preferably may not originate from the same protein. The term also refers to a non-naturally occurring molecule which means that it is manmade. The term "fused to", and other grammatical equivalents, such as "covalently linked", "connected", "attached", "ligated", "conjugated", and as specifically used herein 'inserted in' when referring to a chimeric or fusion polypeptide (as defined herein) refers to any chemical or recombinant mechanism for linking two or more polypeptide components. The fusion of the two or more polypeptide components may be a direct fusion of the sequences or it may be an indirect fusion, e.g. with intervening amino acid sequences or linker sequences, or chemical linkers. The fusion of amino acid residues or (poly)peptides to an Ena protein or insertion into an Ena protein sequence, or to another protein of interest as described herein, may be a covalent peptide bond, or also refer to a fusion obtained by chemical linking. The term "fused to", as used herein, and interchangeably used herein as "connected to", "conjugated to", "ligated to" refers, in particular, to "genetic fusion", e.g., by recombinant DNA technology, as well as to "chemical and / or enzymatic conjugation" resulting in a stable covalent link.

[0053] As used herein, the term "protein complex" or "protein assembly" or "multimer" refers to a group of two or more associated macromolecules, whereby at least one of the macromolecules is a protein. A protein complex or assembly, as used herein, typically refers to binding or associations of macromolecules that can be formed under physiological conditions. Individual members of a protein complex, such as protein subunits or protomers, are linked by non-covalent or covalent interactions.

[0054] It will be understood that a protein complex can be multimeric. Protein complex assembly can result in the formation of homo-multimeric or hetero-multimeric complexes. Moreover, interactions can be HaRe / ENAcoformulant / 875-PCT stable or transient. The term "multimer(s)", "multimeric complex", or "multimeric protein(s) or assemblies" comprises a plurality of identical or heterologous polypeptide monomers. Polypeptides can be capable of self-assembling into multimeric assemblies (i.e.: dimers, trimers, pentamers, hexamers, heptamers, octamers, etc.) formed from self-assembly of a plurality of a single polypeptide monomers (i.e., "homo-multimeric assemblies") or from self-assembly of a plurality of different polypeptide monomers (i.e. "hetero-multimeric assemblies"). As used herein, a "plurality" means 2 or more. The multimeric assembly comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or preferably more polypeptide monomers. The multimeric assemblies can be used for any purpose and provide a way to develop a wide array of protein "nanomaterials." In addition to the finite, cage-like or shell-like protein assemblies, they may be designed by choosing an appropriate target symmetric architecture. The monomers or protomers and / or multimeric assemblies of the invention can be used in the design of higher order assemblies, such as protofibrils, fibrillar assemblies or fibrils and further fibrils, with the attendant advantages of hierarchical assembly. The resulting multimeric or fibrous assemblies are highly ordered materials with superior rigidity and monodispersity, and can be functional as a multimer or fibrous structure itself, or form the basis of advanced functional materials, such as modified surfaces containing multimeric assemblies or fibrillar structures, and custom-designed molecular machines with wide-ranging applications. More specifically, a multimer as used herein refers to homo- or heteromultimeric, or homo- or heteropolymeric protein complexes which are associated with each other to form an organized structure, though non-covalent and / or covalent interactions; and / or further modified to grow or develop into self-assembling or triggered formation of nanofibrils. Said multimeric assemblies may contain monomeric ENA fusion proteins as defined herein, or ENA protein variants, mutant and / or engineered ENA proteins, such as those exemplified and described herein, as well as other proteins that may associate to said ENA protein-based, or in particular A-ENA-based multimers, called engineered multimers, thereby expanding said multimer towards further modifications required for certain applications.

[0055] By "recombinant polypeptide" is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide, which may be obtained in vitro and / or in a cellular context. When the protein nanofibril, or a chimeric polypeptide, or a fusion polypeptide, or biologically active (i.e. functional) portion thereof, is recombinantly produced, it is also preferably enriched, purified or made substantially free of culture medium, i.e., the impurities represent less than about 20 %, more preferably less than about 10 %, and most preferably less than about 5 % of the volume of the protein preparation. By "isolated" or "purified" is meant material that is substantially or essentially free from components that normally accompany it in its native state. More general, the term "heterologous" is defined herein as a sequence or molecule or protein that is different in its origin. The HaRe / ENAcoformulant / 875-PCT term "exogenous" refers to the presence of a material such as a protein or protein nanofibril that is not natively or endogenous to the host or biological system referred to for combining said material with.

[0056] "Homologue", "Homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and / or insertions relative to the unmodified or wild-type protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. The term "amino acid identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met, also indicated in the conventionally known one-letter code herein) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. A "substitution", or "mutation" as used herein, results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental protein or a fragment thereof. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity. The percentage of amino acid identity as provided herein is preferably in view of a window of comparison corresponding to the total length of the native or natural wild-type protein, or of the specific amino acid sequence referred to.

[0057] The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source, or included in a cell, cell line or organism. A wild-type gene or gene product is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene or gene product a observed in nature. In contrast, the term "modified", "engineered", "mutant" or "variant" or "mutant variant" refers to a gene or gene product that displays modifications in sequence, post-translational modifications and / or functional properties (i.e. altered characteristics) when compared to the wild-type or naturally-occurring gene or gene product. A knock-out refers to a modified or mutant or deleted gene as to provide for non-functional gene product and / or function. It is noted that naturally occurring mutants or variants may be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product, and a different sequence as compared to the reference gene or protein. With the term 'functional homologue' or 'functional variant' is herein referred to a modified, engineered or mutant gene product with retained functionality as compared to the wild type or original sequence on which the functional homologue or functional HaRe / ENAcoformulant / 875-PCT variant is based for introduction of modifications or mutations. For instance an A-ENA functional variant differs in its sequence or structure or may have additional parts in its sequence or structure as compared to wild type A-ENA proteins, but would still retain its function in self-assembling into protein nanofibrils. Further, the term 'insecticidal mutant variant' in view of a variant of the agriculturally bioactive substance defined herein refers to an insecticidal agent, such as a Bt strain or spore or Bt-containing composition, which is altered (e.g. mutant as compared to the wild type genome), but still retained its insecticidal functionality.

[0058] "Host cells" can be either prokaryotic or eukaryotic. The cells can be transiently or stably transfected. Such transfection of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. For all standard techniques see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016). Recombinant host cells, in the present context, are those which have been genetically modified to contain an isolated DNA molecule, nucleic acid molecule or expression construct or vector of the invention. The DNA can be introduced by any means known to the art which are appropriate for the particular type of cell, including without limitation, transformation, lipofection, electroporation or viral mediated transduction. A DNA construct capable of enabling the expression of the chimeric protein of the invention can be easily prepared by the art-known techniques such as cloning, hybridization screening and Polymerase Chain Reaction (PCR). Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (2012), Wu (ed.) (1993) and Ausubel et al. (2016). Representative host cells that may be used with the invention include, but are not limited to, bacterial cells, yeast cells, plant cells and animal cells. Bacterial host cells suitable for use with the invention include Escherichia spp. cells, Bacillus spp. cells, Pasteuria spp. cells, Corynebacterium spp., Aspergillus spp., Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells, Pseudomonas spp. cells, Lactococcus spp. cells, Lactobacillus spp. cells, and Salmonella spp. cells. Animal host cells suitable for use with the invention include insect cells and mammalian cells (most particularly derived from Chinese hamster (e.g. CHO), and human cell lines, such as HeLa. Yeast host cells suitable for use with the invention include species within Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g. Pichia pastoris), Hansenula (e.g. Hansenula polymorpha), Yarrowia, Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces and the HaRe / ENAcoformulant / 875-PCT like. Saccharomyces cerevisiae, S. carlsbergensis and K. lactis are the most commonly used yeast hosts, and are convenient fungal hosts. The host cells may be provided in suspension or flask cultures, tissue cultures, organ cultures and the like. Alternatively, the host cells may also be transgenic animals, or plants or parts or materials derived thereof.

[0059] The term 'microbial' herein refers to the origin being found in small microorganisms which are typically limited to single cell organisms such as bacteria, archaea, fungi or protozoa.

[0060] A "composition" relates to a combination of one or more active molecules, such as an agriculturally bioactive molecule or substance, and, according to the present invention preferably including one or more protein nanofibrils, for instance acting as a coformulant or additive, and said composition may further include solutions and / or solutes such as pH buffering substances, water, saline, physiological salt solutions, glycerol, preservatives, etc. for which a person skilled in the art is aware of the suitability to obtain optimal performance. Suitable conditions as used herein could also refer to conditions for using, applying or administering an active molecule or a protein nanofibril as described herein, in view of the composition described herein, including the agriculturally bioactive substance in the composition. The composition may include microbial culture or endospore materials or components (including proteins, toxins, metabolites, among others) or remains from microbial cultures representing the agricultural bioactive substance, and / or remnants or remains of the purified of isolated protein nanofibrils that are included in the composition as a coformulant. Since the composition of the present invention refers to a combination of an agriculturally bioactive substance and A-ENA protein nanofibrils, the latter typically present in a multitude of fibrils for acting as additive or coformulant, the presence of spores or microbial components or remnants may thus be part of the agriculturally bioactive substance and / or of the A-ENA protein nanofibril material, though it is not meant that both the agriculturally bioactive substance and A-ENA protein nanofibrils are provided in the composition as a single 'material', meaning that the presence of for instance Bt strains producing A-ENA endogenously is not sufficient to come to the composition of the present invention. At least one exogenously added A-ENA protein nanofibril has to be present in said composition.

[0061] The "composition" is a biologically active composition comprising the biologically active agents or substance provided by the present invention, referred to herein as an agriculturally bioactive substance, and at least one exogenously added A-ENA or A-ENA-based protein nanofibril as coformulant or additive, and optionally comprising a carrier, diluent or excipient. A "carrier" or "adjuvant", is any suitable excipient, diluent, carrier and / or adjuvant which, by themselves, do not have activity nor do they elicit protection. Exemplified adjuvants in the art include those for instance listed in Brar et al. (2006; Process Biochemistry 41, 323-342). The term "excipient", as used herein, is intended to include all substances HaRe / ENAcoformulant / 875-PCT which may be present in a composition and which are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffer substances, stabilizing agents, flavouring agents or colorants. A "diluent" includes vehicles such as water, saline, physiological salt solutions, glycerol, ethanol, etc. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, or preservatives may be included in such vehicles. An 'effective amount' of the composition of the invention is preferably that amount which produces a result or exerts an influence on the particular activity aimed for.

[0062] The term "agriculturally bioactive material" or "agriculturally bioactive substance" as used interchangeably herein refers to at least one active compound or active ingredient which may be a chemical substance, protein, a crystal, a microbial metabolite, or a biological system such as a microorganism or a spore-containing suspension, as present in any one of the classes selected from the groups of pesticides, fungicides, insecticides, nematicides, biocides, herbicides, herbicide safeners, plant growth regulators; and / or plant health promoters biostimulants, biofertilizers, and / or combinations thereof.

[0063] A 'pesticide1is defined herein as a chemical substance or microorganism intended to prevent, destroy, or control a harmful organism ('pest') or disease, or to protect plants or plant products during production, storage and transport. They include, amongst others: herbicides, fungicides, insecticides, acaricides, nematicides, molluscicides, growth regulators, repellents, rodenticides, algicides, miticides, piscicides, slimicides, and biocides. A 'biocide' is defined as a chemical substance or microorganism intended to destroy, deter, render harmless, or exert a controlling effect on any harmful organism, or as "a diverse group of poisonous substances including preservatives, insecticides, disinfectants, and pesticides used for the control of organisms that are harmful to human or animal health or that cause damage to natural or manufactured products". The terms "biocides" and "pesticides" are regularly interchanged, and often confused with "plant protection products". To clarify this, pesticides include both biocides and plant protection products, where the former refers to substances for non-food and feed purposes and the latter refers to substances for food and feed purposes.

[0064] "Bacillus thuringiensis" (Bt) is an insecticidal, Gram-positive, aerobic endospore-forming bacterium of the Bacillus cereus sensu lato group, found in soil, dust, and on plant surfaces. Bt spores are used as established biopesticides in agriculture, private residential use and vector-borne disease control due to their remarkable capacity to kill larvae of various insect pests from the Coleoptera, Lepidoptera, Hymenoptera and Diptera orders, including Aedes and Anopheles mosquitos - vectors for zika, dengue and malaria. Since Bt strains target specific insect groups with minimal impact on humans and non-target HaRe / ENAcoformulant / 875-PCT organisms (e.g. wildlife, pollinators and other beneficial insects), they can form the basis of biopesticides that offer a safe and eco-friendly alternative to non-discriminatory, environmentally persistent and potentially toxic insecticidal chemicals. Bt adopts its entomopathogenic lifestyle by the characteristic production of vegetative insecticidal toxins (Vip) or delta-endotoxins (Cry and Cyt) during sporulation. Bt toxins exhibit a distinctive composition unique to each strain, resulting in a remarkable selectivity for specific insect genera. The toxins are produced as pro-toxins that assemble into pure or mixed crystalline condensates referred to as parasporal bodies or crystals (PSB). Upon ingestion of Bt spores, the associated pro-toxin crystals dissolve and exert a pore-forming activity in the insect gut, followed by intestinal sepsis, starvation and host death. In the process, germinated vegetative Bt cells colonize the insect until available nutrients in the carcass become depleted, then inducing sporulation to ensure further dissemination and survival of adverse conditions until the spores are taken up by e.g. a new host to restart the cycle.

