Silencing Ditylenchus destructor cathepsin L-like cysteine protease has negative pleiotropic effect on nematode ontogenesis.

Ditylenchus destructor is a migratory plant-parasitic nematode that severely harms many agriculturally important crops. The control of this pest is difficult, thus efficient strategies for its management in agricultural production are urgently required. Cathepsin L-like cysteine protease (CPL) is one important protease that has been shown to participate in various physiological and pathological processes. Here we decided to characterize the CPL gene (Dd-cpl-1) from D. destructor. Analysis of Dd-cpl-1 gene showed that Dd-cpl-1 gene contains a signal peptide, an I29 inhibitor domain with ERFNIN and GNFD motifs, and a peptidase C1 domain with four conserved active residues, showing evolutionary conservation with other nematode CPLs. RT-qPCR revealed that Dd-cpl-1 gene displayed high expression in third-stage juveniles (J3s) and female adults. In situ hybridization analysis demonstrated that Dd-cpl-1 was expressed in the digestive system and reproductive organs. Silencing Dd-cpl-1 in 1-cell stage eggs of D. destructor by RNAi resulted in a severely delay in development or even in abortive morphogenesis during embryogenesis. The RNAi-mediated silencing of Dd-cpl-1 in J2s and J3s resulted in a developmental arrest phenotype in J3 stage. In addition, silencing Dd-cpl-1 gene expression in female adults led to a 57.43% decrease in egg production. Finally, Dd-cpl-1 RNAi-treated nematodes showed a significant reduction in host colonization and infection. Overall, our results indicate that Dd-CPL-1 plays multiple roles in D. destructor ontogenesis and could serve as a new potential target for controlling D. destructor.

Using kin discrimination to construct synthetic microbial communities of Bacillus subtilis strains impacts the growth of black soldier fly larvae.

Using synthetic microbial communities to promote host growth is an effective approach. However, the construction of such communities lacks theoretical guidance. Kin discrimination is an effective means by which strains can recognize themselves from non-self, and construct competitive microbial communities to produce more secondary metabolites. However, the construction of cooperative communities benefits from the widespread use of beneficial microorganisms. We used kin discrimination to construct synthetic communities (SCs) comprising 13 Bacillus subtilis strains from the surface and gut of black soldier fly (BSF) larvae. We assessed larval growth promotion in a pigeon manure system and found that the synthetic community comprising 4 strains (SC 4) had the most profound effect. Genomic analyses of these 4 strains revealed that their complementary functional genes underpinned the robust functionality of the cooperative synthetic community, highlighting the importance of strain diversity. After analyzing the bacterial composition of BSF larvae and the pigeon manure substrate, we observed that SC 4 altered the bacterial abundance in both the larval gut and pigeon manure. This also influenced microbial metabolic functions and co-occurrence network complexity. Kin discrimination facilitates the rapid construction of synthetic communities. The positive effects of SC 4 on larval weight gain resulted from the functional redundancy and complementarity among the strains. Furthermore, SC 4 may enhance larval growth by inducing shifts in the bacterial composition of the larval gut and pigeon manure. This elucidated how the SC promoted larval growth by regulating bacterial composition and provided theoretical guidance for the construction of SCs.

Unzipped chromosome-level genomes reveal allopolyploid nematode origin pattern as unreduced gamete hybridization.

The formation and consequences of polyploidization in animals with clonal reproduction remain largely unknown. Clade I root-knot nematodes (RKNs), characterized by parthenogenesis and allopolyploidy, show a widespread geographical distribution and extensive agricultural destruction. Here, we generated 4 unzipped polyploid RKN genomes and identified a putative novel alternative telomeric element. Then we reconstructed 4 chromosome-level assemblies and resolved their genome structures as AAB for triploid and AABB for tetraploid. The phylogeny of subgenomes revealed polyploid RKN origin patterns as hybridization between haploid and unreduced gametes. We also observed extensive chromosomal fusions and homologous gene expression decrease after polyploidization, which might offset the disadvantages of clonal reproduction and increase fitness in polyploid RKNs. Our results reveal a rare pathway of polyploidization in parthenogenic polyploid animals and provide a large number of high-precision genetic resources that could be used for RKN prevention and control.

Aminopeptidase MNP-1 triggers intestine protease production by activating daf-16 nuclear location to degrade pore-forming toxins in Caenorhabditis elegans.

