Bacillus thuringiensis (Bt) strains produce a variety of Cry proteins with nematicidal properties. Among these, Cry5B has shown toxicity against plant-parasitic nematodes of the genus Meloidogyne. In this study, the potential of a Bt strain producing Cry5B to colonize cucumber plants and suppress the root-knot nematode Meloidogyne javanica infestation was evaluated. The strain was applied by irrigation as a suspension of spores and crystals to cucumber seedlings. Bt was subsequently detected after plating extracts of surface-sterilized roots, cotyledons, stems, and leaves, demonstrating systemic movement and stable endophytic colonization for at least eight weeks, although Bt concentrations in leaves decreased progressively toward the most distal parts of the plant. The presence of vegetative cells in all tissues (assessed by comparing the CFU obtained from plant extracts before and after heat-shock treatments) further supported the ability of the strain to grow endophytically within cucumber. Bt-treated plants irrigated once or twice with 1 × 1010 spores and crystals challenged with 350 M. javanica juveniles failed to reduce nematode infection or reproduction. The number of egg masses and emerging J2 per plant were similar to those observed in plants irrigated with the Cry5B-deficient Bt strain. These results indicate that, although Cry5B displays potent nematicidal activity in vitro, endophytic colonization of cucumber plants by the applied producing strain did not confer effective protection against M. javanica under the experimental conditions tested. Understanding the factors that limit the in planta efficacy of Bt may help guide the development of more consistent Bt-based biological control strategies against plant-parasitic nematodes.

The insecticidal Cry proteins from Bacillus thuringiensis are used in biopesticides or transgenic crops for pest control. The Cry1I protein family has unique characteristics of being produced during the vegetative rather than sporulation phase, its protoxins forming dimers in solution, and exhibiting dual toxicity against lepidopteran and coleopteran pests. The Cry1Ia protoxin undergoes sequential proteolysis from the N- and C-terminal ends, producing intermediate forms with insecticidal activity, while in some cases, the fully processed toxin is inactive. We investigated the oligomerization and toxicity of Cry1Ia intermediate forms generated through trypsinization (T-Int) and larval gut fluid (GF-Int) treatments, as well as the fully trypsinized protein (toxin). Heterologously expressed intermediate forms assembled into oligomers and showed similar toxicity to Cry1Ia protoxin against Ostrinia nubilalis (European corn borer) larvae, while the toxin form was ~30 times less toxic. In contrast, bioassays with Leptinotarsa decemlineata (Colorado potato beetle) larvae did not show significant differences in toxicity among Cry1Ia protoxin, T-Int, GF-Int, and fully processed toxin. These results suggest that the Cry1I mode of action differs by insect order, with N-terminal cleavage affecting toxicity against lepidopteran but not coleopteran larvae. This knowledge is essential for designing pest control strategies using Cry1I insecticidal proteins.

Nymphs of the ambar mutant of Orius laevigatus (Fieber) are orange-colored instead of the yellowish color of the wild-type individuals. Since there were no previous studies of the pigments of this species, we searched for differences in pigments of the pteridine family between both strains. Fluorescent compounds from nymph extracts were separated by cellulose thin-layer chromatography (TLC) and by size exclusion chromatography, followed by LC/MS/MS. The present study has allowed the identification for the first time in O. laevigatus of erythropterin, leucopterin, 7-methylxanthopterin, xanthopterin, isoxanthopterin, pterin, and biopterin. The quantification was performed by fluorometry after elution of the pteridines previously separated by TLC. The results showed that the orange color in the ambar nymphs was due to the accumulation of the orange pigment erythropterin. Additionally, mutant nymphs exhibited significantly elevated levels of pterin and reduced levels of leucopterin. The possibility that these differences were due to differences in xanthine dehydrogenase (XDH) activity was tested; the results indicated that XDH deficiency is unlikely to be responsible for the mutant phenotype. Considering that the ambar mutation is recessive, the mutant phenotype should, most likely, be due to a disruption in downstream metabolic steps involved in erythropterin processing.

