The molecular mechanism for carbon catabolite repression of the chitin response in Vibrio cholerae.

Vibrio cholerae is a facultative pathogen that primarily occupies marine environments. In this niche, V. cholerae commonly interacts with the chitinous shells of crustacean zooplankton. As a chitinolytic microbe, V. cholerae degrades insoluble chitin into soluble oligosaccharides. Chitin oligosaccharides serve as both a nutrient source and an environmental cue that induces a strong transcriptional response in V. cholerae. Namely, these oligosaccharides induce the chitin sensor, ChiS, to activate the genes required for chitin utilization and horizontal gene transfer by natural transformation. Thus, interactions with chitin impact the survival of V. cholerae in marine environments. Chitin is a complex carbon source for V. cholerae to degrade and consume, and the presence of more energetically favorable carbon sources can inhibit chitin utilization. This phenomenon, known as carbon catabolite repression (CCR), is mediated by the glucose-specific Enzyme IIA (EIIAGlc) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). In the presence of glucose, EIIAGlc becomes dephosphorylated, which inhibits ChiS transcriptional activity by an unknown mechanism. Here, we show that dephosphorylated EIIAGlc interacts with ChiS. We also isolate ChiS suppressor mutants that evade EIIAGlc-dependent repression and demonstrate that these alleles no longer interact with EIIAGlc. These findings suggest that EIIAGlc must interact with ChiS to exert its repressive effect. Importantly, the ChiS suppressor mutations we isolated also relieve repression of chitin utilization and natural transformation by EIIAGlc, suggesting that CCR of these behaviors is primarily regulated through ChiS. Together, our results reveal how nutrient conditions impact the fitness of an important human pathogen in its environmental reservoir.

Coordination of two regulators SscA and VosA in Aspergillus nidulans conidia.

Airborne fungal spores are a major cause of fungal diseases in humans, animals, and plants as well as contamination of foods. Previous studies found a variety of regulators including VosA, VelB, WetA, and SscA for sporogenesis and the long-term viability in Aspergillus nidulans. To gain a mechanistic understanding of the complex regulatory mechanisms in asexual spores, here, we focused on the relationship between VosA and SscA using comparative transcriptomic analysis and phenotypic studies. The ΔsscA ΔvosA double-mutant conidia have lower spore viability and stress tolerance compared to the ΔsscA or ΔvosA single mutant conidia. Deletion of sscA or vosA affects chitin levels and mRNA levels of chitin biosynthetic genes in conidia. In addition, SscA and VosA are required for the dormant state of conidia and conidial germination by modulating the mRNA levels of the cytoskeleton and development-associated genes. Overall, these results suggest that SscA and VosA play interdependent roles in governing spore maturation, dormancy, and germination in A. nidulans.

Characterization of a lytic polysaccharide monooxygenase from Aspergillus fumigatus shows functional variation among family AA11 fungal LPMOs.

The discovery of oxidative cleavage of recalcitrant polysaccharides by lytic polysaccharide monooxygenases (LPMOs) has affected the study and industrial application of enzymatic biomass processing. Despite being widespread in fungi, LPMOs belonging to the auxiliary activity (AA) family AA11 have been understudied. While these LPMOs are considered chitin active, some family members have little or no activity toward chitin, and the only available crystal structure of an AA11 LPMO lacks features found in bacterial chitin-active AA10 LPMOs. Here, we report structural and functional characteristics of a single-domain AA11 LPMO from Aspergillus fumigatus, AfAA11A. The crystal structure shows a substrate-binding surface with features resembling those of known chitin-active LPMOs. Indeed, despite the absence of a carbohydrate-binding module, AfAA11A has considerable affinity for α-chitin and, more so, ß-chitin. AfAA11A is active toward both these chitin allomorphs and enhances chitin degradation by an endoacting chitinase, in particular for α-chitin. The catalytic activity of AfAA11A on chitin increases when supplying reactions with hydrogen peroxide, showing that, like LPMOs from other families, AfAA11A has peroxygenase activity. These results show that, in stark contrast to the previously characterized AfAA11B from the same organism, AfAA11A likely plays a role in fungal chitin turnover. Thus, members of the hitherto rather enigmatic family of AA11 LPMOs show considerable structural and functional differences and may have multiple roles in fungal physiology.