Pore-forming toxins (PFTs) are effective tools for pathogens infection. By disrupting epithelial barriers and killing immune cells, PFTs promotes the colonization and reproduction of pathogenic microorganisms in their host. In turn, the host triggers defense responses, such as endocytosis, exocytosis, or autophagy. Bacillus thuringiensis (Bt) bacteria produce PFT, known as crystal proteins (Cry) which damage the intestinal cells of insects or nematodes, eventually killing them. In insects, aminopeptidase N (APN) has been shown to act as an important receptor for Cry toxins. Here, using the nematode Caenorhabditis elegans as model, an extensive screening of APN gene family was performed to analyze the potential role of these proteins in the mode of action of Cry5Ba against the nematode. We found that one APN, MNP-1, participate in the toxin defense response, since the mnp-1(ok2434) mutant showed a Cry5Ba hypersensitive phenotype. Gene expression analysis in mnp-1(ok2434) mutant revealed the involvement of two protease genes, F19C6.4 and R03G8.6, that participate in Cry5Ba degradation. Finally, analysis of the transduction pathway involved in F19C6.4 and R03G8.6 expression revealed that upon Cry5Ba exposure, the worms up regulated both protease genes through the activation of the FOXO transcription factor DAF-16, which was translocated into the nucleus. The nuclear location of DAF-16 was found to be dependent on mnp-1 under Cry5Ba treatment. Our work provides evidence of new host responses against PFTs produced by an enteric pathogenic bacterium, resulting in activation of host intestinal proteases that degrade the PFT in the intestine.

Isolated and identified pathogenic bacteria from black soldier fly larvae with "soft rot" reared in mass production facilities and its incidence characteristics.

The black soldier fly larvae (BSFL) can transform organic waste into high-end proteins, lipids, chitin, biodiesel, and melanin at an industrial scale. But scaling up of its production capacity has also posed health risks to the insect itself. In this investigation, larval "soft rot" which is occurring in mass production facilities that cause larval developmental inhibition and a certain degree of death was reported. Responsible pathogen GX6 was isolated from BSFL with "soft rot" and identified to be Paenibacillus thiaminolyticus. No obvious impact on larval growth was observed when treated with GX6 spores, whereas mortality of 6-day-old BSFL increased up to 29.33% ± 2.05% when GX6 vegetative cells (1 × 106 cfu/g) were inoculated into the medium. Moreover, higher temperature further enhanced the BSFL mortality and suppressed larval development, but increasing substrate moisture showed the opposite effect. The middle intestine of infected larvae became swollen and transparent after dissection and examination. Transmission electron microscopy (TEM) observation indicated that GX6 had destroyed the peritrophic matrix and intestinal microvilli and damaged epithelial cells of larval gut. Furthermore, 16S rRNA gene sequencing analysis of intestinal samples revealed that gut microflora composition was significantly altered by GX6 infection as well. It can be noticed that Dysgonomonas, Morganella, Myroides, and Providencia bacteria became more numerous in the intestines of GX6-infected BSFL as compared to controls. This study will lay foundations for efficient control of "soft rot" and promote healthy development of the BSFL industry to contribute to organic waste management and circular economy.

Characteristics and complete genome analysis of the novel virulent phage Bfsp1 infecting Cytobacillus firmus.

We isolated, identified, and characterised Bfsp1, a novel virulent phage of Cytobacillus firmus. Morphologically, Bfsp1 is similar to phi29-like phages. The linear, double-stranded DNA genome of Bfsp1 is 22,320 bp in length, has a GC content of 36.06%, and has 10-bp inverted terminal repeats. The genome contains 33 open reading frames, and functions of 15 of them were predicted. Comparative genome analysis showed that Bfsp1 is distinct from other known phages, and this was confirmed by phylogenetic analysis. Morphological, genomic, and phylogenetic data indicated that Bfsp1 is a novel member of the family Salasmaviridae.

Multiple Receptors Contribute to the Attractive Response of Caenorhabditis elegans to Pathogenic Bacteria.