Bt-cadherins, a particular type of midgut cadherins, act as receptors for Bacillus thuringiensis Cry1A pesticidal proteins. The aim of this work was to identify and validate the Cry1A cadherin receptor in Grapholita molesta (GmCad1). The GmCad1 gene was annotated by Blast genome mining using lepidopteran Bt-related cadherins. The phylogenetic analyses grouped GmCad1 with other Torticidae cadherins, in a different clade than the lepidopteran Bt-related cadherins. The in silico analysis of the GmCad1 showed a structure similar to that of the Bt-related cadherins, with 11 cadherin repeats (CRs), a transmembrane region, and an intracellular domain. The full-length mRNA sequencing confirmed GmCad1 expression in vivo in G. molesta guts. To validate the binding ability of Cry1A proteins to GmCad1, a cadherin fragment (CR7-CR11) was expressed and in vitro binding was studied demonstrating that Cry1Aa, Cry1Ab, and Cry1Ac bind to this region in a dose-dependent manner. In silico molecular docking analysis suggested that the interaction may involve mainly the Domains II of Cry1Ab and Cry1Ac, and the Domain III of Cry1Aa. This study provides the first evidence of a 99-C cadherin serving as a receptor for Cry1A in G. molesta . ### Competing Interest Statement The authors have declared no competing interest. Ministerio de Ciencia, Innovación y Universidades, PID2021-122914OB-100 (co-funded by EU FEDER funds) Ministerio de Ciencia, Innovación y Universidades, RYC2021-033098-I Ministerio de Ciencia, Innovación y Universidades, https://ror.org/05r0vyz12, PRE2019-089628

Bacillus thuringiensis (Bt) is a Gram-positive, spore-forming, and ubiquitous bacterium harboring plasmids encoding a variety of proteins with insecticidal activity, but also with activity against nematodes. The aim of this work was to perform the genome sequencing and analysis of a native Bt strain showing bipyramidal parasporal crystals and designated V-CO3.3, which was isolated from the dust of a grain storehouse in Córdoba (Spain). Its genome comprised 99 high-quality assembled contigs accounting for a total size of 5.2 Mb and 35.1% G + C. Phylogenetic analyses suggested that this strain should be renamed as Bacillus cereus s.s. biovar Thuringiensis. Gene annotation revealed a total of 5495 genes, among which, 1 was identified as encoding a Cry5Ba homolog protein with well-documented toxicity against nematodes. These results suggest that this Bt strain has interesting potential for nematode biocontrol.

The global imperative to enhance crop protection while preserving the environment has increased interest in the application of biological pesticides. Bacillus thuringiensis (Bt) is a Gram-positive bacterium that can produce nematicidal proteins and accumulate them in parasporal crystals. Root-knot nematodes are obligate root plant parasitic which are distributed worldwide, causing severe damages to the infested plants and, consequently, large yield reductions. In this work, we have evaluated the toxicity of the Bt crystal proteins Cry5, Cry21, App6, and Xpp55 against two root-knot nematodes belonging to the Meloidogyne genus (M. incognita and M. javanica). The results show that all four proteins, when solubilized, were highly toxic for both nematode species. To check the potential of using Bt strains producing nematicidal crystal proteins as biopesticides to control root-knot nematodes in the field, in planta assays were conducted, using two wild Bt strains which produced Cry5 or a combination of App6 and Cry5 proteins. The tests were carried out with cucumber or with tomato plants infested with M. javanica J2, irrigated with spore + cristal mixtures of the respective strains. The results showed that the effectiveness of the nematicidal activity was plant-dependent, as Bt was able to reduce emerged J2 in tomato plants but not in cucumber plants. In addition, the toxicity observed in the in planta assays was much lower than expected, highlighting the difficulty of the proteins supplied as crystals to exert their toxicity. This emphasizes the delivery of the Bt proteins as crucial for its use to control root-knot nematodes. • Solubilized Cry5, Cry21, App6 and Xpp55 Bt proteins are toxic to M. javanica. • Cry21 toxicity to M. incognita is similar to that of Cry5, App6, and Xpp55 proteins. • The Cry5 and App6 toxicities on M. javanica after Bt irrigation is crop dependent.