A secreted LysM effector protects fungal hyphae through chitin-dependent homodimer polymerization.

Plants trigger immune responses upon recognition of fungal cell wall chitin, followed by the release of various antimicrobials, including chitinase enzymes that hydrolyze chitin. In turn, many fungal pathogens secrete LysM effectors that prevent chitin recognition by the host through scavenging of chitin oligomers. We previously showed that intrachain LysM dimerization of the Cladosporium fulvum effector Ecp6 confers an ultrahigh-affinity binding groove that competitively sequesters chitin oligomers from host immune receptors. Additionally, particular LysM effectors are found to protect fungal hyphae against chitinase hydrolysis during host colonization. However, the molecular basis for the protection of fungal cell walls against hydrolysis remained unclear. Here, we determined a crystal structure of the single LysM domain-containing effector Mg1LysM of the wheat pathogen Zymoseptoria tritici and reveal that Mg1LysM is involved in the formation of two kinds of dimers; a chitin-dependent dimer as well as a chitin-independent homodimer. In this manner, Mg1LysM gains the capacity to form a supramolecular structure by chitin-induced oligomerization of chitin-independent Mg1LysM homodimers, a property that confers protection to fungal cell walls against host chitinases.

Combinatorial pathway engineering of Bacillus subtilis for production of structurally defined and homogeneous chitooligosaccharides.

Chitooligosaccharides (COSs) have a widespread range of biological functions and an incredible potential for various pharmaceutical and agricultural applications. Although several physical, chemical, and biological techniques have been reported for COSs production, it is still a challenge to obtain structurally defined COSs with defined polymerization (DP) and acetylation patterns, which hampers the specific characterization and application of COSs. Herein, we achieved the de novo production of structurally defined COSs using combinatorial pathway engineering in Bacillus subtilis. Specifically, the COSs synthase NodC from Azorhizobium caulinodans was overexpressed in B. subtilis, leading to 30 ± 0.86 mg/L of chitin oligosaccharides (CTOSs), the homo-oligomers of N-acetylglucosamine (GlcNAc) with a well-defined DP lower than 6. Then introduction of a GlcNAc synthesis module to promote the supply of the sugar acceptor GlcNAc, reduced CTOSs production, which suggested that the activity of COSs synthase NodC and the supply of sugar donor UDP-GlcNAc may be the limiting steps for CTOSs synthesis. Therefore, 6 exogenous COSs synthase candidates were examined, and the nodCM from Mesorhizobium loti yielded the highest CTOSs titer of 560 ± 16 mg/L. Finally, both the de novo pathway and the salvage pathway of UDP-GlcNAc were engineered to further promote the biosynthesis of CTOSs. The titer of CTOSs in 3-L fed-batch bioreactor reached 4.82 ± 0.11 g/L (85.6% CTOS5, 7.5% CTOS4, 5.3% CTOS3 and 1.6% CTOS2), which was the highest ever reported. This is the first report proving the feasibility of the de novo production of structurally defined CTOSs by synthetic biology, and provides a good starting point for further engineering to achieve the commercial production.

Molecular cloning, heterologous expression, and in silico sequence analysis of Enterobacter GH19 class I chitinase (chiRAM gene).