[0065] With 'animal pests' as used herein these may refer to pests from the order of Lepidoptera including for instance Acronicta major, Aedia leucomelas, Agrotis spp., Alabama argillacea, Anticarsia spp., Barathra brassicae, Bucculatrix thurberiella, Bupalus piniarius, Cacoecia podana, Capua reticulana, Carpocapsa pomonella, Cheimatobia brumata, Chile spp., Choristoneura fumiferana, Clysia ambiguella, Cnaphalocerus spp., Earias insulana, Ephestia kuehniella, Euproctis chrysorrhoea, Euxoa spp., Feltia spp., Galleria mellonella, Helicoverpa spp., Heliothis spp., Hofmannophila pseudospretella, Homona magnanima, Hyponomeuta padella, Laphygma spp., Lithocolletis blancardella, Lithophane antennata, Loxagrotis albicosta, Lymantria spp., Malacosoma neustria, Mamestra brassicae, Mods repanda, Mythimna separata, Oria spp., Oulema oryzae, Panolis flammea, Pectinophora gossypiella, Phyllocn istis citrella, Pieris spp., Plutella xylostella, Prodenia spp., Pseudaletia spp., Pseudoplusia includens, Pyrausta nubilalis, Spodoptera spp., Thermesia gemmatalis, Tinea pellionella, Tineola bisselliella, Tortrix viridana, or Trichoplusia spp. In one embodiment, the animal pest is Spodoptera exigua, Plutella xylostella, or Trichoplusia ni. In another embodiment, said animal pests are mosquitos such as Aedes aegypti, midges, such as Chironomidae).

[0066] "Coformulants" as defined herein are part of the mixtures contained in compositions of pesticide products, serving to enhance potency, product efficiency and usability. Examples of existing coformulants are for instance surfactants, anti-foaming agents, solvents or wetting agents. The present invention provides for a novel proteinaceous type of coformulant which is a polymer based on selfassembled A-ENA protein structures forming nanofibrils. Coformulants are considered as "biologically inactive" components of pesticide products, in contrast to the "active ingredient", the component that is declared as acting against the targeted 'harmful1organism. A coformulant can make up more than 50% of a product formulation or of a composition or pesticidal product. Just as for active substances, HaRe / ENAcoformulant / 875-PCT coformulants are defined as having no harmful effect on human and animal health and no unacceptable effect on the environment.

[0067] In agriculture, an "additive" is any non-fertilizer substance or mixture intended to improve the physical, chemical, or biological properties of a substance, or to otherwise enhance crop or animal production, product quality, or yield.

[0068] When referred to a 'plant' or 'crop' herein, these may be selected from the list of different types of plants including but not limited to: turf, vines, cereals, for example wheat, barley, rye, oats, rice, maize and millet / sorghum; beet, for example sugar beet and fodder beet; fruits, for example pome fruit, stone fruit and soft fruit, for example apples, pears, plums, peaches, almonds, cherries and berries, for example strawberries, raspberries, blackberries; legumes, for example beans, lentils, peas and soybeans; oil crops, for example oilseed rape, mustard, poppies, olives, sunflowers, coconuts, castor oil plants, cacao and peanuts; cucurbits, for example pumpkin / squash, cucumbers and melons; fibre plants, for example cotton, flax, hemp and jute; citrus fruit, for example oranges, lemons, grapefruit and tangerines; vegetables, for example spinach, lettuce, asparagus, cabbage species, carrots, onions, tomatoes, potatoes and bell peppers; Lauraceae, for example avocado, Cinnamomum, camphor, or else plants such as tobacco, nuts, coffee, aubergine, sugar cane, tea, pepper, grapevines, hops, bananas, latex plants and ornamentals, for example flowers, shrubs, deciduous trees and coniferous trees. Further plants are considered to be particularly suitable target crops for applying compositions and methods of the present invention: cotton, aubergine, turf, pome fruit, stone fruit, soft fruit, maize, wheat, barley, cucumber, tobacco, vines, rice, cereals, pear, beans, soybeans, oilseed rape, tomato, bell pepper, melons, cabbage, potato, apple; as well as leguminous plants, such as lentils, peas, alfalfa or soybeans; fiber plants, such as cotton, flax, hemp or jute; citrus fruit, such as oranges, lemons, grapefruits or mandarins; vegetables, such as broccoli, spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes, cucurbits or paprika; lauraceous plants, such as avocados, cinnamon or camphor; natural rubber plants or ornamental and forestry plants, such as flowers, shrubs, broad leaved trees or evergreens, each in its natural or genetically modified form; and on the plant parts and plant propagation material, such as seeds, and the crop material of these plants. The term "parts" or "parts of plants" or "plant parts" are understood as meaning all aerial and subterranean parts and organs of the plants, such as shoot, leaf, flower and root, examples which may be mentioned being leaves, needles, stalks, stems, flowers, fruiting bodies, fruits and seeds, and also roots, tubers and rhizomes. HaRe / ENAcoformulant / 875-PCT

[0069] Detailed description

[0070] The present invention is based on the demonstration that recombinantly produced self-assembled A- ENA-based nanofibrils, exogenously added to a Bt strain culture or spore-(PSB)-containing suspension production, but more importantly, even when the spore-PSB product is derived from a Bacillus sp. lacking endogenous A-ENA, results in induced clustering of spores and PSBs, hence promoting endospore clustering and Cry toxin retention in Bacillus thuringiensis, through the provision of biofilm-like aggregates, thereby increasing the Bacillus killing activity. Furthermore, the strain for which this effect has been exemplified herein, is the widely used biocontrol agent Bacillus thuringiensis Sv. Kurstaki (Btk), anticipating that there is huge potential for improving the Btk-containing agents currently on the market, by applying the exogenously co-formulated A-ENA nanofibrils. Hence we provide for a novel platform for non-GMO improvements to widely used bacterial biopesticide agents, which will be relevant to healthcare, biotech and agro industry, as these agents are involved in the control of vector borne disease as well as agricultural and horticultural pests.

[0071] The novel finding that A-ENA nanofibrils form the basis of a biofilm inducing extrasporal matrix ensuring spore - toxin unison and enhancing virulence leads to the invention presented herein relating to a composition comprising said A-ENA protein nanofibril as an additive or coformulant combined with an active ingredient, which is a biologically active material or substance, also described herein as an agriculturally bioactive substance, preferably an active ingredient or active substance of a pesticide, more preferably a spore-containing pesticide, such as a B. thuringiensis-based substance.

[0072] Moreover, phylogenetic analysis strongly indicates that A-ENA is a widespread virulence factor in sporulating and toxin-producing arthropod and nematode pathogens of the Bacil lota phylum. In many species, A-ENA subunits can be decorated with additional domains, some of which suspected to play a role in host adherence. So further to the co-formulant role in spore-containing bacterial suspensions with pesticidal activity, the A-ENA protein-based material may act as a coformulant for nematicidal or biocidal agents.

[0073] In a further aspect of the invention, a composition is provided comprising an agriculturally bioactive substance, such as a pesticide or an active ingredient of a pesticide, and an exogenous A-ENA proteinbased nanofibril, wherein said A-ENA protein-based nanofibril comprises an engineered A-ENA protein and / or is a modified A-ENA protein nanofibril in that is engineered, contains modified or chimeric A-ENA proteins, e.g. A-ENA fusions to heterologous protein subunits, and / or a functionalized A-ENA nanofibril, wherein said functionalization may be induced by the modified or engineered A-ENA proteins present in said A-ENA protein-based nanofibril or wherein said functionalization may be added, for instance through conjugation or post-treatment, after self-assembly of the A-ENA protein-based nanofibril, as to HaRe / ENAcoformulant / 875-PCT obtain an even more effective A-ENA protein nanofibril. In specific embodiments such modification or functionalization is intended to provide for a coformulant that increases the retention time of the pesticide on the plant area where it is applied on, as a non-limiting example.

[0074] Alphahelical Endospore Appendage (A-ENA) proteins

[0075] The present invention comprises a composition comprising or consisting of a bioactive ingredient, such as a Bt strain, and an A-ENA protein nanofibril, wherein said A-ENA protein nanofibril comprises and A- ENA protein or an engineered or modified A-ENA protein, which upon self-assembly forms rigid protein fibrils. The invention is thus based on the promiscuous properties of these self-assembling A-ENA protein nanofibrils, which can be recombinantly produced and isolated from a production host simply by heterologous expression of the A-ENA protein or engineered or modified A-ENA protein or A-ENA-like protein.

[0076] This novel type of A-ENA proteins has been established in Remaut H. et al. (2025. PCT / EP2024 / 074370), and is also defined therein and incorporated by reference herein. More specifically, the definition of A- ENA proteins and A-ENA protein nanofibrils is further provided herein with regards to the present invention of applying said exogenous recombinantly produced A-ENA protein(-based) nanofibrils as a coformulant.

[0077] The A-ENA protein is a new type of Endospore appendage (ENA) protein, in particular Alpha-helical Endospore Appendage protein, or A-ENA, which was thus found to function as self-assembling protein forming multimeric fibrous structures associated with Bacillus thuringiensis (Bt) endospores and parasporal bodies, and contributing to the pathogenicity of these bacterial spores. These fibrous structures appearing in Bt endospore cultures were resolved revealing a proteinaceous content constituting A-ENA proteins, appearing in a highly-organized, compelling protein fibril structure spontaneously folded to provide for an extremely high stability and rigidity to strongly associate the spore with the parasporal body. The rigidity, thermal and mechanical stability and tensile strength of those A-ENA based protein fibrils appeared to derive from the presence of several isopeptidic covalent iriteractions present within the fibrils, resulting in a biological structured material with huge potential in different application areas. However, ex vivo isolation Of A-ENA fibrils separated from parasporal bodies was principally impossible since these naturally occurring A-ENA-based fibrils, as appearing in Bt spore cultures, always strongly associated with parasporal bodies among potentially further components of said sporulated cultures, thereby / playing a key role in the toxicity of Bt spores to insects, though complicating their isolation and structural analysis. Finally, through more advanced technical improvements in the CryO-EM field, the structural analysis of Bacillus thuringiensis israelensis (Bti) spore cultures allowed to identify these Bti fibrils aS A-ENA protein-based fibrils, represented in the genome HaRe / ENAcoformulant / 875-PCT of this Bacillus species by two A-ENA genes encoding two proteins (depicted A-ENA -SEQ ID NO: 1, and A- ENA-1). Further comparative analysis of homologous bacterial protein sequences, and based on the novel structufal insights, allowed to define the structural features of this newly annotated 'A-ENA' protein family, and in addition, for convenience of the reader, an overview of the non-limiting protein family members has been provided herein in Table 1 and 3.

[0078] The A-ENA proteins are defined herein as protein monomers which appear as self-assembling alphahelical (a-helical) antiparallel coiled-coil monomers providing for a helical pattern in its structure which is determined by its amino acid sequence, as known for coiled-coil Structured proteins, and as further defined herein, consisting of a consensus sequence determined by the fragments NTL-helix 1, L, helix 2, CT, wherein each helix is defined by the sequence at least 5 heptad elements (Hl-5), wherein each heptad element contains 7 amino acids, of which the side chains are defined as 'abcdefg', and as further defined herein. Furthermore, in addition to their sequence-based alpha-helical coiled-coil structural features, A-ENA proteins are characterized by the presence of a number of amino acid residues that, within said self-assembled antiparallel coiled-coil hydrophobic environmerit, position geometrically as such that autocatalytic formation of one or more isopeptide covalent bonds within Or with other A-ENA monomers is facilitated, and resulting in a 'functional' A-ENA protein, which is capable of spontaneously forming a fibrillar assembly, as described herein.