Nematodes feed mainly on bacteria and sense volatile signals through their chemosensory system to distinguish food from pathogens. Although nematodes recognizing bacteria by volatile metabolites are ubiquitous, little is known of the associated molecular mechanism. Here, we show that the antinematode bacterium Paenibacillus polymyxa KM2501-1 exhibits an attractive effect on Caenorhabditis elegans via volatile metabolites, of which furfural acetone (FAc) acts as a broad-spectrum nematode attractant. We show that the attractive response toward FAc requires both the G-protein-coupled receptors STR-2 in AWC neurons and SRA-13 in AWA and AWC neurons. In the downstream olfactory signaling cascades, both the transient receptor potential vanilloid channel and the cyclic nucleotide-gated channel are necessary for FAc sensation. These results indicate that multiple receptors and subsequent signaling cascades contribute to the attractive response of C. elegans to FAc, and FAc is the first reported ligand of SRA-13. Our current work discovers that P. polymyxa KM2501-1 exhibits an attractive effect on nematodes by secreting volatile metabolites, especially FAc and 2-heptanone, broadening our understanding of the interactions between bacterial pathogens and nematodes. IMPORTANCE Nematodes feed on nontoxic bacteria as a food resource and avoid toxic bacteria; they distinguish them through their volatile metabolites. However, the mechanism of how nematodes recognize bacteria by volatile metabolites is not fully understood. Here, the antinematode bacterium Paenibacillus polymyxa KM2501-1 is found to exhibit an attractive effect on Caenorhabditis elegans via volatile metabolites, including FAc. We further reveal that the attractive response of C. elegans toward FAc requires multiple G-protein-coupled receptors and downstream olfactory signaling cascades in AWA and AWC neurons. This study highlights the important role of volatile metabolites in the interaction between nematodes and bacteria and confirms that multiple G-protein-coupled receptors on different olfactory neurons of C. elegans can jointly sense bacterial volatile signals.

Three novel leaderless bacteriocins have antimicrobial activity against gram-positive bacteria to serve as promising food biopreservative.

BACKGROUND: Due to the detrimental effects of chemical preservatives, there has been an increasing demand for safer, healthier and natural bio-preservatives. Bacteriocins have attracted increasing interest because of their potential as natural bio-preservatives. RESULTS: We screened a large number of Bacillus thuringiensis strains and isolated one strain (B. thuringiensis P86) with antimicrobial activity against several foodborne pathogens. Three novel leaderless bacteriocins, including thucin A1, thucin A2 and thucin A3, were purified and identified from the culture supernatant of B. thuringiensis P86, whose molecular masses were 5552.02, 5578.07 and 5609.06 Da, respectively. Thucin A1 was then selected as a representative to be tested, and it exhibited potent inhibitory activity against all tested gram-positive bacteria. More importantly, thucin A1 showed stronger antimicrobial activity than nisin A against two important foodborne pathogens Bacillus cereus and Listeria monocytogenes. In addition, thucin A1 exhibited strong acid-base adaptability (pH 2-11), high endurance to heat, good stability to trypsin and pepsin, no hemolysis activity and cytotoxicity, and could effectively inhibit or eliminate Bacillus cereus and Listeria monocytogenes in skim milk. CONCLUSIONS: Our findings indicate that these novel leaderless bacteriocins are potentially promising food biopreservatives.

Isolation and Characterization of Novel Lytic Bacteriophage vB_RsoP_BMB50 infecting Ralstonia solanacearum.

Ralstonia solanacearum is a soil-borne phytopathogen, and it can cause bacterial wilt disease in a variety of key crops around the world, thus resulting in enormous financial losses. However, there is a lack of effective, green, and safe prevention and control measures against increasingly devastating bacterial wilt disease. Bacteriophages (phages) are considered as potential biocontrol agents against bacterial wilt disease. Although many phages infecting R. solanacearum have been isolated, so far, these Ralstonia phages are still insufficient to deal with the diversity of the bacteria of R. solanacearum. In this study, a novel lytic bacteriophage vB_RsoP_BMB50 infecting multiple R. solanacearum was isolated from tomato fields in Dalian, China. Transmission electron microscopy and genomics analysis indicated that vB_RsoP_BMB50 belonged to the subfamily Okabevirinae, Autographiviridae family, and order Caudovirales, and it comprised a double-stranded DNA with a full length of 43,665 bp and a mean G+C content of 61.79%, containing 53 open reading frames (ORFs). This novel phage exhibited a large burst size, high temperature stability (4-50 °C), and strong pH tolerance (pH 5-10). Comparative analyses and phylogenetic analyses revealed that vB_RsoP_BMB50 represented a novel Ralstonia phage genus since it exhibited a low sequence similarity to other phages in the GenBank database. Due to its broad lytic spectrum, high thermal stability, and strong pH tolerance, vB_RsoP_BMB50 is considered as an effective candidate biocontrol agent against bacterial wilt disease caused by R. solanacearum.