We report the draft genome of Bacillus thuringiensis strain V-AB8.18, comprising 308 contigs totaling 6,182,614 bp, with 35% G + C content. It contains 6,151 putative protein-coding genes, including App6 and Cry5-like crystal proteins, exhibiting 99% pairwise identity to nematicidal proteins App6Aa2 and Cry5Ba2, active against Meloidogyne incognita and Meloidogyne hapla.

Nanotechnology is a promising way to enhance the stability of Bacillus thuringiensis (Bt) insecticidal proteins under envi-ronmental conditions. In this work, two emulsions were prepared through the Pickering emulsion technique, stabilized by Cu2+-SQDs/S-CN nanocomposites and by GO nanosheets. In addition, a pH-sensitive polymer was incorporated into these emulsions, allowing the Bt protein, Cry1Ab, to be released in an alkaline pH environment, as it occurs in the lepidopteran pests' gut. The effectiveness of these two nanomaterials in protecting Cry1Ab from degradation, and therefore enhancing its pesticidal activity, was assessed by exposing samples of the purified unprotected protein and encapsulated protein to high-intensity UV light and 40 degrees C temperature treatments. The UV treatment results were evaluated using SDS-PAGE analysis and pointed out that Cry1Ab could be structurally protected by the emulsions. The bioassays with first instar larvae of the lepidopteran pest Ostrinia nubilalis confirm the nanomaterial protection to UV and temperature treatments, i.e., decreasing about half the degradation rate and increasing up to 12-fold the residual activity after UV treatment. Our results indicate that encapsulation could be an effective strategy to improve the effectiveness of Cry1Ab under environmental conditions.

Modern agriculture makes use of bacterial genes to protect crops from the attack of insects. Bt-crops are commercialized and express cry and vip3Aa genes from Bacillus thuringiensis, which confer protection against insect pests. Vip3A proteins are highly active against lepidopterans and the fact that they do not share binding sites with Cry proteins makes them perfect "partners" to be combined in insect-protected crops. The structure of Vip3Aa is composed of five well differentiated domains, and in solution the protein spontaneously forms a pyramid-shaped tetramer protoxin. The N-terminal domains of the protein (Domains I and II) are very conserved, mainly formed by a-helices, and are critical for the formation and maintenance of the tetrameric structure. The C-terminal domains (Domains III, IV and V) are highly variable, mainly consisting of beta-sheets and thought of being responsible for binding to specific receptors (Domain III) and to glycosylated molecules (Domains IV and V). The activation of the protoxin involves a profound conformational change in which Domains I are reorganized forming a coiled-coil structure that resembles a long "needle". The tip of this needle inserts into the epithelial membrane eventually causing cell death. To date, no resistance to Vip3Aa proteins has been reported in the field, though laboratory selection and F2 screens have succeeded in obtaining resistant colonies. In contrast to resistance to Cry proteins, no case of alteration of the binding sites has been found in Vip3-resistant insects so far.

Abstract Vip3 proteins are produced by Bacillus thuringiensis and are toxic against lepidopterans, reason why the vip3Aa gene has been introduced into cotton and corn to control agricultural pests. Recently, the structure of Vip3 proteins has been determined and consists of a tetramer where each monomer is composed of five structural domains. The transition from protoxin to the trypsin‐activated form involves a major conformational change of the N‐terminal Domain I, which is remodelled into a tetrameric coiled‐coil structure that is thought to insert into the apical membrane of the midgut cells. To better understand the relevance of this major change in Domain I for the insecticidal activity, we have generated several mutants aimed to alter the activity and remodelling capacity of this central region to understand its function. These mutants have been characterized by proteolytic processing, negative staining electron microscopy, and toxicity bioassays against Spodoptera exigua. The results show the crucial role of helix α1 for the insecticidal activity and in restraining the Domain I in the protoxin conformation, the importance of the remodelling of helices α2 and α3, the proteolytic processing that takes place between Domains I and II, and the role of the C‐t Domains IV and V to sustain the conformational change necessary for toxicity.