[0079] 'A-ENA protein nanofibrils' built from or consisting of two protofibrils, wherein each protofibril contains two or more monomer protein subunits, based on or derived from or consisting of A-ENA protein subunits, which spontaneously fold as alpha-helical antiparallel coiled-coil structured monomers and have an amino acid sequence which consists of protein fragments according to the formula: NTL- helixl(hl)-L-helix2(h2)-CT, wherein each fragment NTL, L, and CT, contains at least 1, 1 and 4 amino acids, respectively, and wherein fragments hl and h2 (also called helix al and helix a2) contain at least 5 heptad elements to allow formation of the helix hairpin, and wherein the amino acids are defined as further detailed below, and wherein said monomeric protein subunits are interconnected through at least one or more isopeptidic bonds (IPBs). Said self-assembling monomer protein subunits are A-ENA proteins, as defined herein, or are A-ENA-based proteins or proteins derived from A-ENA protein subunits, as also further defined herein, wherein said protein fibril thus constitutes the basic A-ENA protein structure scaffold as defined herein, and when A-ENA-based or derived protein monomer sequences are applied for forming the fibril, these are also functional in assembling into the fibril similar as for wild type A-ENA proteins, but may contain modifications, such as insertions, mutations, fusions, or conjugations to the A-ENA monomer (therefore called an 'A-ENA-based' monomer or 'A-ENA derived' monomer sequence, i.e. functional homologues, or functional variants, or engineered A-ENA proteins), which are provided by engineering the A-ENA monomer sequences or proteins. HaRe / ENAcoformulant / 875-PCT

[0080] The monomeric protein subunit of the protein nanofibril provided herein is called Alpha-helical- Endospore appendage (A-ENA) polypeptide, a protein family originating from bacteria, particularly from sporulating bacteria, most particularly from Bacillus, and is defined herein as a spontaneously folding monomeric protein subunit forming 2 alpha-helices which assemble in an antiparallel coiled-coil structure, packed as knobs into holes, with an amino acid sequence comprising fragments according to the following formula NTL-hl-L-h2-CT, which are covalently connected via peptide bonds to form one polypeptide chain, and wherein:

[0081] NTL refers to the N-terminal lock, or N-terminal amino acid sequence, which contains at least 1 amino acid preceding the sequence constituting helix 1, and which provides for a variable fragment; L refers to the linker fragment which forms a loop or turn in the monomer 3D structure, and comprises at least four amino acid residues, located between the helix 1 and helix 2, providing the covalent connection (through peptidic bonds) to allow an alpha-helical intramolecular coiled-coil, and typically said linker is variable in its sequence;

[0082] CT refers the C-terminal tail or C-terminal end of the polypeptide of the A-ENA monomer and contains at least one amino acid residue following the sequence constituting helix 2, and which provides for a variable fragment; and hl and h2 referring to helix 1 and helix 2, resp., also referred to as al helix or a2 helix, of said coiled- coil, which are further defined by the present of (at least) 5 heptad elements (depicted Hl-5) in each of said helix 1 and helix 2, and are defined for the A-ENA protein family as follows:

[0083] Helix 1 comprises or consists of Hl-1 -Hl-2 -Hl-3 -Hl-4 -Hl-5 wherein said heptad elements are defined by residues 'abcdefg' and connected in this particular order from N-terminal end to C- terminal end of said helix, and wherein the consensus amino acid sequence 'a-b-c-d-e-f-g' of each heptad element (Hl-1 to Hl-5) is, or respectively corresponds to,:

[0084] - X-X-X- -X-X- for Heptad 1-1, -X-Qj- -Qj- -y for Heptadl-2,

[0085] S-X-QJ- -QJ-X- for Heptadl-3, -N-Qj-y-Qj-y- 6 for Heptadl-4, -s-X- - -X-X for Heptadl-5,

[0086] Helix 2 comprises or consists of H2-1 -H2-2 -H2-3 -H2-4 -H2-5 wherein said heptad elements are defined by residues 'abcdefg' and connected in this particular order from N-terminal end to C- terminal end of said helix, and wherein the consensus amino acid sequence 'a-b-c-d-e-f-g' of each heptad element is, or respectively corresponds to,: -X-X- - -X- for Heptad2-1, HaRe / ENAcoformulant / 875-PCT

[0087] - 8-X-X- -X-X-X for Heptad2-2, -X-X- - -6-6 for Heptad2-3, s- - - -s-X- 6 for Heptad2-4, -y-X- - -X-X for Heptad2-5, and wherein said consensus amino acid sequence residues are defined as follows:

[0088] ' ' represents a hydrophobic amino acid residue selected form the list of M, V, I, L, A, G, H, W, Y, F for at least 70 % of said O> positions indicated in said heptad elements; preferably from the list of L, M, I, V, F and W;

[0089] 'tp' represents a short side chain amino acid residue selected from the list of V, C, G, A, P, S, T, N, D; preferably from the list of A, G, S, and T;

[0090] Y is an amino acid residue for isopeptide interactions functioning as acid / base catalyst, selected from E , or D, for at least one or more of said y positions indicated in said heptad elements; preferably the amino acid representing y is E;

[0091] '6' is an amino acid residue for isopeptide interactions functioning as 'donor' or nucleophile, which is preferably a Lysine residue (K), for at least one or more of said 6 positions indicated in said heptad elements;

[0092] 's' is an amino acid residue for isopeptide interactions functioning as 'acceptor' or electrophile, selected from the list of E, Q, D, and N, for at least one or more of said s positions indicated in said heptad elements; preferably from the list of E, Q. and N; and wherein X represents an amino acid that can be any type of amino acid residue known in the art, and of which the identity is not restrictive for the functionality of the A-ENA protein.

[0093] Amino acids 'X' or 'any type of amino acid residue' as used herein may thus be defined as any one of the 20 naturally occurring amino acid residues conventionally known and listed herein (below), as well as different isomeric forms or enantiomers thereof and synthetic analogues or variants thereof. The amino acids are presented herein by their 3- or 1-lettercode nomenclature as defined and provided also in the IUPAC-IUB Joint Commission on Biochemical Nomenclature (Nomenclature and Symbolism for Amino Acids and Peptides. Eur. J. Biochem. 138: 9-37 (1984)); as follows: Alanine (A or Ala), Cysteine (C or Cys), Aspartic acid (D or Asp), Glutamic acid (E or Glu), Phenylalanine (F or Phe), Glycine (G or Gly), Histidine (H or His), Isoleucine (I or He), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q. or Gin), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Vai), Tryptophan (W or Trp), and Tyrosine (Y or Tyr). HaRe / ENAcoformulant / 875-PCT

[0094] Specifically for more detailed definition on how to establish the identity of an A-ENA protein sequence, we refer to Remaut et al. (WO2025 / 046122) and Table 1 herein, which summarizes the A-ENA protein class as defined therein, and more specifically, for the known family members, as described therein, to any of the sequences of A-ENA protein provided therein, as well as the accession numbers provided in Table 3 herein. As previously defined in Remaut et al. WO2025 / 046122, the A-ENA proteins are conserved in their structural formula and helical composition defined by the sequence-based motifs, though this structural alignment is less obvious when comparing primary amino acid sequences, resulting in A-ENA protein classifications with low primary sequence similarity. In view of the structural alignment, a group of A-ENA-like or A-ENA type proteins was defined as listed in Table 3 here below (obtained from previous determination in Remaut et al. WO2025 / 046122). So the A-ENA proteins referred to herein relate to the protein class and sequences as defined in Table 1 herein, and as provided in Table 3 herein.

[0095] Said A-ENA proteins are applied for forming an isolated protein nanofibril to provide as a component of the composition of the present invention. Said isolated protein nanofibril may thus be obtained from a host wherein said A-ENA proteins are produced recombinantly by expression of a nucleic acid molecule encoding said A-ENA protein.

[0096] Table 1. Self-assembling A-ENA fibril-forming proteins. HaRe / ENAcoformulant / 875-PCT

[0097] A-ENA protein fibrils

[0098] Upon recombinant production, an 11 kDa two-helix bundle protein that is extensively covalently crosslinked to its nearest neighbors via iso-peptide bonds formed the basis of the protein nanofibril constituting A-ENA subunits spontaneously and efficiently polymerized within the host, in particular in E. coli , through a dock-and-lock self-assembly mechanism wherein subunits first rendezvous driven by hydrophobics, followed by enzyme-free, autocatalytic iso-peptide bond formation, yielding robust, micrometer-long covalent, flexible nanofibrils. These recombinantly produced A-ENA fibrils were shown to enhance spore clustering and toxin retention, resulting in an increased killing activity towards the agricultural pest Trichoplusia ni. A-ENA was thus shown herein to provide for (i) a virulence factor that exerts its function by ensuring close proximity of the infectious particle (spore) to its toxic payload (PSB) and (ii) a role outside its native Bti context due to its aspecific mode of binding to spore and PSB surfaces, therefore ideally positioned for application in a more widespread market including other (Bt) systems that are naturally devoid of A-ENA but otherwise characterized by the presence of extracellular PSBs, or moreover in non-Bt systems of pathogenic sporulating species, or further animal pests that gain by the presence of A-ENA, in analogy to their natural A-ENA-like endogenous counterparts, such as in Pasteuria species.

[0099] The term 'nanofibril' or 'protein nanofibril' is defined herein as a fibrous assembly with a diameter in the nanoscale, preferably 5-200 nm, or 5-100 nm, or 5-80 nm, or 10-200 nm, or 10-100nm, or 10-50 nm, and significantly of a length up to several micrometers. The term 'fibril', 'fibrous assembly', or 'fibrous structure', as used interchangeably herein, refers to structured biochemical compounds, such as protein assemblies or protein-based assemblies, preferably composed of protein material, forming long-shaped HaRe / ENAcoformulant / 875-PCT ordered structures with diameters up to 100 to 200 nanometers, and potentially part of larger hierarchical structures. In view of the terminology used herein for defining (nano)fibrils, we refer herein to 'fibers' as a potential plurality of nanofibrils, wherein fibers are generally considered to represent rather larger diameter (in the micro- to m il li-sca le) structures as compared to fibrils, and wherein 'fibers' thus preferably provide for a higher-ordered hierarchical structure of said plurality of fibrils. In a specific embodiment, such a fiber comprises a plurality of A-ENA nanofibrils, wherein each fibril makes further lateral associations to another fibril, to provide for a structured fiber. A further example of a fiber is a woven assembly of protein nanofibrils. Nanofibrillar structures provide for unique and interesting characteristics to form subject of investigation in several areas of research and applications, such as microbiology, biomechanics, and material science. The present invention provides for protein nanofibrils which are obtained upon the spontaneous intertwining of at least two protofibril lar structures, wherein the term 'protofibril' refers to a fibrous self-assembly of multimers or polymers, which is fundamentally composed of the monomeric protein subunits as described herein, called A-ENA proteins or A-ENA-based proteins, comprising protein fragments according to the formula defined herein as NTL-helixl(hl)-L- helix2(h2)-CT, as further detailed herein, interconnected through at least one or more isopeptidic bonds, i.e. wherein said units are auto-cross-linked. In particular said protofibrils are composed of functional A- ENA proteins, or orthologues thereof, with functional referring to their capability to self-assemble as coiled-coil structures into protofibrillar structures that intertwine forming the nanofibril as described herein.

[0100] From a phenotypic point of view, the A-ENA fibrils are thus easily distinguished from any other fibrils in bacterial or more specifically bacillus endospore samples by simply microscopically analyzing them. Indeed, when inspecting a 2D image of a microscopic analysis, the A-ENA nanofibrils reveal a highly ordered unique fibril structure.

[0101] In a further specific embodiment, said protein nanofibril is an isolated protein nanofibril, wherein 'isolated' refers to the nanofibril being devoid of other compounds such as materials from bacterial spore culture or from bacterial constituents like parasporal bodies, spores, or other bacterially derived material. In reality, ex v / 'vo extraction or isolation of an A-ENA fibril from naturally occurring bacterial endospores was not possible. In hindsight, this is considered as caused by their particular structure and the presence of the isopeptidic bonds. Since the A-ENA nanofibrils cannot be obtained from the bacterial spores 'as such', so the fibrils isolated from any further bacterial compounds (e.g. without parasporal body or spores attached), the protein nanofibrils of the present invention are isolated or obtained from other means, such as from recombinantly produced fibrils, allowing isolation without sporulation and presence of the associating bacterial compounds. So in a specific embodiment, the protein nanofibrils of 1 HaRe / ENAcoformulant / 875-PCT the present invention, also called the A-ENA protein nanofibril or A-ENA-based protein nanofibril, is a recombinantly produced A-ENA nanofibril or A-ENA-based nanofibril.

[0102] Engineered, modified and / or functionalized A-ENA protein assemblies

[0103] The A-ENA proteins may be provided as A-ENA proteins according to the formula NTL-hl-L-h2-CT, as defined and described herein above, wherein the monomer is modified or engineered as to obtain a 'modified A-ENA protein', or 'A-ENA-based monomeric protein', which thus refer to A-ENA proteins which are based on naturally-occurring or native A-ENA protein sequences, or at least are defined by the A-ENA definitions as provided herein for their sequence and functionality, but which are modified or engineered to result in an A-ENA protein that functionalized the spontaneously assembled A-ENA protein nanofibrils upon production in a host. Said modifications or engineering strategies are further exemplified and discussed in PCT / EP2024 / 074370, in a non-limiting manner, and serve as support to enable the skilled person in designing such modified A-ENA proteins. With 'modified' or 'engineered' A- ENA protein, or 'A-ENA-based protein' (as used interchangeably herein) is thus meant herein that the reference A-ENA protein to which the 'modified' A-ENA protein is compared to is a protein subunit with a sequence comprising the interlinked fragments according to the formula NTL-hl-L-h2-CT as previously described herein, wherein the at least 5 heptad elements of the hl and h2 helices are defined as described herein and thus allow for a spontaneous folding of the monomer in a helix hairpin, with the Linker forming a loop connecting the two antiparallel helices, and the NTL and CT present at the surface, and which further self-assembles into fibrous structures as described herein, wherein each monomer is involved in at least one intermolecularly formed IPB, to optimally 10 IPBs, with further monomers (which may be identical or different in sequence from each other).

[0104] It is envisaged herein that modifications which involve an A-ENA fusion or conjugation with or to heterologous or further peptides, proteins, protein domains, tags, labels or other functional moieties, are provided to adapt or improve the A-ENA nanofibril in its function as a coformulant as part of the composition of the present invention. Said modifications may be obtained by connecting said heterologous moiety directly or through the use of a linker to the A-ENA subunit(s). In a specific embodiment disclosed herein, the A-ENA subunit is fused or conjugated to a tag or protein binding partner, for post-fibril formation of a specific tag-protein binding interaction on the fibril surface. In further specific embodiments, said tag / protein binding partner pair comprises a protein and tag which are capable to covalently interact.