C. elegans monitor energy status via the AMPK pathway to trigger innate immune responses against bacterial pathogens.

Pathogen recognition and the triggering of host innate immune system are critical to understanding pathogen-host interaction. Cellular surveillance systems have been identified as an important strategy for the identification of microbial infection. In the present study, using Bacillus thuringiensis-Caenorhabditis elegans as a model, we found an approach for surveillance systems to sense pathogens. We report that Bacillus thuringiensis Cry5Ba, a typical pore-forming toxin, caused mitochondrial damage and energy imbalance by triggering potassium ion leakage, instead of directly targeting mitochondria. Interestingly, we find C. elegans can monitor intracellular energy status to trigger innate immune responses via AMP-activated protein kinase (AMPK), secreting multiple effectors to defend against pathogenic attacks. Our study indicates that the imbalance of energy status is a prevalent side effect of pathogen infection. Furthermore, the AMPK-dependent surveillance system may serve as a practicable strategy for the host to recognize and defense against pathogens.

BtToxin_Digger: a comprehensive and high-throughput pipeline for mining toxin protein genes from Bacillus thuringiensis.

SUMMARY: Bacillus thuringiensis (Bt) has been used as the most successful microbial pesticide for decades. Its toxin genes are used for the development of genetically modified crops against pests. We previously developed a web-based insecticidal gene mining tool BtToxin_scanner. It has been frequently used by many researchers worldwide. However, it can only handle the genome one by one online. To facilitate efficiently mining toxin genes from large-scale sequence data, we re-designed this tool with a new workflow and the novel bacterial pesticidal protein database. Here, we present BtToxin_Digger, a comprehensive and high-throughput Bt toxin mining tool. It can be used to predict Bt toxin genes from thousands of raw genome and metagenome data, and provides accurate results for downstream analysis and experiment testing. Moreover, it can also be used to mine other targeting genes from large-scale genome and metagenome data with the replacement of the database. AVAILABILITY AND IMPLEMENTATION: The BtToxin_Digger codes and web services are freely available at https://github.com/BMBGenomics/BtToxin_Digger and https://bcam.hzau.edu.cn/BtToxin_Digger, respectively. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Isolation, Characterization, and Genome Sequence Analysis of a Novel Lytic Phage, Xoo-sp15 Infecting Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (X. oryzae) is a bacterial pathovar of rice diseases all over the world. Owing to emerging antibacterial resistance, phage therapies have gained significant attention to treat various bacterial infections. Nevertheless, comprehensive research is needed for their use as a safe biocontrol agent. In this study, isolation and characterization of a novel phage Xoo-sp15, that infects X. oryzae was ascertained through experimental and bioinformatics analyses to determine its virulent potency and reliability. High throughput sequencing demonstrated that Xoo-sp15 has a dsDNA genome with a total size of 157,091 bp and 39.9% GC content lower than its host (63.6%). Morphological and phylogenetic analyses characterized it as a new member of the Bastille-like group within the family Herelleviridae. In silico analysis revealed that it contains 229 open reading frames and 16 tRNAs. Additionally, this novel phage does not contain any resistant determinants and can infect nine X. oryzae strains. Therefore, Xoo-sp15 has the potential to serve as a novel candidate for phage therapy.

Genomic analysis of bacteriophage Xoo-sp13 infecting Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae is a bacterial pathogen that gives rise to diseases in rice all over the world. A bacteriophage infecting this bacterium was isolated from rice fields in China. Here, we report the complete genome sequence of this phage, which has a linear dsDNA genome of 309,023 bp and a G + C content of 42.43%. It contains 401 open reading frames and encodes 28 tRNAs. It belongs to the family Myoviridae and has a broad host range, making it a possible candidate for phage therapy.

Sequence Analysis of a Jumbo Bacteriophage, Xoo-sp14, That Infects Xanthomonas oryzae pv. oryzae.

A jumbo bacteriophage, Xoo-sp14, infecting Xanthomonas oryzae pv. oryzae was isolated from rice fields in China. Here, we report the complete genome sequence of this phage, revealing that it had a linear double-stranded DNA (dsDNA) molecule 232,104 bp long, with a G+C content of 58%. It has 251 annotated protein-coding sequences.

Multi-copy alpha-amylase genes are crucial for Ditylenchus destructor to parasitize the plant host.