BACKGROUND: Using in silico sequence analyses, the present study aims to clone and express the gene-encoding sequence of a GH19 chitinase from Enterobacter sp. in Escherichia coli. METHODS AND RESULTS: The putative open reading frame of a GH19 chitinase from Enterobacter sp. strain EGY1 was cloned and expressed into pGEM®-T and pET-28a (+) vectors, respectively using a degenerate primer. The isolated nucleotide sequence (1821 bp, GenBank accession no.: MK533791.2) was translated to a chiRAM protein (606 amino acids, UniProt accession no.: A0A4D6J2L9). The in silico protein sequence analysis of chiRAM revealed a class I GH19 chitinase: an N-terminus signal peptide (Met1-Ala23), a catalytic domain (Val83-Glu347 and the catalytic triad Glu149, Glu171, and Ser218), a proline-rich hinge region (Pro414 -Pro450), a polycystic kidney disease protein motif (Gly 465-Ser 533), a C-terminus chitin-binding domain (Ala553- Glu593), and conserved class I motifs (NYNY and AQETGG). A three-dimensional model was constructed by LOMETS MODELLER of PDB template: 2dkvA (class I chitinase of Oryza sativa L. japonica). Recombinant chiRAM was overexpressed as inclusion bodies (IBs) (~ 72 kDa; SDS-PAGE) in 1.0 mM IPTG induced E. coli BL21 (DE3) Rosetta strain at room temperature 18 h after induction. Optimized expression yielded active chiRAM with 1.974 ± 0.0002 U/mL, on shrimp colloidal chitin (SCC), in induced E. coli BL21 (DE3) Rosetta cells growing in SB medium. LC-MS/MS identified a band of 72 kDa in the soluble fraction with a 52.3% coverage sequence exclusive to the GH19 chitinase of Enterobacter cloacae (WP_063869339.1). CONCLUSIONS: Although chiRAM of Enterobacter sp. was successfully cloned and expressed in E. coli with appreciable chitinase activity, future studies should focus on minimizing IBs to facilitate chiRAM purification and characterization.

Conserved function of the matriptase-prostasin proteolytic cascade during epithelial morphogenesis.

Extracellular matrix (ECM) assembly and remodelling is critical during development and organ morphogenesis. Dysregulation of ECM is implicated in many pathogenic conditions, including cancer. The type II transmembrane serine protease matriptase and the serine protease prostasin are key factors in a proteolytic cascade that regulates epithelial ECM differentiation during development in vertebrates. Here, we show by rescue experiments that the Drosophila proteases Notopleural (Np) and Tracheal-prostasin (Tpr) are functional homologues of matriptase and prostasin, respectively. Np mediates morphogenesis and remodelling of apical ECM during tracheal system development and is essential for maintenance of the transepithelial barrier function. Both Np and Tpr degrade the zona pellucida-domain (ZP-domain) protein Dumpy, a component of the transient tracheal apical ECM. Furthermore, we demonstrate that Tpr zymogen and the ZP domain of the ECM protein Piopio are cleaved by Np and matriptase in vitro. Our data indicate that the evolutionarily conserved ZP domain, present in many ECM proteins of vertebrates and invertebrates, is a novel target of the conserved matriptase-prostasin proteolytic cascade.

Single-channel properties, sugar specificity, and role of chitoporin in adaptive survival of Vibrio cholerae type strain O1.

Vibrio cholerae is a Gram-negative, facultative anaerobic bacterial species that causes serious disease and can grow on various carbon sources, including chitin polysaccharides. In saltwater, its attachment to chitin surfaces not only serves as the initial step of nutrient recruitment but is also a crucial mechanism underlying cholera epidemics. In this study, we report the first characterization of a chitooligosaccharide-specific chitoporin, VcChiP, from the cell envelope of the V. cholerae type strain O1. We modeled the structure of VcChiP, revealing a trimeric cylinder that forms single channels in phospholipid bilayers. The membrane-reconstituted VcChiP channel was highly dynamic and voltage induced. Substate openings O1', O2', and O3', between the fully open states O1, O2, and O3, were polarity selective, with nonohmic conductance profiles. Results of liposome-swelling assays suggested that VcChiP can transport monosaccharides, as well as chitooligosaccharides, but not other oligosaccharides. Of note, an outer-membrane porin (omp)-deficient strain of Escherichia coli expressing heterologous VcChiP could grow on M9 minimal medium supplemented with small chitooligosaccharides. These results support a crucial role of chitoporin in the adaptive survival of bacteria on chitinous nutrients. Our findings also suggest a promising means of vaccine development based on surface-exposed outer-membrane proteins and the design of novel anticholera agents based on chitooligosaccharide-mimicking analogs.