[0105] In specific embodiments, said protein nanofibril comprises a modified A-ENA monomer containing a heterologous protein domain, wherein said heterologous protein domain is fused to said A-ENA monomer via its N- or C-terminal end or inserted in the linker region of A-ENA, resulting in a 3D protein HaRe / ENAcoformulant / 875-PCT structure wherein the heterologous protein domain is displayed on the surface of the nanofibril. Specifically in view of the role of A-ENA protein nanofibrils as coformulant for a pesticide composition, said heterologous protein domain may comprise properties that are important for increasing the lifespan of the composition or of the pesticidal active ingredient. Alternatively, the A-ENA protein nanofibrils function in the compositions as an additive, to further stabilize, enhance or maintain the activity of a plant beneficial substance such as a plant growth regulator, a biostimulant or a biofertilizer. For instance, plant-binding peptides or proteins or antigen-binding domains may be incorporated into a chimeric A- ENA protein fusion as to provide for protein nanofibrillar coformulants or additives that increase retention time on plants or plant parts or that increase affinity or avidity for sticking onto the plants or plant areas where the composition is applied on; or agents that improve resistance against UV damage; or agents facilitating the uptake by the pest.

[0106] Further examples of modified A-ENA protein nanofibrils for functionalization may as well include those engineered A-ENA monomers wherein aromatic amino acid residues (e.g. phenylalanine, tyrosine, and tryptophan) are added or substituted as compared to the wild type A-ENA sequence, preferably in its C-terminal part, providing for Ultraviolet light absorbing residues due to the resonance in their aromatic rings.

[0107] So such engineered A-ENA protein nanofibrils will provide for UV protection of the agriculturally bioactive substance. This A-ENA-based functionalization may easily be determined by the skilled person through testing the UV damage to the Cry toxins, or by doing a spore viability assay, on the composition of the invention including the engineered A-ENA protein nanofibrils versus a negative control (with wildtype A-ENA protein nanofibrils or without A-ENA protein nanofibrils).

[0108] To perform such assessments, first a controlled UV-dosing is performed using a Dymax Redicure UV-light at 2 cm height and 100 % power on a small amount of dried preparations of spore-PSB mixtures (Figure 12). Afterwards, the mixtures are redissolved with water and can be prepared for SDS-PAGE analysis or spore viability assays.

[0109] For the detection of UV-induced damage on Cry toxins an SDS-PAGE or western blot analysis is performed which reveals that upon extended UV treatment, degradation of the toxins is witnessed by a decrease in intensity of Cry toxin bands on SDS-PAGE as compared to the input material, as well as the appearance of a high MW band (likely indicative for aggregating / inactivated Cry toxin proteins) is observed. The main band visible on the SDS-PAGE of Figure 12 is the 70kDa band corresponding to the size of the Cry toxin proteins, and this band is reduced in intensity, and correspondingly there appears a smaller band just below, indicating of the UV-degrading effect, as well as a higher MW band, indicating UV-induced Cry protein aggregation. Using UV-antennas on A-ENA delays such degradation. HaRe / ENAcoformulant / 875-PCT

[0110] For testing the spore viability after UV-dosing, dilutions of the spore mixture are plated and monitored through performing CFU counting. Additionally, ODgoo breakthrough experiments are performed to account for dilution aberrations. For this, a small amount of the treated spore-PSB mixture is added to a fresh LB medium and the timing of ODgoo increase above a threshold value of 0.5 is monitored.

[0111] Production ofA-ENA nanofibrils and production of the composition comprising the same

[0112] A further aspect relates to a production method for recombinantly producing the A-ENA or A-ENA-based protein nanofibril as described herein, comprising the steps of: a) Introducing the nucleic acid molecule encoding the A-ENA protein, or A-ENA-based or modified A-ENA protein as described herein for recombinant expression of the A-ENA monomeric protein in a host cell, and / or b) Incubating the host cell expressing said A-ENA protein in suitable conditions for cultivating the production of the A-ENA proteins or A-ENA-based or modified A-ENA proteins self-assembling, and preferentially spontaneously forming protein nanofibrils, in said host cell, or in said host cell culture, c) Releasing said A-ENA protein assemblies from said host cell, preferably through cell lysis, and / or d) Isolating the self-assembled protein nanofibrils, preferably through resuspension from the insoluble fraction and / or further purification from the cell lysate.

[0113] In a preferred embodiment said introducing in step a) is obtained by transforming said host cell with a chimeric gene comprising said A-ENA (or modified A-ENA) protein-encoding sequence. In another embodiment, said host cell is a microbial cell, in particular a bacterial or a fungal cell, in a more preferred embodiment a microbial production host such as E.coli for cytosolic expression of the A-ENA protein nanofibril, or such as Lactococcus lactis for secretion of the A-ENA protein nanofibril in the cultivation medium.

[0114] In another preferred embodiment, said host is a spore-forming strain, more preferably a Bacillus strain, more preferably a Bt strain or a Bacillus subtilis strain. In another preferred embodiment, the A-ENA protein produced in said bacterial strain, preferably in said Bacillus strain, is a heterologous A-ENA protein. Another embodiment provides for a host wherein said Bacillus strain expresses an endogenous A-ENA protein, which may be used to introduce a heterologous A-ENA protein, as to produce the (heteropolymeric) protein nanofibril described herein.

[0115] The disclosure further provides for recombinantly produced protein nanofibrils, preferably A-ENA or A- ENA-based or modified-A-ENA protein nanofibrils, obtainable by or from said method to produce the nanofibril as described herein. The invention further provides for compositions containing said A-ENA protein nanofibrils, which are isolated A-ENA or A-ENA-based or modified-A-ENA protein nanofibrils, HaRe / ENAcoformulant / 875-PCT obtainable by or isolated from said host as provided by the method to produce the nanofibril as described herein.

[0116] As disclosed herein, a number of Bacillus strains lacking an endogenous or native gene for producing an A-ENA protein or protein nanofibril have been identified. Hence, in a specific embodiment, recombinantly (or exogenously) introducing or expressing an A-ENA protein or A-ENA-based protein in said bacterial strain or cell lacking the gene or capacity to produce a native A-ENA is provided herein, for the production of the A-ENA protein nanofibril as described herein.

[0117] Thus, in a specific embodiment, said protein nanofibril as described herein, and preferably as recombinantly produced by the method described herein, provides for an additional or synergistical virulence when applying pesticidal spore substances to their respective pests.

[0118] The protein nanofibril of the present invention thus further provides for an alternative use in agricultural crop treatments or crop improvements and may contribute to future improved pest management strategies.

[0119] Agriculturally bioactive substances

[0120] The term "agriculturally bioactive material" or "agriculturally bioactive substance" or "bioactive substance" as used interchangeably herein and as defined herein refers to at least one active ingredient, more specifically it is in the context of the invention represented by materials or components of biological nature, such as a microbial or a spore-containing suspension, or a microorganism, or an active component derivable or obtained from a microorganism, including for instance one ore more cells, spores, toxins, proteins, crystals, and / or metabolites, possibly among other of its microorganisms' components, provided by pathogenic and / or sporulating microbial species, or plant-beneficial microorganisms.

[0121] The pesticidal or nematicidal activity of substances obtained from or derived from said pathogens hence involves an agricultural bioactive substance of interest to apply in the composition of the present invention. Alternatively, several spore-forming taxa of the Bacillota phylum (formerly Firmicutes), including Bacillus thuringiensis, Paenibacillus sp., Pasteuria sp., Brevibacillus laterosporus, Clostridium bifermentans, Lysinibacillus sphaericus and others, adopt a lifestyle as facultative or obligate parasites of arthropods (i.e. insect larvae, crustaceans) and nematodes, with killing activity associated with their 'parasporal bodies' (PSBs) that consist of crystals of lytic toxins. Furthermore, the composition may further comprise a bioactive substance from for instance, but not limited to Bacillus wiedmannii, B. bombysepticus, B. sphaericus, B. licheniformis, B. amyloliquefaciens, B.subtilis, or B. mojavensisan. Hence the agriculturally bioactive substance may specifically relate to substances originating from or derived HaRe / ENAcoformulant / 875-PCT from said species, which in combination with A-ENA protein nanofibrils constitute the composition of the present invention.

[0122] The activity of such pathogens relies on the ingestion of PSBs and spores by the target host (pest) resulting in sepsis, spore germination, followed by vegetative replication until resources in the cadaver run out and a new sporulation and starting the dissemination cycle. The potent and often specific killing activity of these 'agriculturally bioactive substances', specifically here said bacterial toxins, evolved to widely used biocontrol agents of spore - PSB preparations, isolated toxins or transgenic toxins (i.e. Bt crops) of these species. Most notably, Bacillus thuringiensis has a widespread use as biopesticide agent in agriculture and household horticulture (i.e. preventing crop damage by Lepidoptera and Coleoptera larvae) and vector-borne disease control (i.e. mosquito larvae of the Dengue, Yellow fever and Malaria vectors Aedes and Anopheles).

[0123] In an alternative embodiment said agriculturally bioactive substance may also specifically refer to a plant growth regulator, a biostimulant or a biofertilizer active component, and / or any other agriculturally bioactive substance selected from the group consisting of repellents, attractants, semiochemicals, postharvest treatment compounds, soil amendments, vertebrate control agents, and / or combinations thereof.

[0124] The present invention thus provides for a composition comprising any of said bioactive substance, and a coformulant or additive, which is provided by the A-ENA protein(-based) nanofibrils, and optionally further agents, such as preservatives, structuring agents or surfactants, buffers, dyes, anti-foaming agents, wetting agents, or freeze-thaw stabilizing agents, among others know to the skilled person in view of formulating the composition to obtain a commercially interesting product.

[0125] The A-ENA protein nanofibril material's primary role in said composition is thus to enhance the bioactive substance its efficacy by improving the toxic effect, such as the spore-Cry protein interactions in Bt- related substances, or by improving the delivery or stability of a bioactive substance. For the latter roles, beyond its use with Bt or alike, the A-ENA protein nanofibril coformulant or additive holds broader potential as in other (bio)pesticides, biostimulants, or biofertilizers, to improve the lifespan of the active ingredient. This improvement of the lifespan may be obtained through applying the nanofibril as to optimize the delivery, stability, or efficacy of various active agents, offering innovative and sustainable solutions across the agricultural sector, and therefore applicable to the broad range of 'agriculturally bioactive substances' defined herein.

[0126] Bacillus thuringiensis (Bt) is an insecticidal, Gram-positive, aerobic endospore-forming bacterium of the Bacillus cereus sensu lato group, found in soil, dust, and on plant surfaces. Bt spores are used as established biopesticides in agriculture, private residential use and vector-borne disease control due to HaRe / ENAcoformulant / 875-PCT their remarkable capacity to kill larvae of various insect pests from the Coleoptera, Lepidoptera, Hymenoptera and Diptera orders, including Aedes and Anopheles mosquitos - vectors for zika (Musso et al., 2019), dengue (Jansen and Beebe, 2010) and malaria (Phillips et al., 2017). Since Bt strains target specific insect groups with minimal impact on humans and non-target organisms (e.g. wildlife, pollinators and other beneficial insects), they can form the basis of biopesticides that offer a safe and eco-friendly alternative to non-discriminatory, environmentally persistent and potentially toxic insecticidal chemicals (Koch et al., 2015). Bt adopts its entomopathogenic lifestyle by the characteristic production of vegetative insecticidal toxins (Vip) (Schnepf et al., 1998) or delta-endotoxins (Cry and Cyt) during sporulation. Bt toxins exhibit a distinctive composition unique to each strain, resulting in a remarkable selectivity for specific insect genera. The toxins are produced as pro-toxins that assemble into pure or mixed crystalline condensates referred to as parasporal bodies or crystals (PSB). Upon ingestion of Bt spores, the associated pro-toxin crystals dissolve and exert a poreforming activity in the insect gut, followed by intestinal sepsis, starvation and host death. In the process, germinated vegetative Bt cells colonize the insect until available nutrients in the carcass become depleted, then inducing sporulation to ensure further dissemination and survival of adverse conditions until the spores are taken up by e.g. a new host to restart the cycle.

[0127] Compositions of the present invention can be obtained by culturing Bacillus thuringiensis strains or an insecticidal mutant (strain) derived therefrom according to methods well known in the art, optionally further treated to isolate parts thereof, such as spore-preparations thereof, and combining that with the A-ENA protein nanofibrils as described herein.

[0128] For Bt culturing the conventional large-scale microbial culture processes include submerged fermentation, solid state fermentation, or liquid surface culture. Towards the end of fermentation, as nutrients are depleted, cells begin the transition from growth phase to sporulation phase, such that the final product of fermentation is largely spores, metabolites and residual fermentation medium. Sporulation is part of the natural life cycle of Bacillus thuringiensis and is generally initiated by the cell in response to nutrient limitation. Fermentation is configured to obtain high levels of colony forming units and to promote sporulation. The bacterial cells, spores and metabolites in culture media resulting from fermentation may be used directly or concentrated by conventional industrial methods, such as centrifugation, tangential-flow filtration, depth filtration, and evaporation. Compositions of the present invention may include fermentation products. Alternatively, a "broth concentrate," may be used, referring to whole broth (fermentation broth) that has been concentrated by conventional industrial methods, but remains in liquid form; or alternatively "fermentation solid," as used herein, referring to the solid material that remains after the fermentation broth is dried; alternatively "fermentation product," is used, referring to whole broth, broth concentrate and / or fermentation solids. The HaRe / ENAcoformulant / 875-PCT fermentation broth or broth concentrate can be dried with or without the addition of carriers using conventional drying processes or methods such as spray drying, freeze drying, tray drying, fluidized-bed drying, drum drying, or evaporation. The resulting dry products may be further processed, such as by milling or granulation, to achieve a specific particle size or physical format. Cell-free preparations of fermentation broth of the strains can be obtained by any means known in the art, such as extraction, centrifugation and / or filtration of fermentation broth. Those of skill in the art will appreciate that so- called cell-free preparations may not be devoid of cells but rather are largely cell-free or essentially cell- free, depending on the technique used (e.g., speed of centrifugation) to remove the cells. The resulting cell-free preparation may be dried and / or formulated with components that aid in its application to plants or to plant growth media. Concentration methods and drying techniques described above for fermentation broth are also applicable to cell-free preparations. The fermentation product may also be envisaged as a freeze-dried powder or a spray-dried powder.

