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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Green et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Chitin, a major component of insect cuticles, plays a critical role in insect molting and morphogenesis. Thus, coordination of chitin remodeling during insect development requires tight transcriptional control of the chitin metabolism genes involved in chitin synthesis, assembly and degradation. However, the molecular mechanism underlying transcriptional coordination of chitin metabolism genes during beetle development is not yet completely understood., Results: We cloned the full-length cDNA encoding hormone receptor 3 (TcHR3) from Tribolium castaneum and showed a critical role of TcHR3 in modulating chitin metabolism gene expression during molting. Genome-wide transcriptome analysis of HR3-deficient old larvae using RNA sequencing analysis revealed a positive correlation between TcHR3 and transcription of chitin metabolism genes involved in chitin synthesis and degradation. In addition, HR3 overexpression significantly induced the gene promoter activity of N-acetylglucosaminidase 1 (NAG1) involved in chitin degradation and UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) involved in chitin synthesis. Chromatin immunoprecipitation analysis revealed that HR3 could directly bind to HR3-response element of NAG1 and UAP1 promoters. Finally, HR3-deficient late instar larvae and prepupae exhibited defects in larval-larval and larval-pupal molting, respectively, leading to eventual larval death because developing larvae were trapped inside the old cuticle as a result of abnormal chitin metabolism., Conclusion: TcHR3 is a transcriptional regulator of chitin metabolic genes for molting of T. castaneum. Controlling the molting system by TcHR3 might be a new management strategy for selective control of red flour beetle infestation. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Chitin is a kind of insoluble structural polysaccharide and plays different roles in different species. In crustaceans, it forms the structural components in the exoskeleton. In our previous studies, novel mud crab hybrids have been produced from the interspecific hybridization of Scylla serrata ♀ × S. paramamosain ♂. Some of the hybrid crabs have been found to be morphologically (eyestalk) abnormal, but the genetic mechanism remains unknown. To address this question, we performed whole-transcriptome RNA sequencing on the control group (normal hybrids), abnormal hybrids, and S. paramamosain to uncover the genetic basis underlying this morphological abnormality. A total of 695 mRNAs, 10 miRNAs, 44 circRNAs, and 1957 lncRNAs were differentially expressed between normal and abnormal hybrids. Several differentially expressed genes (DEGs) associated with chitin and cuticle metabolism were identified, including chitin synthase, chitinase, chitin deacetylase, β-N-acetylglucosaminidase, β-1,4-endoglucanase, N-alpha-acetyltransferase, cuticle proprotein, early cuticle protein, and arthrodial cuticle protein. Functional analysis showed that DE miRNAs, DE circRNAs, DE lncRNAs, and lncRNA/circRNA-miRNA-mRNA network were enriched in pathways related to the amino acid, carbohydrate, and glycogen metabolism. Considering the importance of the chitin and cuticle in exoskeleton formation, it can be concluded that the changes in the chitin and cuticle biosynthesis might have caused the eyestalk abnormality in hybrid crabs. These findings can lay the solid foundation for a better understanding of the important roles of chitin and cuticle related genes and the development of hybridization techniques in crustaceans., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

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 (Met 1 -Ala 23 ), a catalytic domain (Val 83 -Glu 347 and the catalytic triad Glu 149 , Glu 171 , and Ser 218 ), a proline-rich hinge region (Pro 414 -Pro 450 ), a polycystic kidney disease protein motif (Gly 465 -Ser 533 ), a C-terminus chitin-binding domain (Ala 553 - Glu 593 ), 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., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

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, β-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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

A novel chitinase (P1724) was discovered from a Qinghai-Tibetan plateau microbial metagenome. P1724 contains two GH18 family catalytic domains and is phylogenetically distant from any of the chitinases studied. P1724 and its truncated versions, P1724(∆cGH18) and P1724(∆nGH18), were produced in Escherichia coli and characterized. Using colloidal chitin as substrate, the three recombinant proteins showed maximum hydrolytic activities at 40 °C, pH 5.0-6.0 and 0-0.5 M NaCl, and were cold adaptive, as they remained active at 4 °C; their chitinase activities were decreased with the presence of Cu 2+ and EDTA, but increased with Ba 2+ and Ca 2+ ; they all showed both chitobiosidase and endochitinase activities. Compared to P1724(∆nGH18), P1724 and P1724(∆cGH18) shared more similarities in temperature and pH stabilities, NaCl tolerance, and substrate affinity, suggesting the N-terminal GH18 domain contributed more than the C-terminal GH18 did in biochemical characteristics of P1724. k cat /K m value of P1724 was significantly higher than the sum values of P1724(∆cGH18) and P1724(∆nGH18), which indicated that two GH18 domains of P1724 worked cooperatively in degrading chitin. This study has not only broadened the understanding of unknown chitinases in nature but also discussed the strategy of adding additional catalytic domains in enzyme engineering., (Copyright © 2021 Elsevier B.V. All rights reserved.)

β-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 β-chitin-derived high-value enzymes and products.

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America.)