Reduction of OsMPK6 activity by a R89K mutation induces cell death and bacterial blight resistance in rice.

KEY MESSAGE: The R89 is essential for the kinase activity of OsMPK6 which negatively regulates cell death and defense response in rice. Mitogen-activated protein kinase cascade plays critical roles in various vital activities, including the plant immune response, but the mechanisms remain elusive. Here, we identified and characterized a rice lesion mimic mutant osmpk6 which displayed hypersensitive response-like lesions in company with cell death and hydrogen peroxide hyperaccumulation. Map-based cloning and complementation demonstrated that a G702A single-base substitution in the second exon of OsMPK6 led to the lesion mimic phenotype of the osmpk6 mutant. OsMPK6 encodes a cytoplasm and nucleus-targeted mitogen-activated protein kinase and is expressed in the various organs. Compared with wild type, the osmpk6 mutant exhibited high resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), likely due to the increased ROS production induced by flg22 and chitin and up-regulated expression of genes involved in pathogenesis, as well as activation of SA and JA signaling pathways after inoculation. By contrast, the OsMPK6-overexpression line (OE-1) was found to be susceptible to the bacterial pathogens, indicating that OsMPK6 negatively regulated Xoo resistance. Furthermore, the G702A single-base substitution caused a R89K mutation at both polypeptide substrate-binding site and active site of OsMPK6, and kinase activity assay revealed that the R89K mutation led to reduction of OsMPK6 activity, suggesting that the R89 is essential for the function of OsMPK6. Our findings provide insight into a vital role of the R89 of OsMPK6 in regulating cell death and defense response in rice.

Full-Length Transcriptome of Thalassiosira weissflogii as a Reference Resource and Mining of Chitin-Related Genes.

ß-Chitin produced by diatoms is expected to have significant economic and ecological value due to its structure, which consists of parallel chains of chitin, its properties and the high abundance of diatoms. Nevertheless, few studies have functionally characterised chitin-related genes in diatoms owing to the lack of omics-based information. In this study, we first compared the chitin content of three representative Thalassiosira species. Cell wall glycosidic linkage analysis and chitin/chitosan staining assays showed that Thalassiosira weissflogii was an appropriate candidate chitin producer. A full-length (FL) transcriptome of T. weissflogii was obtained via PacBio sequencing. In total, the FL transcriptome comprised 23,362 annotated unigenes, 710 long non-coding RNAs (lncRNAs), 363 transcription factors (TFs), 3113 alternative splicing (AS) events and 3295 simple sequence repeats (SSRs). More specifically, 234 genes related to chitin metabolism were identified and the complete biosynthetic pathways of chitin and chitosan were explored. The information presented here will facilitate T. weissflogii molecular research and the exploitation of ß-chitin-derived high-value enzymes and products.

Chitin Synthesis and Degradation in Crustaceans: A Genomic View and Application.

Chitin is among the most important components of the crustacean cuticular exoskeleton and intestinal peritrophic matrix. With the progress of genomics and sequencing technology, a large number of gene sequences related to chitin metabolism have been deposited in the GenBank database in recent years. Here, we summarized the genes and pathways associated with the biosynthesis and degradation of chitins in crustaceans based on genomic analyses. We found that chitin biosynthesis genes typically occur in single or two copies, whereas chitin degradation genes are all multiple copies. Moreover, the chitinase genes are significantly expanded in most crustacean genomes. The gene structure and expression pattern of these genes are similar to those of insects, albeit with some specific characteristics. Additionally, the potential applications of the chitin metabolism genes in molting regulation and immune defense, as well as industrial chitin degradation and production, are also summarized in this review.