[0129] The formulation comprising the composition of the present invention may be in the presence of a typical wetting agent, extender, solvent, spontaneity promoter, emulsifier, dispersant, frost protectant, thickener, and / or an adjuvant.

[0130] Preferred formulations for the composition of the invention are provided as a tank mix formulation, a wettable granule formulation, a spray formulation, or a floatable briquettes formulation. As used herein, "tank mix" refers to a composition prepared by mixing at least one pesticidal ingredient with an adjuvant or coformulant, in the composition of the present invention, and optionally a quantity of water in a tank by a user immediately before application.

[0131] Method for control of plant health

[0132] In a method according to the invention a composition containing an agriculturally bioactive substance, such as a Bacillus strain or an insecticidal mutant thereof , and an A-ENA protein nanofibril, can be applied to any plant or any part of any plant grown in any type of media used to grow plants (e.g., soil, vermiculite, shredded cardboard, and water) or applied to plants or the parts of plants grown aerially, such as orchids or staghorn ferns. The composition may for instance be applied by spraying, atomizing, vaporizing, scattering, dusting, watering, squirting, sprinkling, pouring or fumigating. Application may be carried out at any desired location where the plant of interest is positioned, such as agricultural, horticultural, forest, plantation, orchard, nursery, organically grown crops, turfgrass and urban environments. The treatment of the plants and plant parts with the compositions according to the invention is carried out directly or by acting on the environment, habitat or storage space using customary treatment methods, for example by dipping, spraying, atomizing, misting, evaporating, dusting, fogging, scattering, foaming, painting on, spreading, injecting, drenching, trickle irrigation and, HaRe / ENAcoformulant / 875-PCT in the case of propagation material, in particular in the case of seed, furthermore by the dry seed treatment method, the wet seed treatment method, the slurry treatment method, by encrusting, by coating with one or more coats and the like. It is furthermore possible to apply the active substances by the ultra-low volume method or to inject the active substance preparation or the active substance itself into the soil.

[0133] A preferred direct treatment of the plants is the leaf application treatment, i.e., compositions according to the invention are applied to the foliage, it being possible for the treatment frequency and the application rate to be matched to the infection pressure of the pathogen in question.

[0134] In the case of systemically active agents, the compositions according to the invention reach the plants via the root system. In this case, the treatment of the plants is effected by allowing the compositions according to the invention to act on the environment of the plant. This can be done for example by drenching, incorporating in the soil or into the nutrient solution, i.e., the location of the plant (for example the soil or hydroponic systems) is impregnated with a liquid form of the compositions according to the invention, or by soil application, i.e., the compositions according to the invention are incorporated into the location of the plants in solid form (for example in the form of granules).

[0135] The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.

[0136] EXAMPLES

[0137] Example 1. A-ENA promotes spore clustering and retention of parasporal bodies (PSBs).

[0138] The biological function of A-ENA was investigated by inactivating the ATN07_33990 (a-ena) in Bt. Israelensis, and spores of the resulting Bti Aa-ena lost recognition by a polyclonal serum raised against recombinant A-ENA, and lacked discernible sporesilk fibrils when inspected by nsEM (Figure Id). To evaluate if a-ena expression would alter PSB content, OD-normalized sporeprepfractions were treated at pHll and analysed by SDS-PAGE (Figure lb and 2a). SDS-PAGE showed bands at ~28, ~70 and ~130 kDa, identified, respectively, as the CytlA, CryllAal and Cry4Aal / Cry4Abl toxins by MS fingerprinting, as well as minor bands around 55 and 38 kDa found to represent CryllAal breakdown products (Figure 2a). A quantitative comparison of the released bands showed sporep repinputs of Bti WT and Aa-ena mutant showed no discernible difference in the of amounts of Cry toxins.

[0139] Our microscopy experiments reveal Bti spore preparations consist of microcolonies with clustered spores and PSBs, embedded in a dense extrasporal matrix of A-ENA (Figure 3). During sporulation, the endospore and PSB are formed in the daughter and mother cell compartment, respectively (Figure 3a) HaRe / ENAcoformulant / 875-PCT

[0140] (Schnepf et al., 1998). We investigated if A-ENA fibrils are implicated in spore clustering and may serve to ensure spores and PSBs remain associated after mother cell lysis and during the infection process. To do so, spore preparation of Bti WT and Aa-ena were subjected to cushioned density centrifugation (1 hr at 100g or 10.000g) with three phases corresponding to low density buffer or supernatant ('SN') layer with a density of approximately p=lg / ml (plow), a medium density layer of 40% Histodenz with a p= 1.21 g / ml (pmedlum);and a high density layer of 60% Histodenz with p=1.32 g / ml (phlgh). Sedimentation fractions were qualitatively followed by light microscopy (Figure la), and quantitatively monitored by SDS-PAGE of the pHll-released fraction to follow PSBs (Figure lb) and by ODgoo to track the fractionation of spores (Figure lc). At a low g force of ~1 pN (i.e. calculating a volume and density of "'0.5 pm3and 1.21 - 1.32 g / ml for individual spores), PSBs of WT Bti were found primarily in the plowfraction, with spores in a 50 / 50 ratio in the plowand pmedlumfractions. Microscopy showed the plowand pmedlumfractions contained clusters of agglutinated spores with high and low PSB content, respectively. Individual spores or PSBs were sparse. In contrast, for the Aa-ena mutant, roughly 60 % of PSBs and > 90 % of spores fractionated to the pmedlumfraction, and light microscopy showed dispersed spores and PSBs with no apparent spore - PSB association. At higher g force (~100 pN), the agglutinated WT Bti spore-PSB fraction partially broke up into smaller clusters that fractionated to the pmedlumzone (~100% of PSBs; 70% of spores) and individual spores pelleted in the phlghzone (0 % PSB; 30 % of spores). The Aa-ena mutant showed a similar distribution, though again lacked an apparent clustering of PSBs and spores. Thus, WT Bti spores and PSBs are found in biofilm-like microcolonies that are agglutinated by an extrasporal matrix of A-ENA nanofibrils. In absence of A-ENA, spores and PSBs are found in a dispersed suspension.

[0141] Spurred on by this striking result, we also followed a gain-of-function approach, where we introduced a- ena in Bt kurstaki (Btk), which naturally lacks pAM65-52-4-128K or A-ENA homologs. Light microscopy and nsTEM imaging of Btk WT showed mostly dispersed spores and PSBs (Figure le), with the presence of spore-attached S-ENA fibrils (Figure 2d; Pradhan et al., 2021), but a lack of A-ENA-like fibrils or an extensive extrasporal matrix as seen for Bti (Figure 2d). In sharp contrast, Btk pAS_o-eno showed a rich network of A-ENA fibrils, resulting in the clustering of Btk spores into multibody biofilms (Figure le, Figure 2c, d). Moreover, typical bipyramidal Btk toxin crystals (referred to as parasporal crystals or PSCs) were seen to be enclosed in the Btk pAS_o-eno biofilms. Large A-ENA bundles could be seen to grace the PSC crystal surface as well as making contact with neighbouring spores, seemingly functioning as a molecular tether mimicking the spore-PSB association seen in Bti. Similar to Bti, we found no significant difference in the PSC formation for WT and Btk pAS_o-eno (Figure 2a).

[0142] Example 2. Recombinantly produced purified A-ENA as aide in spore clustering.

[0143] Based on the observations in the Btk gain of function engineered strain, and taking into account that A- ENA fibrils can be produced recombinantly, as provided in PCT / EP2024 / 074370 and as described herein, HaRe / ENAcoformulant / 875-PCT it was intended to apply purified A-ENA fibrils to the spore cultures in view of the biological role in spore clustering and PSB retention. Remarkably, indeed, exogenous addition of recombinant A-ENA nanofibrils ('+A-ENA') purified from E. coli (Figure le, Figure 2c, d) to said Btk strain, revealed a very similar A-ENA dependent spore and PSB clustering showing the agglutinating activity does not require A-ENA expression by the sporulating cells, and can be induced post-sporulation. When WT Btk, pAS_o-eno and +A-ENA spore preps were subjected to cushioned density centrifugation, we observed that the spore - PSB agglutination induced by recombinant expression, or exogenous addition of A-ENA results in the increased retention of spores and PSBs in the low density fraction (Figure 2b, e). Even at high g-forces, as much as 50% of PSBs were retained in a low density agglutinated biofilm fraction. Moreover, whereas WT Btk spores and PSB separated over a 70% sucrose cushion, as previously reported (Thomas and Ellar, 1983), in Btk pAS_o-eno PSBs would co-pellet with the spores, demonstrating the encapsulation in A- ENA matrix ensured a robust spore - PSB association (Figure 2f).

[0144] Example 3. A-ENA increases insecticidal activity of B. thuringiensis Sv. Kurstaki.

[0145] The prior observation that A-ENA forms a virulence factor for the insecticidal activity of B. thuringiensis Sv. Israelensis, as shown in PCT / EP2024 / 074370, through showing that Bti WT versus Bti A-ENA KO increased the survival time of Chironomus aprillinus larvae treated with these spores. Next, recombinant expression of A-ENA in Bt Sv. Kurstaki was tested in view of its potential enhancing virulence and thus efficacy in pest control, in an insect assay. Trichoplusia ni, commonly known as the cabbage looper, a highly destructive leaf feeder with a devastating impact on cruciferous crops such as cabbage and broccoli was used as an insect model , as it is a natural target of Btk. The above-described Btk strain expressing A-ENA on a plasmid (gain of function approach) was tested and compared in their survival of the larvae feeding on medium supplemented with spores of Btk WT or the Btk WT strain recombinantly expressing A-ENA (Btk+A-ENA). Although Bti naturally contains A-ENA, its PSBs are not effective against T. ni, thus serving as a negative control (Figure 4c). We used 1-week-old larvae and followed their survival over a week after adding a suspension of spore on their solid feeding media. Results clearly showed that ~50 % (15 / 28) of larvae were still alive on day four in the Btk WT strain, whereas this number dropped to ~10 % (23 / 26) for the BTK strain expressing A-ENA (Figure 4c). The clustering activity of A-ENA seemingly enhances virulence, so as a next step, we investigated whether adding purified A-ENA nanofibrils (recombinantly produced in E. coli) to WT Btk spores would maintain that effect. ns-EM showed that the addition of purified A-ENA mimics the aggregation of spore and PSBs, similar to what is seen for Btk recombinantly expressing A-ENA. The results demonstrate that the addition of recombinant A-ENA fibrils effectively clusters Btk spores and PSBs (Figure 4a). We then repeated the killing assay with WT Btk spores and PSB suspension, with and without the addition of recombinant A-ENA fibrils. Consistent with our earlier findings, approximately 50 % (8 / 16) of larvae remained alive on day four in HaRe / ENAcoformulant / 875-PCT the WT Btk strain, whereas survival dropped to approximately 18% (3 / 16) when recombinant A-ENA fibrils were present (Figure 4d). As a negative control, we included recombinant A-ENA, confirming that in themselves, the A-ENA fibrils do not exert a direct toxic effect.

[0146] Example 4. Phylogenetic analysis suggests biological functions beyond spore / PSB coupling.

[0147] Phylogenetic analysis of A-ENA prevalence across the Firmicutes phylum shows that A-ENA homologues are found across both the Clostridia and Bacillales clades. Due to the genetic association between A-ENA and cyt / cry-toxins in Bti, we focused on the A-ENA prevalence across the Bacillus cereus group sensu lato. A-ENA is found in 496 of 5976 (8%) tested genomes of the Btyper database, and although found in all GTDB Bacillus species, it is predominantly found in B. thuringiensis (156 genomes), B. mycoides (124 genomes), B. wiedmanni (96 genomes) and to a lesser extent in B.anthracis / paranthracis / cereus (39 genomes). Moreover, out of the 496 genomes containing A-ENA, 351 (71%) genomes contained one or more type of Bt toxin. If we compare that to the Bt-toxin prevalence across the Btyper database -only 1071 genomes (18%) were Bt positive, showing that A-ENA and Bt are positively correlated, reinforcing the notion that the main biological function of A-ENA is related to the presence of toxins, and by extension parasporal bodies. Interestingly, 350 genomes (70%) carried more than 2 A-ENA-like genes (as is the case for Bti) with 83 (17%) genomes harboring 5 or more homologues (e.g. the Bacillus cereus sensu stricto strain AFS036381 has 19 A-ENA-like genes). We performed a gene cluster analysis and found that many A-ENA-like genes are organized in (putative) operons and / or gene clusters. Looking at these gene clusters in further detail, we discern two types of A-ENA homologues, (i) those constructed of a single A-ENA domain (shown in orange in Figure 5) and (ii) those carrying an additional domain (A-ENA fusion, shown in blue in Figure 5). After performing an interproscan analysis on the dataset of A-ENA homologue sequences, we identified 5 classes of passenger domain types. The gene architecture of the most abundant class (type 1) consists of an A-ENA domain, followed by a collagen-like region and a Clq domain. Other less frequently observed passenger domain types are SdrD_B (PF17210) / OmcB (PF01345) or 4Fe-4S cluster domain (PF13370). We recently showed that collagen-like proteins can form trimeric tip fibrillae (ruffles) on the termini of S- and L-type ENA fibrils found on Bacillus cereus and showed that these ruffles are involved in spore-spore clustering (Pradhan et al., 2021; Sleutel et al., 2024). We reasoned that a similar mechanism could be at play for the A-ENA / Clq homologues and modelled a heterohexamer of WP_277491740.1 (putative A-ENA ruffle) of a Bacillus cereus strain SIBC65 isolated from raw bulk milk in complex with WP_000075910.1 (single A-ENA domain) which is the presumed major subunit of an A-ENA type pilus (Figure 5a, b). AF3 predicts an A-ENA / ruffle complex wherein the A-ENA domains of the ruffle dock onto the single domain A-ENA protomers thereby mimicking the homotypic A-ENA contacts in the fibril (Figure 5c). The residues involved in IPB formation are conserved in both WP_277491740.1 and WP_000075910.1 (Figure 5d), suggesting that the ruffle is HaRe / ENAcoformulant / 875-PCT seamlessly integrated into the fibril or covalently tethered to the ter inus of the A-ENA fibril (Figure 5e). Clues to a putative function of A-ENA ruffles can be found for Pasteuria ramosa, an endoparasitic bacterium of aquatic microcrustaceans (e.g. water flea Daphnia). Recently, Huessy et al demonstrated that the collagen-like protein Pci 7 of P. Ramose functions as an adhesin to mediate spore attachment to the epithelium of the oesophagus of the host Daphnia (Huessy et al., 2024). Here we show that although Pcl7 is a CLP lacking an N-terminal A-ENA domain, it is embedded in a 3 gene operon flanked by two additional CLPs (ORFs 01218, 01220) one of which (01218) has an N-terminal A-ENA domain (Figure 5f,g). We also identify another 3 gene cluster (ORFs 00694, 00695 and 00696) which encode for 3 A-ENA-like genes. AF3 predicts a heterohexameric structure of the three CLPs in complex with the 3 putative A-ENA major subunits that is topologically similar to the ruffle described above (Figure 5h,i).