Angiosperm cell adhesion is dependent on interactions between pectin polysaccharides which make up a significant portion of the plant cell wall. Cell adhesion in Arabidopsis may also be regulated through a pectin-related signaling cascade mediated by a putative O-fucosyltransferase ESMERALDA1 (ESMD1), and the Epidermal Growth Factor (EGF) domains of the pectin binding Wall associated Kinases (WAKs) are a primary candidate substrate for ESMD1 activity. Genetic interactions between WAKs and ESMD1 were examined using a dominant hyperactive allele of WAK2, WAK2cTAP, and a mutant of the putative O-fucosyltransferase ESMD1. WAK2cTAP expression results in a dwarf phenotype and activation of the stress response and reactive oxygen species (ROS) production, while esmd1 is a suppressor of a pectin deficiency induced loss of adhesion. Here we find that esmd1 suppresses the WAK2cTAP dwarf and stress response phenotype, including ROS accumulation and gene expression. Additional analysis suggests that mutations of the potential WAK EGF O-fucosylation site also abate the WAK2cTAP phenotype, yet only evidence for an N-linked but not O-linked sugar addition can be found. Moreover, a WAK locus deletion allele has no effect on the ability of esmd1 to suppress an adhesion deficiency, indicating WAKs and their modification are not a required component of the potential ESMD1 signaling mechanism involved in the control of cell adhesion. The WAK locus deletion does however affect the induction of ROS but not the transcriptional response induced by the elicitors Flagellin, Chitin and oligogalacturonides (OGs)., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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.

Glutamine:fructose-6-phosphate aminotransferase (GFAT) and phosphofructokinase (PFK) are enzymes related to chitin metabolism. RNA interference (RNAi) technology was used to explore the role of these two enzyme genes in chitin metabolism. In this study, we found that GFAT and PFK were highly expressed in the wing bud of Nilaparvata lugens and were increased significantly during molting. RNAi of GFAT and PFK both caused severe malformation rates and mortality rates in N. lugens. GFAT inhibition also downregulated GFAT, GNPNA, PGM1, PGM2, UAP, CHS1, CHS1a, CHS1b, Cht1-10, and ENGase. PFK inhibition significantly downregulated GFAT; upregulated GNPNA, PGM2, UAP, Cht2-4, Cht6-7 at 48 h and then downregulated them at 72 h; upregulated Cht5, Cht8, Cht10, and ENGase; downregulated Cht9 at 48 h and then upregulated it at 72 h; and upregulated CHS1, CHS1a, and CHS1b. In conclusion, GFAT and PFK regulated chitin degradation and remodeling by regulating the expression of genes related to the chitin metabolism and exert opposite effects on these genes. These results may be beneficial to develop new chitin synthesis inhibitors for pest control.

Short chain chitooligosaccharides (COs) are chitin derivative molecules involved in plant-fungus signaling during arbuscular mycorrhizal (AM) interactions. In host plants, COs activate a symbiotic signalling pathway that regulates AM-related gene expression. Furthermore, exogenous CO application was shown to promote AM establishment, with a major interest for agricultural applications of AM fungi as biofertilizers. Currently, the main source of commercial COs is from the shrimp processing industry, but purification costs and environmental concerns limit the convenience of this approach. In an attempt to find a low cost and low impact alternative, this work aimed to isolate, characterize and test the bioactivity of COs from selected strains of phylogenetically distant filamentous fungi: Pleurotus ostreatus, Cunninghamella bertholletiae and Trichoderma viride. Our optimized protocol successfully isolated short chain COs from lyophilized fungal biomass. Fungal COs were more acetylated and displayed a higher biological activity compared to shrimp-derived COs, a feature that-alongside low production costs-opens promising perspectives for the large scale use of COs in agriculture.

Trehalose-6-phosphate synthase (TPS) and trehalase (TRE) directly regulate trehalose metabolism and indirectly regulate chitin metabolism in insects. Real-time quantitative PCR (RT-qPCR) and RNA interference (RNAi) were used to detect the expressions and functions of the ApTPS and ApTRE genes. Abnormal phenotypes were found after RNAi of ApTRE in the Acyrthosiphon pisum. The molting deformities were observed in two color morphs, while wing deformities were only observed in the red morphs. The RNAi of ApTPS significantly down-regulated the expression of chitin metabolism-related genes, UDP-N-acetyglucosamine pyrophosphorylase (ApUAP), chitin synthase 2 (Apchs-2), Chitinase 2, 5 (ApCht2, 5), endo-beta-N-acetylglucosaminidase (ApENGase) and chitin deacetylase (ApCDA) genes at 24 h and 48 h; The RNAi of ApTRE significantly down-regulated the expression of ApUAP, ApCht1, 2, 8 and ApCDA at 24 h and 48 h, and up-regulated the expression of glucose-6-phosphate isomerase (ApGPI) and Knickkopf protein (ApKNK) genes at 48 h. The RNAi of ApTRE and ApTPS not only altered the expression of chitin metabolism-related genes but also decreased the content of chitin. These results demonstrated that ApTPS and ApTRE can regulate the chitin metabolism, deepen our understanding of the biological functions, and provide a foundation for better understanding the molecular mechanism of insect metamorphosis.