Identification and RNAi-Based Functional Analysis of Four Chitin Deacetylase Genes in Sogatella furcifera (Hemiptera: Delphacidae).

Chitin deacetylases (CDAs) are chitin-degrading enzymes that play a key role in insect molting. In this study, we identified and characterized four full-length cDNAs of CDAs from Sogatella furcifera (Horváth). Developmental expression showed that SfCDA1 and SfCDA2 were expressed at all nymph developmental stages, SfCDA3 and SfCDA4 were mainly expressed in the third-instar to fifth-instar nymph stages, whereas tissue-specific analyses indicated that four CDA genes were mainly high expressed in the integument and head during the fifth-instar nymph. RNA interference (RNAi) results revealed that SfCDA1, SfCDA2, and SfCDA4 are associated with molting defect and high mortality with nymph-adult molting. Furthermore, transcripts of chitin synthase 1 variants (SfCHS1, SfCHS1a, and SfCHS1b) were significantly downregulated and causing significant changes in the expression levels of trehalases (TRE1 and TRE2) in the SfCDA1, SfCDA2, and SfCDA4 dsRNA treatment groups. By contrast, no significant phenotypic characteristics were observed after dsSfCDA3 injection. Taken together, our results suggest that SfCDA1, SfCDA2, and SfCDA4 play a vital role in nymph-adult transition, and these genes could regulate chitin biosynthesis expression levels.

A Tale of Two Transcriptomic Responses in Agricultural Pests via Host Defenses and Viral Replication.

Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) is a baculovirus that causes systemic infections in many arthropod pests. The specific molecular processes underlying the biocidal activity of AcMNPV on its insect hosts are largely unknown. We describe the transcriptional responses in two major pests, Spodoptera frugiperda (fall armyworm) and Trichoplusia ni (cabbage looper), to determine the host-pathogen responses during systemic infection, concurrently with the viral response to the host. We assembled species-specific transcriptomes of the hemolymph to identify host transcriptional responses during systemic infection and assessed the viral transcript abundance in infected hemolymph from both species. We found transcriptional suppression of chitin metabolism and tracheal development in infected hosts. Synergistic transcriptional support was observed to suggest suppression of immune responses and induction of oxidative stress indicating disease progression in the host. The entire AcMNPV core genome was expressed in the infected host hemolymph with a proportional high abundance detected for viral transcripts associated with replication, structure, and movement. Interestingly, several of the host genes that were targeted by AcMNPV as revealed by our study are also targets of chemical insecticides currently used commercially to control arthropod pests. Our results reveal an extensive overlap between biological processes represented by transcriptional responses in both hosts, as well as convergence on highly abundant viral genes expressed in the two hosts, providing an overview of the host-pathogen transcriptomic landscape during systemic infection.

From sporadic single genes to a broader transcriptomic approach: Insights into the formation of the biomineralized exoskeleton in decapod crustaceans.

One fundamental character common to pancrustaceans (Crustacea and Hexapoda) is a mineralized rigid exoskeleton whose principal organic components are chitin and proteins. In contrast to traditional research in the field that has been devoted to the structural and physicochemical aspects of biomineralization, the present study explores transcriptomic aspects of biomineralization as a first step towards adding a complementary molecular layer to this field. The rigidity of the exoskeleton in pancrustaceans dictates essential molt cycles enabling morphological changes and growth. Thus, formation and mineralization of the exoskeleton are concomitant to the timeline of the molt cycle. Skeletal proteinaceous toolkit elements have been discovered in previous studies using innovative molt-related binary gene expression patterns derived from transcriptomic libraries representing the major stages comprising the molt cycle of the decapod crustacean Cherax quadricarinatus. Here, we revisited some prominent exoskeleton-related structural proteins encoding and, using the above molt-related binary pattern methodology, enlarged the transcriptomic database of C. quadricarinatus. The latter was done by establishing a new transcriptomic library of the cuticle forming epithelium and molar tooth at four different molt stages (i.e., inter-molt, early pre-molt, late pre-molt and post-molt) and incorporating it to a previous transcriptome derived from the gastroliths and mandible. The wider multigenic approach facilitated by the newly expanded transcriptomic database not only revisited single genes of the molecular toolkit, but also provided both scattered and specific information that broaden the overview of proteins and gene clusters which are involved in the construction and biomineralization of the exoskeleton in decapod crustaceans.