[0148] Taken altogether, it seems plausible that the (short) hair-like appendages (Duneau et al., 2011) that coat the surface of P. ramosa is (in part) formed by A-ENA like fibrils decorated with adhesive Pci 7 tip fibrillae. Based on our analysis, this might be a recurring feature across the family of A-BclA (i.e. A-ENA + collagen / Clq) carrying strains.

[0149] Example 5. Coformulation of A-ENA nanofibrils with spores and cry toxin crystals of Bacillus thuringiensis Aizawai (Bta) or Bacillus thuringiensis Tenebrionis (Btt) results in Induced Temporary Biofilms (ITB) formation.

[0150] In Bacillus thuringiensis Israelensis (Bti), endogenous A-ENA nanofibers are responsible for the clustering of parasporal bodies and spores into biofilm-like aggregates that enhance the bactericidal activity of the spore preparations. As shown herein, we demonstrate that when purified A-ENA nanofibrils are exogenously added to spore preparations of Bacillus thuringiensis Kurstaki (Btk) this results in an induced clustering of the spores and the cry toxin crystals into biofilm-like aggregates, with A-ENA nanofibrils forming a bonding matrix between spores and the micrometer scale particulate crystals (Figures 1, 2).

[0151] We further explored whether said A-ENA biofilm inducing activity or 'induced temporary biofilms' (ITBs) is specific to species or toxin by supplementing spore preparations of Bta or Btt with exogenous A- ENA, recombinantly produced in E. coli and purified by differential centrifugation, and analysed by light microscopy. Where native, unmodified spore preparation of Btt or Bta are composed of loosely dispersed spores and cry toxin crystals (Figure 6a, b), preparations with exogenously added A-ENA nanofibers shows spores and crystals clump together into biofilm like clusters of multiple tens to hundreds of particulates (Figure 6a, b), demonstrating ITB formation by coformulation with A-ENA is non-specific and independent of the composition of cry toxin crystals or the genetic background of Bt spores. HaRe / ENAcoformulant / 875-PCT

[0152] So, hereby we provide a generic platform for the agglutination or clustering of micrometer scaled particulates such as microbial spores, specifically exemplified herein by different Bt spores, but also clustering, aggregation or biofilm formation of particulates such as bacterial cells, protein crystals and combinations are envisaged as to be bound together by a matrix of A-ENA nanofibrils.

[0153] Indeed, to assess the bonding capacity of such ITBs, spore preparations of Btt with and without the exogenous addition of recombinant A-ENA nanofibers purified from E. coli were subjected to cushioned density centrifugation (1 hr at 100g) with three phases corresponding to a low density buffer or supernatant (‘SN’) layer with a p = 1 .00g / ml (plow), a medium density layer of 40% Histodenz with a p = 1 .21 g / ml (pmedium), and a high density layer of 60% Histodenz with p = 1 .32 g / ml (phigh). Sedimentation fractions were quantitatively monitored by SDS-PAGE of the high pH-released fraction to follow PSBs (Figure 6c). PSBs of WT Btt were primarily retained in the pmediumfraction (i.e. >95%). The addition of exogenous A-ENA nanofibrils, however, induces spore - PSB agglutination into ITBs with increased spore - toxin association, as witnessed by the increased retention of PSBs in the plowdensity fraction (SN).

[0154] Based on the obtained results in several Bt species, we conclude that the agglutinating activity of recombinant A-ENA fibrils is not limited to specific spores or toxin crystals, and can rather be considered a generic technology to induce temporary biofilms by their exogenous addition. It is conceived that the application of A-ENA nanofibrils as an exogenous coformulant, inducing particle clustering and biofilm formation is of temporary nature, since this activity will be lost upon germination and / or replication of the spores or microbial cells embedded in the A-ENA matrix. Said "Induced temporary biofilms" or 'ITBs' can find useful application in the formulation of preparations composed of microbial spores, cells, protein microcrystals, metabolites, and micrometer scaled particulates in general, and combinations thereof (Figure 7). The formation of said microbial ITBs can thus enhance the insecticidal activity as herein disclosed for various Bacillus thuringiensis subspecies. Similar to endogenous biofilms, ITBs can aid in the persistence and protection of embedded cells and spores from external stresses such drought, heath, UV light, reactive oxygen and hazardous chemicals. Furthermore, alike endogenous biofilms, ITBs can find application by aiding in the attached and persistence of the embedded particulates such as spores, cells, proteins, metabolites, ... to biological and non-biological surfaces (Philipp et al. 2024).

[0155] Example 6. ITBs of Bacillus thuringiensis Aizawai (Bta) or Bacillus thuringiensis Tenebrionis (Btt) formed with exogenous A-ENA nanofibrils show increased adherence to plant leaf surfaces.

[0156] Native microbial biofilms are well documented for their increased adherence and persistence on biological and non-biological surfaces. Such biofilms are the result of endogenous or recombinant expression of fibrous extracellular matrix components such as environmental DNA (eDNA), HaRe / ENAcoformulant / 875-PCT polysaccharides (i.e. cellulose and derivatives) and various protein fibrils (i.e. functional amyloids such as curli and different pilus types such as type 1 pili, bundle forming pili, TasA, among others) by the cells that become embedded in the biofilm. These extracellular matrix components encapsulate and agglutinate the embedded cells, and often enhance the adherence onto various biological and non- biological surfaces, in the scope of the present invention, we demonstrate the formation of 'Induced Temporary Biofilms' by coformulation of microbial cells and / or spores with exogenously added A-ENA protein nanofibrils, and further demonstrate that such A-ENA-based ITBs can endow the embedded cells, spores and / or particulate materials with an increased adherence onto various surfaces.

[0157] Indeed, as an example of applying ITBs to increase attachment to biological surfaces, we here demonstrate the use of A-ENA ITBs for the protection of Bt spore preparations against run-off from leaf surfaces. To test so, A-ENA ITBs were formed by the exogenous addition of purified A-ENA nanofibrils to spore and toxin preparations of native Bacillus thuringiensis Aizawai (Bta) or Bacillus thuringiensis Tenebrionis (Btt). To asses the ability of A-ENA ITBs to enhance binding and retention of Bt spores and toxins onto plant leaf surfaces, Bta and Btt spore preparations with and without the exogenous addition of purified A-ENA nanofibrils were spotted onto tomato leaf discs, allowed to dry, and subjected to consecutive washes with 5 mL of deionized water. To assess the retention of native spore preps or their respective A-ENA ITBs, leaf discs were then placed in Cry toxin solubilizing Laemli buffers and subjected SDS-PAGE. The SDS-PAGE analysis demonstrates the loss of over 70 % of Cry toxins for both Btt or Bta preparations following 5 subsequent washes in water (Figure 8). Remarkably, the coformulations with purified A-ENA increased leaf retention, as demonstrated by the retained signal for Cry3Aa (Btt) and CrylAb (Bta) under repetitive washes. Thus, ITB formation of Bt preparations increases on leaf persistence and protects spray-on formulations of Bt spores and toxins against run-off.

[0158] As such, the use of A-ENA as a coformulant for ITB formation of microbial preparations can find broad technological applications, providing inducible and temporary formulations with biofilm-endowed properties, but without the risk of adverse effects of longer term and propagating biofilms such as found in endogenous biofilm-inducing pathways.

[0159] Example 7. Co-administration of A-ENA and Bt kurstaki in a Spodoptera model.

[0160] Next, in view of validating the increased virulence of exogenously added A-ENA to Bt spore formulations, insect assays were conducted in vitro, as to further investigate the potential of recombinant purified A- ENA fibrils as insecticide coformulant to develop novel pesticides to combat agricultural pests. Spodoptera species are amongst the most significant agricultural pests affecting a wide range of crops globally, and Bacillus thuringiensis has been extensively used to manage these pests through both Bt- HaRe / ENAcoformulant / 875-PCT based biopesticides and transgenic Bt crops. Larvicidal bioassays were conducted with (among others) the beet armyworm, Spodoptera exigua. An agar-based artificial diet is prepared and poured into a multiwell plate. Different preparation approaches allow to evaluate the impact of A-ENA on Btk spore toxicity, including serial dilutions of Btk spores alone, spores combined with a fixed concentration of A-ENA, Btk crystals alone, and BTK crystals combined with A-ENA. An additional control with A-ENA only was included to isolate its effects.

[0161] After cooling and solidification, 0.15 ml of test solution is added to each well. The plates are placed on a rocking platform, and as soon as the diet is dried or absorbed in the diet, individual LI larvae (~25 larvae per assay) are placed on the diet in each well. A folded paper tissue is placed on top, and the plate is closed with a lid. The plates are then incubated at 25°C, 60% RH and 16:8 L:D. Larval survival is scored after at least 3 days over a range of concentrations allowing to determine dose response curves, after which data are analyzed with Probit analysis.

[0162] These bioassays were performed to determine the lethal concentration (LC) of Bacillus thuringiensis in presence or absence of exogenously added A-ENA nanofibrils, affecting the spore-parasporal bodies clustering of Bt in order to lower the effective lethal concentration and / or time to achieve lethality.

[0163] Example 8. Lepidoptera bio assays using different Bt strains.

[0164] The bio assays as presented in Figures 9-11, using Spodoptera exigua or Plutella xylostella, were performed as follows. Fresh eggs, deposited on sheet substrate, were collected from cages containing artificial-diet-reared adults, and placed inside a ventilated container with agar-based artificial diet. The eggs were allowed to hatch under controlled conditions, and larvae were collected for the experiments when reaching the required stage. Artificial diet was poured into multi-well plates and the liquid spore- PSB preparation was then added on top of the solidified diet. The plates were placed on a rocking platform and as soon as the diet surface had dried (absorption and / or liquid evaporation), larvae were transferred to the wells. For Plutella, generally ten specimens were placed in each well, while for the bigger Spodoptera species, each well housed a single larva (20 larvae per multi-well plate). Four such replicates were included for each test condition. The plates with larvae were incubated under controlled conditions, and larval survival and pupation were scored on a regular basis. The experiment continued until at least 90 % of the surviving larvae in the control condition had reached the pupal stage.

[0165] As shown in Figure 9, the pupation was reduced for Spodoptera exigua larvae when A-ENA nanofibrils were present together with (low concentrations of) Bt kurstaki, indicative for the increase in virulence. A further indicative result is obtained by scoring the morality of Plutella xylostella larvae using Bt kurstaki strain ABTS-351 spore suspension (Figure 10a) or using Dipel, which is a commercial formulation containing Bt kurstaki strain ABTS-351 (Figure 10b), with or without A-ENA exogenously added. Although HaRe / ENAcoformulant / 875-PCT these results are not statistically significant, the closing and reproducibility of the assays is further optimized.

[0166] In addition, testing of the Plutella mortality using alternative Bt species, such as Bt aizawai clearly indicate the need for further testing dose response, and show (Figure 11) that a highly significant improvement in mortality is obtained in the present of A-ENA protein nanofibrils for higher Bt concentrations (as compared to the initial Spodoptera bio assays). So the trend that A-ENA had a beneficial effect on the virulence of the Bt suspension is clear from all assays, though for commercial formulations of A-ENA compositions as coformulant and / or ITB agent, optimization is required.