Bacillus thuringiensis Cry1I insecticidal proteins are structurally similar to other three-domain Cry proteins, although their size, activity spectrum, and expression at the stationary phase are unique among other members of the Cry1 family. The mode of action of Cry1 proteins is not completely understood but the existence of an activation step prior to specific binding is widely accepted. In this study, we attempted to characterize and determine the importance of the activation process in the mode of action of Cry1I, as Cry1Ia protoxin or its partially processed form showed significantly higher toxicity to Ostrinia nubilalis than the fully processed protein either activated with trypsin or with O . nubilalis midgut juice. Oligomerization studies showed that Cry1Ia protoxin, in solution, formed dimers spontaneously, and the incubation of Cry1Ia protoxin with O . nubilalis brush border membrane vesicles (BBMV) promoted the formation of dimers of the partially processed form. While no oligomerization of fully activated proteins after incubation with BBMV was detected. The results of the in vitro competition assays showed that both the Cry1Ia protoxin and the approx. 50 kDa activated proteins bind specifically to the O . nubilalis BBMV and compete for the same binding sites. Accordingly, the in vivo binding competition assays show a decrease in toxicity following the addition of an excess of 50 kDa activated protein. Consequently, as full activation of Cry1I protein diminishes its toxicity against lepidopterans, preventing or decelerating proteolysis might increase the efficacy of this protein in Bt-based products. Key points • Processing Cry1I to a 50 kDa stable core impairs its full toxicity to O. nubilalis • Partially processed Cry1Ia protoxin retains the toxicity of protoxin vs O. nubilalis • Protoxin and its final processed forms compete for the same functional binding sites

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that accumulates pesticidal proteins (Cry and Cyt) in parasporal crystals. Proteins from the Cry5, App6 (formerly Cry6), Cry12, Cry13, Cry14, Cry21, and Xpp55 (formerly Cry55) families have been identified as toxic to nematodes. In this study, a total of 846 Bt strains belonging to four collections were analyzed to determine the diversity and distribution of the Bt Cry nematicidal protein genes. We analyzed their presence by PCR, and positives were confirmed by sequencing. As a result, 164 Bt isolates (20%) contained at least one gene coding for nematicidal Cry proteins. The cry5 and cry21 genes were enriched in collection 1 and were often found together in the same strain. Differently, in collection 4, obtained from similar habitats but after 10 years, cry14 was the gene most frequently found. In collection 2, cry5 and app6 were the most abundant genes, and collection 3 had a low incidence of any of these genes. The results point to high variability in the frequencies of the studied genes depending on the timing, geographical origins, and sources. The occurrence of cry1A, cry2, and cry3 genes was also analyzed and showed that the nematicidal Cry protein genes were frequently accompanied by cry1A + cry2. The expression of the genes was assessed by mass spectrometry showing that only 14% of the positive strains produced nematicidal proteins. To our knowledge, this is the first comprehensive screening that examines the presence and expression of genes from the seven known Bt Cry nematicidal families.

Plant-parasitic nematodes are ubiquitous in nature and cause large losses in agriculture. The current concerns regarding the use of chemical pesticides have increased the interest in new control alternatives. One of these is the one based on Bacillus thuringiensis (Bt). These Gram-positive bacteria have the ability to synthesize pesticide proteins during sporulation. Some of these proteins have nematicidal properties. Studies have shown that preparations of certain strains of Bt can prevent or slow down the infestation of phytonematodes. The expression of some Bt nematicidal genes in transgenic plants has also demonstrated their effectiveness. Bt is nowadays an effective ecological alternative for controlling plant-parasitic nematodes.

Bacillus thuringiensis (Bt)-based products are the most successful microbial insecticides to date [...].

Bacillus thuringiensis Vip3 ( V egetative I nsecticidal P rotein 3 ) toxins are widely used in biotech crops to control Lepidopteran pests. These proteins are produced as inactive protoxins that need to be activated by midgut proteases to trigger cell death. However, little is known about their three-dimensional organization and activation mechanism at the molecular level. Here, we have determined the structures of the protoxin and the protease-activated state of Vip3Aa at 2.9 Å using cryo-electron microscopy. The reconstructions show that the protoxin assembles into a pyramid-shaped tetramer with the C-terminal domains exposed to the solvent and the N-terminal region folded into a spring-loaded apex that, after protease activation, drastically remodels into an extended needle by a mechanism akin to that of influenza haemagglutinin. These results provide the molecular basis for Vip3 activation and function, and serves as a strong foundation for the development of more efficient insecticidal proteins.