Identification and Characterization of a Newly Isolated Chitinase-Producing Strain Bacillus licheniformis SSCL-10 for Chitin Degradation.

Chitinases or chitinolytic enzymes have different applications in the field of medicine, agriculture, and industry. The present study is aimed at developing an effective hyperchitinase-producing mutant strain of novel Bacillus licheniformis. A simple and rapid methodology was used for screening potential chitinolytic microbiota by chemical mutagenesis with ethylmethane sulfonate and irradiation with UV. There were 16 mutant strains exhibiting chitinase activity. Out of the chitinase-producing strains, the strain with maximum chitinase activity was selected, the protein was partially purified by SDS-PAGE, and the strain was identified as Bacillus licheniformis (SSCL-10) with the highest specific activity of 3.4 U/mL. The induced mutation model has been successfully implemented in the mutant EMS-13 (20.2 U/mL) that produces 5-6-fold higher yield of chitinase, whereas the mutant UV-11 (13.3 U/mL) has 3-4-fold greater chitinase activity compared to the wild strain. The partially purified chitinase has a molecular weight of 66 kDa. The wild strain (SSCL-10) was identified as Bacillus licheniformis using 16S rRNA sequence analysis. This study explores the potential applications of hyperchitinase-producing bacteria in recycling and processing chitin wastes from crustaceans and shrimp, thereby adding value to the crustacean industry.

miR-2703 regulates the chitin biosynthesis pathway by targeting chitin synthase 1a in Nilaparvata lugens.

The chitin biosynthesis pathway is an important physiology process in arthropods. However, few microRNAs (miRNAs) involved in the regulation of the chitin biosynthesis pathway in insects have been reported until now. In this study, four groups of samples that either upregulated or downregulated the chitin biosynthesis pathway were collected for deep sequencing, and a total of 15 unique mature miRNAs with significantly different expression levels were found, including 11 known miRNAs and four novel miRNAs. Subsequently, we showed that miR-2703 and its new target gene chitin synthase 1a are important for ecdysone-induced chitin biosynthesis in Nilaparvata lugens, a serious insect pest of rice. The nymphs showed an obvious moulting defect phenotype, lower survival rate and significantly reduced chitin content after miR-2703 feeding or injection. Furthermore, we found that the transcription level of miR-2703 was not repressed by 20-hydroxyecdysone signalling after Broad-Complex (BR-C) double-stranded RNA (dsRNA) injection compared with the repressed levels after green fluorescent protein dsRNA injection, suggesting that the involvement of miR-2703 in the 20-hydroxyecdysone pathway contributes to BR-C activity. miR-2703 regulates the chitin biosynthesis pathway by targeting chitin synthase 1a in response to 20-hydroxyecdysone signalling.

Aspergillus fumigatus exoß(1-3)glucanases family GH55 are essential for conidial cell wall morphogenesis.

The cell wall of Aspergillus fumigatus is predominantly composed of polysaccharides. The central fibrillar core of the cell wall is composed of a branched ß(1-3)glucan, to which the chitin and the galactomannan are covalently bound. Softening of the cell wall is an essential event during fungal morphogenesis, wherein rigid cell wall structures are cleaved by glycosyl hydrolases. In this study, we characterised the role of the glycosyl hydrolase GH55 members in A. fumigatus fungal morphogenesis. We showed that deletion of the six genes of the GH55 family stopped conidial cell wall maturation at the beginning of the development process, leading to abrogation of conidial separation: the shape of conidia became ovoid, and germination was delayed. In conclusion, the reorganisation and structuring of the conidial cell wall mediated by members of the GH55 family is essential for their maturation, normal dissemination, and germination.