[0167] Finally, for bio assays using Leptinotarsa decemlineata, leaves with fresh eggs were collected from potted potato plants infested with beetle adults. The eggs were allowed to hatch and the resulting larvae were maintained on detached potato leaves under controlled conditions, until reaching the LI larval stage required for the experiment. Leaf disks (1.8 cm2) or leaflets were submerged in the liquid Bt tenebrionis (NB176 from Novodor) spore-PSB preparation (at increasing concentrations between 0.005 and 0.04 OD, and including surfactant Tween-20 promote adhesion to leaves), with or without lmg / mL A-ENA, during 10 seconds, after which they were left to dry and beetle larvae were introduced. A replicate consisted of 6 individual larvae, while 3 replicates were tested for each test condition. The larvae were allowed to feed on the leaf disks for 48h. After this period, the larvae were provided with a fresh untreated leaf disk on a regular basis, while also keeping the cotton wool moist. Larval mortality was recorded on a regular basis until the end of the experiment.

[0168] Table 2 shows a dose-response curve indicative of the same beneficial trend of A-ENA presence for mortality as observed in previous Lepidopteran assays with alternative Bt strains.

[0169] Table 2. Dose-response analysis.

[0170] Methods

[0171] Bacterial strains and growth conditions. Escherichia coli Stellar™ was used for cloning and was grown in

[0172] Luria-Bertani (LB) broth or agar plates at 37°C supplemented with 50 pg / ml kanamycin or 100 pg / ml HaRe / ENAcoformulant / 875-PCT ampicillin when required. For protein production, E. coli BL21 (DE3) was grown in TB broth supplemented with 100 pg / ml ampicillin and placed at 30°C for A-ENA expression after induction. E. coli Dam- / Dcm- (New England Biolabs) was used to obtain unmethylated plasmids in order to increase the efficiency of transformation in Bt. Bti (WT= Bacillus thuringiensis serovar Israelensis AM65-52; Bolotin et al., 2017), and Btk (WT= Bacillus thuringiensis serovar Kurstaki HD 1; Day et al., 2014) were grown in LB at 30°C with 150 rpm shaking and supplemented with 25 pg / ml kanamycin when required. For sporulation, 1 ml of overnight culture was plated on LB media and placed at 30°C for a week. Spores were scrapped and diluted in miliQ. water and stored at 4°C.

[0173] Cloning for recombinant protein expression in E. coli. Cloning of untagged A-ENA, A-ENA1 and variants were produced with the purpose of cytoplasmic overexpression in E. coli. Water-boiled colonies of Bti were used as a source of gDNA. As a general cloning strategy, all PCR fragments containing the coding sequence were cloned into a linearized pASK-IBA3plus vector (primers 321 and 322) by Gibson assembly using NEBuilder Hi Fi DNA Assembly Cloning Kit (New England Biolabs).

[0174] Cloning for a-ena-STOP using CRISPR / Cas9 based deletion plasmid. Plasmid pJOE-Aa-ena (pJOE899 derivative for A-ENA STOP insertions) was used to knockout a-ena from B. thuringiensis serovar isrealensis. The plasmid is derived from the plasmid pJOE8999 (Altenbuchner, 2016) purchased from Bacillus Genetic Stock Center (BGSC). Oligonucleotides for sgRNA construction were designed using the online sgRNA design tool Cas-Designer (Park et al., 2015). First, pJOE8999 was linearized with primers p551 + p552. pJOE-Aa-ena homology regions were cloned with p588+p591 and three STOP codon mutations were introduced with oligos p961+p962. We used NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs) to insert homology regions. Sequencing and insertion were verified with the pair of oligos p525+p526. Insertion of sgRNA was done by PCR using oligos p594+p595. Primers p531+p579 were used to sequence sgRNA region. Verification of the A-ENA STOP mutant was done by PCR and sequencing using oligos p963+p651 and p964 respectively.

[0175] Construction and isolation of 8. thuringiensis serovar isrealensis mutants. The recombinant pJOE8999- based plasmids described above, were introduced by electroporation into B. thuringiensis serovar isrealensis competent cells. Briefly, B. thuringiensis cells were made electrocompetent by growing in BHI + 0.5% glycerol at 379C until ODgoo reached 0.6. Cells were harvested by centrifugation (5 min at 15 000 xg). Cells were washed 3 times with ice-cold sterile electroporation buffer (1 mM HEPES, 10% glycerol, pH 7.0) and flash-frozen in liquid nitrogen. Up to lpg of unmethylated plasmid was transformed into 200 pl of competent cells using a 0.2 cm gap cuvette (Biorad) and electroporated using the MicroPulser Electroporator device (Biorad). Cells were transferred into 1ml of recovery buffer (BHI with 10% glycerol, 0.4% glucose, and 10 mM MgCL) for lh at 30gC before plating in a 25 pg / ml kanamycin plate. Next day, HaRe / ENAcoformulant / 875-PCT a single colony was inoculated into 10 ml of liquid BHI supplemented with 25 pg / ml kanamycin and incubated with 180 rpm shaking for 3h at 379C. Then, Mannose (Sigma) was added to a final concentration of 0.4% w / v in order to induce the expression of the Cas9 nuclease. After 3 hours, serial dilution of the culture was plated on LB agar plates with 25 pg / ml kanamycin + 0.4% w / v Mannose and incubated at 37gC overnight. Screening of the knockout was done using colony PCR, WT B. thuringiensis serovar isrealensis was used as a control. Positive colonies were sequenced-verified using the same oligos (Eurofins). To curate the pJOE8999-based plasmid, mutant strains were passaged three times in the absence of antibiotics and plate in LB plates at 42gC overnight. The absence of the plasmid was checked by striking the same colony in LB plates with or without Kanamycin.

[0176] Cloning for recombinant protein expression in B. thuringiensis. For ectopic recombinant expression of the different constructs in B. thuringiensis we modified the pJOE8999 plasmid (Altenbuchner, 2016) by removal of the coding sequence for the Cas9 nuclease and its promoter by PCR using oligos p622+p623, resulting in the 'pAS' shuttle expression vector. Complementation of a-ena stop and a-enal was achieved by expressing these constructs in the pAS vector. The a-ena region, including its promoter, ORF, and terminator, was amplified using primers p683 and p684. Colony PCR and sequencing (using primers p631 and p617) confirmed successful insertion, resulting in plasmid pAS-Pa-ena-A-ENA.

[0177] Protein expression and purification. A-ENA was expressed in E. coli BL21 (DE3) in Terrific Broth (TB) supplemented with 100 pg / mL of Ampicillin at 37 °C and induced with 200 pg / L anhydrotetracycline when ODgoo reached 0.5. At this point, the temperature was set at 18 °C and cells were left to express overnight. Cells were harvested by centrifugation (20 min at 5,000g) and incubated with A-ENA lysis buffer (25 mM Hepes pH8, 2.5 mM EDTA, 250 mM NaCI and 20 mg of Lysozyme) at 37 °C for 2 hours under shaking conditions. Next, 1% final SDS concentration was added and the resuspended pellet was boiled for 30 min. A-ENA fibrils were pelleted down by centrifugation at 30,000g for 45 minutes. Pellet was resuspended with a homogenizer in 30 ml deionized H2O and centrifuged for 45 minutes at 30,000g. This washing step was repeated 4 times. After the last centrifugation step, the pellet was resuspended with 3 ml of deionized H2O.

[0178] Spore preparation and Fractionation. Sporulation of Bti, Btk, Btt, or Bta was triggered by nutrient exhaustion on LB-agar plates supplemented with 25 pg / mL of kanamycin when required. A single colony was inoculated in LB media (supplemented with 25 pg / mL of kanamycin when required) and grown overnight at 30°C. Next day, 200 pl of the cell suspension was plated on 245 x 245 mm LB plates (with or without antibiotic). Plates were incubated at 30°C for 7 days under aerobic conditions. Spores were harvested by scraping the LB plates with a cell scraper and resuspended in deionized H2O, spore stock was stored at 4°C. To fractionate Bti, Btk, Btt or Bta spores, the spore suspension was gently vortexed HaRe / ENAcoformulant / 875-PCT and then a discontinuous Histodenz gradient was prepared by layering 500 pl of 40% (w / v) Histodenz over 500 pl of 60% (w / v) Histodenz in 2 mL microcentrifuge tubes. A total of 500 pl of spore stock (OD6oo = 42) was carefully layered on top. Two tubes were prepared per strain: one for SDS-PAGE analysis and the other for optical density (OD) measurements and imaging. Samples were centrifuged at 10,000 x g or 100 x g for 1 hour using a tabletop swinging-bucket rotor (S-24-11-AR, Eppendorf).

[0179] After centrifugation, three fractions (supernatant (SN), 40%, and 60%) were carefully collected into separate tubes. Each fraction was washed 3 times by adding 500 pl of water, vortexed, and centrifuged at 12,000 x g for 5 minutes. The supernatant was removed, and the pellet was resuspended in 100 pl of DX buffer (10 mM CHES pH 9.6, 8M Urea, 1% SDS, lx protease inhibitor tablet (Roche), 2 mM DTT). Samples were boiled for 10 minutes with securely closed lids and subsequently centrifuged at maximum speed for 10 minutes to pellet insoluble spores. A total of 50 pl of the supernatant was mixed with 12.5 pl of blue loading dye, and 5 pl of the final mixture was loaded onto an SDS-PAGE gel for protein analysis.

[0180] For OD measurements and imaging, the SN, 40% Histodenz, and 60% Histodenz fractions were collected into separate tubes. Each fraction was washed as described above. The supernatant was removed, and the pellet was resuspended in 100 pl of water. OD6oo measurements were performed by diluting 50 pl of the sample into 950 pl of water (1:20 dilution) before spectrophotometric analysis.

[0181] Phase contrast microscopy. For imaging spores, a single colony of Bti, Btk, Btt or Bta was inoculated into 10 mL of LB medium with the appropriate antibiotic when required and grown overnight at 30°C. The next day, 250 pL of the culture was plated onto LB agar, with or without antibiotics, and incubated for 5 days at 30°C to allow sporulation. The resulting spore lawn was harvested and resuspended in 5 mL of water. A 3 pL aliquot of the spore suspension was transferred onto an LB-agar strip for microscopy. For time-lapse experiments comparing sporulation, t = 0 was defined as the moment cells were plated on the LB agar plate. At each time point, a sample was collected using a sterile loop, resuspended in 30 pL of PBS, and imaged. Images were acquired in phase contrast using a Leica DMi8 inverted microscope (Leica) equipped with a 100x / 1.32 oil objective (Leica).

[0182] Immunodetection. A polyclonal mouse antiserum raised against purified rec-A-ENA fibrils was obtained from Davids Biotechnologie GmbH (Regensburg, Germany). The following 63 day immunization schedule was followed: immunizations on days 0, 14, 28, 42 and 56. To detect A-ENA on spore samples, we diluted the spore suspension to ODgoo=l and blotted 2 pl onto a nitrocellulose membrane. After 5 minutes of drying at room temperature (RT), the membrane was blocked with 5% (w / v) skimmed milk for 30 minutes. The membrane was incubated with A-ENA antibody mix (dilution 1:1000 in TBS-Tween buffer (Tris-HCI pH8 10 mM; NaCI 150 mM; Tween200,05% (vol / vol)) and incubated at RT for lh. After 3 washes of 5 minutes each, the membrane was incubated in the secondary antibody mix (dilution 1:1000) for an HaRe / ENAcoformulant / 875-PCT extra hour at RT. After 3 washes of 5 minutes each, the membrane was imaged with the LI-COR Odyssey M using the LI-COR Acquisition Software v 2.0.0.86.

[0183] Monitoring the entomopathogenic activity of different Bt strains. For the virulence assessment of Btk strains, we used the insect model Trichoplusia ni. Larvae or eggs were obtained from Frontier Agricultural Sciences (Newark, USA). Btk wild-type and A-ENA-expressing strains (pA-ENA) were sporulated on LB plates at 30°C for one week. Spores and PSBs were harvested and processed as described for Bti strains. To determine the mortality rate, a minimum of 16 two week old T. ni larvae per group were individually placed on 2.5 x 2.5 cm pieces of solid food provided by the supplier. Each food piece was spread with a 50 pl aliquot of the harvested spore suspension at an OD6oo of 0.06. As a negative control, Bti (which has no toxic effect on T. ni) was included. Larval survival was monitored daily for one week. For experiments involving recombinant A-ENA, it was added to Btk WT samples at a final concentration of 4.18 mg / ml and incubated for at least 3 hours under slow rotation. As an additional control, purified recombinant A-ENA was tested at the same final concentration (4.18 mg / ml) under identical conditions. The study was performed in biological triplicates at room temperature.

[0184] Leaf retention assays. For leaf retention experiments, spore preparations of Btt or Bta were diluted to an ODgoo of 7. Exogenous addition of recombinant A-ENA nanofibrils purified from E. coli was performed at a concentration of 5 mg / ml. Tomato leaves were harvested from plants and tissue discs of 8 mm diameter were generated using a leaf tissue punch. 30 pl of the respective spore preparations was added on each leaf disc and left to dry in for 1 h. Treated discs were washed 0 - 5 times by subsequent submerging in 5 ml sterile water, after which they were again left to dry for 1 h. Run-off resistance of the various spore preparations was assessed through SDS-PAGE of the recovered of PSBs when placing the leaf discs in an Eppendorf containing 250 pl of DX buffer (10 mM CHES pH 9.6, 8M Urea, 1% SDS, lx protease inhibitor tablet (Roche), 2 mM DTT) and boiling for 15 minutes with securely closed lids. A total of 15 pl of the solution was mixed with 5 pl of blue loading dye and 15 pl of the final mixture was loaded onto and SDS-PAGE gel for protein analysis.