Discovery and identification of novel insecticidal proteins in Bacillus thuringiensis (Bt) strains are of crucial importance for efficient biological control of pests and better management of insect resistance. In this study, the Bt strain KhF, toxic for Plodia interpunctella and Grapholita molesta larvae, underwent genomics and proteomics analyses to achieve a better understanding of the bases of its pathogenicity. The whole-genome sequencing results revealed that the KhF strain contained nine coding sequences with homologies to Bt insecticidal genes. The lepidopteran toxic mixture of spores and crystals of this Bt strain was subjected to liquid chromatography and tandem mass spectrometry (LC-MS/MS) to assess the protein composition. The results of the proteomic analyses, combined with the toxin gene sequences, revealed that two of the main components of the crystals were two new candidate pesticidal proteins, named KhFA and KhFB. These proteins showed a similarity lower than 36% to the other known Bt toxins. The phylogenetic analysis showed that the KhFA and KhFB grouped with the newly denominated Xpp and Mpp (former ETX/Mtx) pesticidal protein groups, respectively. Altogether, this study has led to the discovery of two novel candidate pesticidal toxins in the lepidopteran toxic KhF strain.

Bacillus thuringiensis (Bt) produces insecticidal proteins that are either secreted during the vegetative growth phase or accumulated in the crystal inclusions (Cry proteins) in the stationary phase. Cry1I proteins share the three domain (3D) structure typical of crystal proteins but are secreted to the media early in the stationary growth phase. In the generally accepted mode of action of 3D Cry proteins (sequential binding model), the formation of an oligomer (tetramer) has been described as a major step, necessary for pore formation and subsequent toxicity. To know if this could be extended to Cry1I proteins, the formation of Cry1Ia oligomers was studied by Western blot, after the incubation of trypsin activated Cry1Ia with insect brush border membrane vesicles (BBMV) or insect cultured cells, using Cry1Ab as control. Our results showed that Cry1Ia oligomers were observed only after incubation with susceptible coleopteran BBMV, but not following incubation with susceptible lepidopteran BBMV or non-susceptible Sf21 insect cells, while Cry1Ab oligomers were persistently detected after incubation with all insect tissues tested, regardless of its host susceptibility. The data suggested oligomerization may not necessarily be a requirement for the toxicity of Cry1I proteins.

Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper) are two important defoliation pests of soybeans. In the present study, we have investigated the susceptibility and brush border membrane-binding properties of both species to Bacillus thuringiensis Cry1Ea toxin. Bioassays performed in first-instar larvae demonstrated potent activity against both soybean pests in terms of mortality or practical mortality. Competition-binding studies carried out with (125)Iodine-labelled Cry1Ea, demonstrated the presence of specific binding sites on the midgut brush border membrane vesicles (BBMV) of both insect species. Heterologous competition-binding experiments indicated that Cry1Ea does not share binding sites with Cry1Ac or Cry1Fa in either soybean pest. This study contributes to the knowledge of Cry1Ea toxicity and midgut binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ea with other Bt proteins aimed at controlling lepidopteran pests in soybeans.

Characterization of new Bacillus thuringiensis strains is a valuable tool to discover novel insecticidal toxins and to manage resistance problems. In this study, seven Iranian Bt strains were selected according to their toxicity against Plodia interpunctella, to be thoroughly characterized based on their toxicity, protein profiling, proteomic analysis, gene content and β-exotoxin production. The toxicity was assessed by insect bioassays and cell viability assays (a less cost, time and material consuming technique), using four lepidopteran pests and four lepidopteran cell lines from Trichoplusia ni (Hi5), Helicoverpa zea (HzGUT), Spodoptera exigua (UCR-SE) and Spodoptera frugiperda (Sf21). The selected Bt strains showed similar protein electrophoretic profiles, but differed in toxicity. LC–MS/MS analysis of solubilized crystal proteins and gene content analyses (PCR screening) were compared and correlated with the toxicity results. Based on our data, three Bt strains could be considered as candidates for development of future bioinsecticides.