Ditylenchus destructor is a migratory plant-parasitic nematode that causes huge damage to global root and tuber production annually. The main plant hosts of D. destructor contain plenty of starch, which makes the parasitic environment of D. destructor to be different from those of most other plant-parasitic nematodes. It is speculated that D. destructor may harbor some unique pathogenesis-related genes to parasitize the starch-rich hosts. Herein, we focused on the multi-copy alpha-amylase genes in D. destructor, which encode a key starch-catalyzing enzyme. Our previously published D. destructor genome showed that it has three alpha-amylase encoding genes, Dd_02440, Dd_11154, and Dd_13225. Comparative analysis of alpha-amylases from different species demonstrated that the other plant-parasitic nematodes, even Ditylenchus dipsaci in the same genus, harbor only one or no alpha-amylase gene, and the three genes from D. destructor were closely clustered in the phylogenetic tree, indicating that there was a unique expansion of the alpha-amylase gene in D. destructor. The enzymatic activity of the three alpha-amylase proteins was verified by an enzyme assay. Quantitative real-time PCR assay showed that the expression of the three alpha-amylase genes in the post-hatching stage of D. destructor was found to be significantly higher than that in eggs. In the in situ hybridization assay, the expression of the genes was localized to the intestine, implying the association of these genes with nematode digestion. An infection assay in sweet potato demonstrated that RNA interference of any one alpha-amylase gene had no influence on the infectivity of D. destructor. Using the multi-target dsRNA cocktail method, it was found that silencing of two of the three genes inhibited nematode infection, and the infectivity of worms treated with three dsRNA simultaneously changed the most, which decreased by 76.6%. Thus, the multi-copy alpha-amylase genes in D. destructor are compensatory and crucial for nematodes to parasitize the plant host.

In Silico Analysis Highlights the Diversity and Novelty of Circular Bacteriocins in Sequenced Microbial Genomes.

Consumer demand for "fresh food" with no chemical preservatives has prompted researchers to pay more attention to natural antimicrobial peptides such as bacteriocins. Nisin is currently the most widely used food biopreservative among the bacteriocins; however, its applications are restricted due to its low stability at neutral and alkaline pH values. Circular bacteriocins have potent antimicrobial activity against foodborne pathogens, show exceptional stability, and have great potential to be developed as biopreservatives. Here, we take advantage of the precursor peptides of 15 reported circular bacteriocins to devise an in silico approach to identify potential circular bacteriocins in sequenced microbial genomes. A total of nearly 7,000 putative precursor peptides were identified from 86 species of bacteria and further classified into 28 groups based on their amino acid similarity. Among the groups, 19 showed low similarity (less than 50%) to any known precursor peptide of circular bacteriocins. One novel circular bacteriocin in group 11, cerecyclin, showed the highest identity (34%) to the known circular bacteriocin enterocin NKR-5-3B and was selected for verification. Cerecyclin showed antimicrobial activity against several Gram-positive bacteria, inhibited the outgrowth of Bacillus cereus spores, and did not exhibit hemolysis activity. Moreover, it showed 4-fold- to 8-fold-higher antimicrobial activity against B. cereus and Listeria monocytogenes than nisin A. Cerecyclin also had increased stability compared to nisin A under neutral or alkaline conditions. This work not only identified a promising food biopreservative but also provided a rich source for novel circular bacteriocins.IMPORTANCE Circular bacteriocins are promising biopreservatives, and it is important to identify more novel circular bacteriocins to enhance the current arsenal of antimicrobials. In this study, we used an in silico approach to identify a large number of novel circular bacteriocins and classified these bacteriocins into 28 groups rather than the 2 groups that were described in previous studies. Nineteen groups were novel and had low similarity (less than 50%) to any known precursor peptides of circular bacteriocins; this finding greatly expands the awareness of the novelty and diversity of circular bacteriocins. A novel circular bacteriocin which we named cerecyclin was identified in the B. cereus group; this circular bacteriocin had great antimicrobial activity against some foodborne pathogens and showed extreme stability. This study not only identified a promising food biopreservative but also provided a rich source for the identification of novel circular bacteriocins and the development of new biopreservatives.

The Caenorhabditis elegans CUB-like-domain containing protein RBT-1 functions as a receptor for Bacillus thuringiensis Cry6Aa toxin.