[0185] Search for orthologs and homologs of A-ENA. To probe for the presence of A-ENA across the Firmicutes phylum, all publicly available Genbank genomes (9880 genomes) belonging to the Bacillus / Clostridium group (taxid 1239; assembly-level complete) were downloaded from the NCBI database. Homo- and orthologs of A-ENA were searched in all assemblies with hmmsearch (Eddy, 2011) (881 genomes) using a hidden Markov model that was generated with hmmbuild using a manually curated multiple sequence alignment of A-ENA sequences obtained from a blastp (Johnson et al., 2008) search using Q8KNV8 as a query. For this, AlphaFold2 models (either retrieved from the AlpfaFold database (Varadi et al., 2024) or predicted using localcolabfold (Mirdita et al., 2022)) of the corresponding blast sequences were manually HaRe / ENAcoformulant / 875-PCT inspected and compared to the A-ENA cryoEM structure to remove any non-A-ENA sequences. The inclusion threshold for hmmsearch was set to an E-value of le-7.

[0186] For the more detailed search of A-ENA homo- and orthologs across the Bacillus cereus group sensu lato, the BtyperDB vl database (5976 genomes) was downloaded from https: / / www.btyper.app / , and hmmsearch was run against all assemblies with an E-value threshold of le-7 (496 genomes). The phylogenetic tree described in BtyperDB (Ramnath et al., 2023) and the corresponding meta-data table was kindly provided by Laura M. Caroll. Gene cluster analysis was performed using cblaster 1.3.18 (Gilchrist et al., 2020) using default settings. Protein domain organization of A-ENA homologs with C- terminal fusions was performed with Interproscan 5.67-99.0 (Jones et al., 2014).

[0187] Table 3: Reference Sequence accession numbers of A-ENA like proteins (n=597) HaRe / ENAcoformulant / 875-PCT

[0188] >WP_126294795.1; >WP_071392824.1; >WP_151701241.1; >WP_210471316.1; >WP_040376149.1;

[0189] >WP_212924438.1; >WP_204668115.1; >WP_212976039.1; >WP_066186838.1; >WP_152657822.1;

[0190] >WP_040982409.1; >WP_188733585.1; >WP_066142189.1; >WP_115749335.1; >WP_020616168.1;

[0191] >WP_128658904.1; >WP_238650419.1; >WP_173140208.1; >WP_010268044.1; >WP_047980762.1;

[0192] >WP_263706280.1; >MBO2505129.1; >WP_016839178.1; >WP_141601888.1; >WP_208650703.1;

[0193] >WP_090774452.1; >WP_090776151.1; >PFB49380.1; >WP_257209516.1; >OQR53105.1;

[0194] >WP_068775901.1; >WP_142506947.1; >TQR39522.1; >WP_119150851.1; >WP_215175558.1;

[0195] >WP_215175557.1; >WP_211350104.1; >WP_175371047.1; >PGQ44283.1; >WP_181538938.1;

[0196] >WP_044896080.1; >MCL6586930.1; >WP_181538937.1; >WP_012957170.1; >KQU17358.1;

[0197] >KRF52451.1; >KRF52448.1; >KQU17360.1; >WP_066154797.1; >WP_155477750.1; >NLX02164.1;

[0198] >NLK52489.1; >NLV22477.1; >WP_134230475.1; >WP_134230476.1; >WP_224876227.1;

[0199] >WP_064094024.1; >WP_224876226.1; >WP_197315488.1; >WP_224809048.1; >WP_066265084.1;

[0200] >WP_270407384.1; >WP_134230544.1; >WP_069644379.1; >WP_069703205.1; >WP_069644377.1;

[0201] >WP_153725722.1; >NLF45454.1; >WP_221860360.1; >WP_019155143.1; >NLT40941.1;

[0202] >MBE3101226.1; >MBE3101227.1; >WP_235222005.1; >OQB14150.1; >MCL2703241.1;

[0203] >WP_242057465.1 / 6-7; >WP_019155144.1; >WP_080630428.1 / 4-4; >WP_183242236.1;

[0204] >WP_042536196.1 / 4-4; >KIP20212.1 / ll-ll; >HBR31256.1; >OON92172.1; >ONI43887.1; >HIS65799.1;

[0205] >HIU33750.1; >NLK86606.1; >WP_090446350.1; >QGU95879.1; >WP_072907480.1;

[0206] >MCL6617755.1 / 104-l; >WP_181520874.1; >WP_055441798.1; >AST05588.1; >WP_035048978.1;

[0207] >WP_184664076.1; >WP_184664075.1; >WP_110609538.1; >WP_093051849.1; >WP_106589654.1;

[0208] >WP_010239793.1; >MCK9536559.1; >WP_078665439.1; >NMB44602.1; >NU90181.1; >OQA15291.1;

[0209] >NLO10347.1; >WP_236914082.1; >WP_252225997.1; >WP_204489239.1; >HHU63352.1;

[0210] >WP_254495860.1; >HBI56858.1; >WP_008908033.1; >SEF39824.1; >WP_242971176.1;

[0211] >WP_122963797.1; >WP_122903756.1; >WP_007783061.1; >WP_122961155.1; >WP_122925796.1;

[0212] >WP_163859565.1; >WP_134757257.1; >WP_173140217.1; >WP_216857486.1; >WP_049741785.1;

[0213] >WP_088910016.1; >WP_106657270.1; >WP_172139574.1; >WP_137031666.1; >WP_016739500.1;

[0214] >WP_106784580.1; >WP_219661378.1; >WP_047070179.1; >TQR34900.1; >WP_087348525.1;

[0215] >WP_199929547.1; >WP_017246715.1; >WP_174226400.1; >WP_144618485.1; >WP_007720341.1;

[0216] >WP_056488977.1; >WP_197936189.1; >WP_010268048.1; >OAB30303.1; >WP_138224403.1;

[0217] >WP_091176138.1; >WP_130607412.1; >WP_087443534.1; >WP_127585513.1; >WP_161411873.1;

[0218] >WP_173215395.1; >WP_079412742.1; >WP_171414263.1; >WP_021254599.1; >WP_270170784.1;

[0219] >OBZ18017.1; >OBZ18016.1; >WP_028562891.1; >WP_209971550.1; >WP_028546586.1;

[0220] >WP_232275159.1; >WP_028595674.1; >WP_258279168.1; >WP_175373344.1; >WP_058303866.1;

[0221] >WP_048744837.1; >CDN41938.1 / 4-4; >WP_036651907.1; >WP_113029034.1; >MBD2860923.1;

[0222] >WP_223836348.1; >WP_144509072.1; >WP_098688323.1; >MBK5491851.1; >WP_252211459.1;

[0223] >PFI81502.1; >PGK33495.1; >WP_098650108.1; >WP_197722729.1; >WP_216636299.1 / 22-;

[0224] >WP_197478852.1 / 21-; >KXG08769.1; >WP_115749336.1; >WP_246020340.1; >WP_104059545.1;

[0225] >WP_151701235.1; >WP_212508844.1; >WP_174521724.1; >WP_191814960.1; >WP_244811852.1;

[0226] >WP_173660201.1; >WP_036198767.1; >WP_208650728.1; >WP_019155127.1; >WP_212924437.1;

[0227] >WP_040982410.1; >WP_229720117.1; >WP_235817440.1; >WP_244704997.1; >WP_244853042.1;

[0228] >WP_239587967.1; >SKB05896.1; >WP_244715222.1; >WP_185959608.1; >NMA90008.1;

[0229] >MBP1969341.1 / 5-5; >WP_209462530.1; >WP_257230192.1; >MBG0967840.1; >WP_236686903.1;

[0230] >WP_019244172.1; >ALP35341.1; >WP_238354028.1; >WP_044648569.1; >WP_212734477.1;

[0231] >WP_211556547.1; >SIS52057.1; >WP_234969477.1; >WP_245629781.1; >AEJ44700.1;

[0232] >WP_237700123.1; >ACV59513.1; >WP_245530800.1; >MCD8500378.1; >WP_071313875.1;

[0233] >WP_089023434.1; >WP_197315495.1; >WP_134230541.1; >WP_064094028.1; >WP_224876222.1;

[0234] >WP_217225283.1; >WP_246421570.1; >WP_242175596.1; >WP_274069240.1; >TQR39524.1;

[0235] >WP_255504946.1; >MCD8503411.1; >WP_166246166.1; >WP_131015779.1; >WP_270407379.1;

[0236] >WP_232317625.1; >NLC94992.1; >REK56576.1; >WP_113805716.1; >WP_078665440.1;

[0237] >WP_188038901.1; >WP_075387276.1; >RBW69966.1 / 7-7; >WP_245947291.1; >WP_171719036.1;

[0238] >WP_209879878.1; >ETT74612.1; >WP_256761495.1; >RXZ80755.1; >WP_119150854.1; HaRe / ENAcoformulant / 875-PCT

[0239] The unique accession code representative for an A-Ena-like protein sequence is provided herein between '>' and

[0240] Sequence Listing

[0241] >SEQ ID NO: 1: Bacillus thuringiensis Sv. Israelensis strain ATCC35646 A-ENA amino acid sequence (UniProt: Q8KNV8 )

[0242] >SEQ ID NO: 2: amino acid sequence N-terminal part of Bacillus cereus WP_277491740.1 protein, as shown in Figure 5

[0243] >SEQ ID NO: 3: amino acid sequence of Bacillus WP_000075910.1 protein, as shown in Figure 5

[0244] >SEQ ID NO: 4: A-ENA amino acid sequence of SEQ ID NO:1 without C-terminal Serine (Figure 5g)

[0245] >SEQ ID NOs: 5: Pasteuria ramosa protein sequence (Figure 5 g : A-ENA-1 / 1-102) HaRe / ENAcoformulant / 875-PCT

[0246] >SEQ ID NOs: 6: Pasteuria ramosa protein sequence (Figure 5 g: A-ENA-1 / 1-103)

[0247] >SEQ ID NOs: 7: Pasteuria ramosa protein sequence (Figure 5 g: A-ENA-1 / 1-104)

[0248] >SEQ ID NOs: 8: Pasteuria ramosa protein sequence (Figure 5 g: AKNPHEEL_01218 / l-4219)

[0249] Table 4. Oligonucleotide primers used herein.

[0250] HaRe / ENAcoformulant / 875-PCT

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Claims

HaRe / ENAcoformulant / 875-PCTCLAIMS1. A composition comprising an agriculturally bioactive substance and a protein nanofibril, wherein said protein nanofibril comprises an A-ENA protein.

2. The composition of claim 1, wherein said agriculturally bioactive substance comprises a pesticidal substance , such as an insecticide, nematicide, biocide, fungicide, herbicide, herbicide safener, or comprises a plant growth regulator, or a biostimulant, or a biofertilizer, and / or combinations of any one thereof.

3. A composition of any of claims 1 to 2, wherein said agriculturally bioactive substance comprises a microbial spore, cell, toxin, protein and / or metabolite.

4. The composition of any one of claims 1 to 3, wherein the agriculturally bioactive substance comprises a Bacillus sp. strain or a Pasteuria sp. strain., preferably a B. thuringiensis strain, or a biocidal mutant variant thereof.

5. The composition of any one of claims 1 to 4, wherein the agriculturally bioactive substance comprises a spore-containing suspension, preferably selected from Bacillus sp., Paenibacillus sp., Pasteuria sp., Brevibacillus laterosporus, Clostridium bifermentans, Lysinibacillus sphaericus, or a biocidal mutant variant thereof.

6. The composition of any one of claims 4 or 5, wherein the Bacillus sp. is selected from Bacillus thuringiensis subsp. Israelensis, Kurstaki, Aizawai, Kenyae, or Tenebrionis, or Bacillus wiedmannii, B. sphaericus, B. bombysepticus, B. licheniformis, B. amyloliguefaciens, B. subtilis, or B. mojavensisan, or a functional variant thereof.

7. The composition of any one of claims 1 to 6, wherein the protein nanofibril comprises an engineered or functionalized A-ENA protein.

8. The composition of claim 7, wherein said engineered or functionalized A-ENA comprises an A-ENA protein fused or conjugated with a plant-binding agent, a UV-protectant agent, or a phagostimulant agent.

9. The composition of any of claims 3 to 8, wherein the A-ENA protein nanofibrils and the microbial spores or cells are present in the composition as a biofilm.

10. The composition of any one of claims 1 to 9, further comprising a preservative, a structuring agent or surfactant, a buffer, a dye, an anti-foam agent, a wetting agent, or a freeze-thaw stabilizing agent.

11. A formulation comprising the composition of any one of claims 1 to 10, which is selected from a tank mix formulation, a wettable granule formulation, a spray formulation, or a floatable briquettes formulation.

12. A method for controlling plant health comprising applying to a plant area an effective amount of the composition of any one of claims 1 to 10, or of the formulation of claim 11.55HaRe / ENAcoformulant / 875-PCT13. The method of claim 12, wherein the plant area comprises a leaf, shoot, seed, or fruit part of said plant.

14. A method for producing the composition of any one of claims 1 to 10, comprising the steps of: a. recombinantly producing the A-ENA or engineered A-ENA protein nanofibrils in a host, b. releasing and isolating the A-ENA or engineered A-ENA protein nanofibrils from the host cell, preferably through cell lysis, and resuspension from the insoluble fraction and / or further purification from the cell lysate, c. combining the purified protein nanofibril with an agriculturally bioactive substance to obtain the composition.

15. The method of claim 14 wherein said A-ENA or engineered A-ENA protein nanofibril is functionalized after purification from the host cell in step b. prior to step c.

16. Use of the composition of any one of claims 1 to 10, or of the formulation of claim 11, for controlling animal pests and / or protecting a plant or part of a plant in need of protection from pest damage.

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