Transcriptome analysis reveals the regulation of the shrimp STAT on host chitin-binding domain containing proteins and energy metabolism process during WSSV infection.

JAK/STAT signaling pathway is suggested to enhance the infection of WSSV in crustaceans. However, the regulation mechanism of this process is not quite clear. Here, comparative transcriptomic analysis was performed among shrimps before and after Litopenaeus vannamei STAT (LvSTAT) was silenced by dsRNA approach during WSSV infection. Differentially expressed genes (DEGs) common in the STAT-interfered groups and control groups at different times after WSSV infection were analyzed to acquire the genes probably regulated by LvSTAT. DEGs annotation and further GO terms enrichment analyses revealed that the identified DEGs mainly contained two categories, chitin-binding domain containing proteins and energy metabolism related genes. The former mainly included cuticle proteins, thrombospondins (TSPs) and peritrophin, while the later mainly included hexose catabolic process and glycolysis related genes. Two cuticle proteins and two TSPs were further studied to learn their expression changes during WSSV infection. They were significantly regulated during WSSV infection, implying the involvement of chitin-binding domain containing protein in the invasion process of WSSV. Systematic analysis on the glycolysis and lipid synthesis pathway demonstrated that silencing of LvSTAT could reduce the glycolysis efficiency and the production of lipids. It could be speculated that a favorable function of LvSTAT for WSSV replication existed by regulating the energy metabolism of the host. Through revealing the main category of genes and biological processes regulated by STAT, our study could shed new light on the roles of JAK/STAT signaling pathway in shrimp during virus infection.

Mechanical Control of Whole Body Shape by a Single Cuticular Protein Obstructor-E in Drosophila melanogaster.

Body shapes are much more variable than body plans. One way to alter body shapes independently of body plans would be to mechanically deform bodies. To what extent body shapes are regulated physically, or molecules involved in physical control of morphogenesis, remain elusive. During fly metamorphosis, the cuticle (exoskeleton) covering the larval body contracts longitudinally and expands laterally to become the ellipsoidal pupal case (puparium). Here we show that Drosophila melanogaster Obstructor-E (Obst-E) is a protein constituent of the larval cuticle that confers the oriented contractility/expandability. In the absence of obst-E function, the larval cuticle fails to undergo metamorphic shape change and finally becomes a twiggy puparium. We present results indicating that Obst-E regulates the arrangement of chitin, a long-chain polysaccharide and a central component of the insect cuticle, and directs the formation of supracellular ridges on the larval cuticle. We further show that Obst-E is locally required for the oriented shape change of the cuticle during metamorphosis, which is associated with changes in the morphology of those ridges. Thus, Obst-E dramatically affects the body shape in a direct, physical manner by controlling the mechanical property of the exoskeleton.

eIF2α Phosphorylation by GCN2 Is Induced in the Presence of Chitin and Plays an Important Role in Plant Defense against B. cinerea Infection.

Translation plays an important role in plant adaptation to different abiotic and biotic stresses; however, the mechanisms involved in translational regulation during each specific response and their effect in translation are poorly understood in plants. In this work, we show that GCN2 promotes eIF2α phosphorylation upon contact with Botrytis cinerea spores, and that this phosphorylation is required for the proper establishment of plant defense against the fungus. In fact, independent gcn2 mutants display an enhanced susceptibility to B. cinerea infection, which is highlighted by an increased cell death and reduced expression of ethylene- and jasmonic-related genes in the gcn2 mutants. eIF2α phosphorylation is not only triggered in the presence of the fungus, but interestingly, is also achieved in the sole presence of the microbe-associated molecular pattern (MAMP) chitin. Moreover, analysis of de novo protein synthesis by 35SMet-35SCys incorporation indicates that chitin treatment promotes a global inhibition of translation. Taken together, these results suggest that eIF2α phosphorylation by GCN2 is promoted in the presence of chitin and plays an important role in plant defense against B. cinerea infection.