Plant-parasitic nematodes cause huge agricultural economic losses. Two major families of Bacillus thuringiensis crystal proteins, Cry5 and Cry6, show nematicidal activity. Previous work showed that binding to midgut receptors is a limiting step in Cry toxin mode of action. In the case of Cry5Ba, certain Caenorhabditis elegans glycolipids were identified as receptors of this toxin. However, the receptors for Cry6 toxin remain unknown. In this study, the C. elegans CUB-like-domain containing protein RBT-1, released by phosphatidylinositol-specific phospholipase C (PI-PLC), was identified as a Cry6Aa binding protein by affinity chromatography. RBT-1 contained a predicted glycosylphosphatidylinositol (GPI) anchor site and was shown to locate in lipid rafts in the surface of the midgut cells. Western ligand blot assays and ELISA binding analysis confirmed the binding interaction between Cry6Aa and RBT-1 showing high affinity and specificity. In addition, the mutation of rbt-1 gene decreased the susceptibility of C. elegans to Cry6Aa but not that of Cry5Ba. Furthermore, RBT-1 mediated the uptake of Cry6Aa into C. elegans gut cells, and was shown to be involved in triggering pore-formation activity, indicating that RBT-1 is required for the interaction of Cry6Aa with the nematode midgut cells. These results support that RBT-1 is a functional receptor for Cry6Aa.

The CRISPR-Cas systems were selectively inactivated during evolution of Bacillus cereus group for adaptation to diverse environments.

CRISPR-Cas systems are considered as barriers to horizontal gene transfer (HGT). However, the influence of such systems on HGT within species is unclear. Also, little is known about the impact of CRISPR-Cas systems on bacterial evolution at the population level. Here, using Bacillus cereus sensu lato as model, we investigate the interplay between CRISPR-Cas systems and HGT at the population scale. We found that only a small fraction of the strains have CRISPR-Cas systems (13.9% of 1871), and most of such systems are defective based on their gene content analysis. Comparative genomic analysis revealed that the CRISPR-Cas systems are barriers to HGT within this group, since strains harboring active systems contain less mobile genetic elements (MGEs), have lower fraction of unique genes and also display limited environmental distributions than strains without active CRISPR-Cas systems. The introduction of a functional CRISPR-Cas system into a strain lacking the system resulted in reduced adaptability to various stresses and decreased pathogenicity of the transformant strain, indicating that B. cereus group strains could benefit from inactivating such systems. Our work provides a large-scale case to support that the CRISPR-Cas systems are barriers to HGT within species, and that in the B. cereus group the inactivation of CRISPR-Cas systems correlated with acquisition of MGEs that could result in better adaptation to diverse environments.

Endophyte Bacillus subtilis evade plant defense by producing lantibiotic subtilomycin to mask self-produced flagellin.

Microbes can enter into healthy plants as endophytes and confer beneficial functions. The entry of commensal microbes into plants involves penetrating plant defense. Most mechanisms about overcoming plant defense are focused on adapted pathogens, while the mechanism involved in beneficial endophyte evades plant defense to achieve harmonious commensalism is unclear. Here, we discover a mechanism that an endophyte bacterium Bacillus subtilis BSn5 reduce to stimulate the plant defensive response by producing lantibiotic subtilomycin to bind self-produced flagellin. Subtilomycin bind with flagellin and affect flg22-induced plant defense, by which means promotes the endophytic colonization in A. thaliana. Subtilomycin also promotes the BSn5 colonization in a distinct plant, Amorphophallus konjac, where the BSn5 was isolated. Our investigation shows more independent subtilomycin/-like producers are isolated from distinct plants. Our work unveils a common strategy that is used for bacterial endophytic colonization.

Bacillus thuringiensis targets the host intestinal epithelial junctions for successful infection of Caenorhabditis elegans.

Pathogenic bacteria use different strategies to infect their hosts, including the simultaneous production of pore forming toxins and several virulence factors that may synergize their pathogenic effects. However, how the pathogenic bacteria are able to break out the host intestinal barrier is poorly understood. The infectious cycle of Bacillus thuringiensis (Bt) bacterium in Caenorhabditis elegans is a powerful model system to study the early stages of the infection process. Bt produces Cry pore-forming toxins during the sporulation phase that are key virulence factors involved in its pathogenesis. In this study, we show that Bt disrupts the intestinal epithelial junctions of C. elegans at early stages of infection allowing Bt bacterium to complete its life cycle in the worm. We further confirmed that the vegetative Bt cells trigger a quorum sensing response that is activated by PlcR regulator, resulting in production of different virulence factors, such as the metalloproteinases ColB and Bmp1, that besides Cry toxins are necessary to disrupt the nematode epithelial junctions causing efficient bacterial host infection and death of the nematode. Our work provides new insights into the pathogenesis of Bt and highlights the importance of breaking down host epithelial junctions for a successful infection. A similar mechanism could be used by other pathogen-host interactions since epithelial junctions are conserved structures from insects to mammals.