Chitin is the second most widely found natural polymer next to cellulose. Chitinases degrade the insoluble chitin to bioactive chitooligomers and monomers for various industrial applications. Based on their function, these enzymes act as biocontrol agents against pathogenic fungi and invasive pests compared with conventional chemical fungicides and insecticides. They have other functional roles in shellfish waste management, fungal protoplast generation, and Single-Cell Protein production. Among the chitinases, thermophilic and thermostable chitinases are gaining popularity in recent years, as they can withstand high temperatures and maintain the enzyme stability for longer periods. Not all chitinases are thermostable; hence, tailor-made thermophilic chitinases are designed to enhance their thermostability by direct evolution, genetic engineering involving mutagenesis, and proteomics approach. Although research has been done extensively on cloning and expression of thermophilic chitinase genes, there are only few papers discussing on the mechanism of chitin degradation using thermophiles. The current review discusses the sources of thermophilic chitinases, improvement of protein stability by gene manipulation, metagenomics approaches, chitin degradation mechanism in thermophiles, and their prospective applications for industrial, agricultural, and pharmaceutical purposes.

A chitosanase (CvCsn46) from Chromobacterium violaceum ATCC 12472 was produced in Escherichia coli, purified, and partially characterized. When subjected to denaturing polyacrylamide gel electrophoresis, the enzyme migrated as two protein bands (38 and 36 kDa apparent molecular masses), which were both identified as CvCsn46 by mass spectrometry. The enzyme hydrolyzed colloidal chitosan, with optimum catalytic activity at 50 °C, and two optimum pH values (at pH 6.0 and pH 11.0). The chitosanolytic activity of CvCsn46 was enhanced by some ions (Ca 2+ , Co 2+ , Cu 2+ , Sr 2+ , Mn 2+ ) and DTT, whereas Fe 2+ , SDS and β-mercaptoethanol completely inhibited its activity. CvCsn46 showed a non-Michaelis-Menten kinetics, characterized by a sigmoidal velocity curve (R 2  = 0.9927) and a Hill coefficient of 3.95. ESI-MS analysis revealed that the hydrolytic action of CvCsn46 on colloidal chitosan generated a mixture of low molecular mass chitooligosaccharides, containing from 2 to 7 hexose residues, as well as D-glucosamine. The chitosan oligomers generated by CvCsn46 inhibited in vitro the mycelial growth of Lasiodiplodia theobromae, significantly reducing mycelium extension and inducing hyphal morphological alterations, as observed by scanning electron microscopy. CvCsn46 was characterized as a versatile biocatalyst that produces well-defined chitooligosaccharides, which have potential to control fungi that cause important crop diseases., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A mutant lager strain resistant to the cell wall-perturbing agent Congo red (CR) was isolated and the genetic alterations underlying CR resistance were investigated by whole genome sequencing. The parental lager strain was found to contain three distinct Saccharomyces cerevisiae (Sc)-type CHS6 (CHitin Synthase-related 6) alleles, two of which have one or two nonsense mutations in the open reading frame, leaving only one functional allele, whereas the functional allele was missing in the isolated CR-resistant strain. On the other hand, the Saccharomyces eubayanus-type CHS6 alleles shared by both the parental and mutant strains appeared to contribute poorly to chitin synthase-activating function. Therefore, the CR resistance of the mutant strain was attributable to the overall compromised activity of CHS6 gene products. The CR-resistant mutant cells exhibited less chitin production on the cell surface and smaller amounts of mannoprotein release into the medium. All these traits, in addition to the CR resistance, were complemented by the functional ScCHS6 gene. It is of great interest whether the frequent nonsense mutations found in ScCHS6 open reading frame in lager yeast strains are a consequence of the domestication process of lager yeast., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.)

Recently discovered Lytic Polysaccharide Mono-Oxygenase (LPMO) enhances the enzymatic deconstruction of complex polysaccharide by oxidation. The present study demonstrates the agricultural waste hydrolyzing capabilities of Paenibacillus dendritiformis CRN18, which exhibits the enzyme activity of exo-glucanase, β-glucosidase, β-glucuronidase, endo-1, 4 β-xylanases, arabinosidase, and α-galactosidase as 0.1U/ml, 0.3U/ml, 0.09U/ml, 0.1U/ml, 0.05U/ml, and 0.41U/ml, respectively. The genome analysis of strain reveals the presence of four LPMO genes, along with lignocellulolytic genes. The gene structure of LPMO and its phylogenetic analysis shows the evolutionary relatedness with the Bacillus LPMO gene. Gene position of LPMOs in the genome of strains shows the close association of two LPMOs with chitin active enzyme GH18, and the other two are associated with hemicellulases (GH39, GH23). Protein-protein interaction and gene networking of LPMO sheds light on the co-occurrence, neighborhood, and interaction of LPMOs with chitinase and xylanase enzymes. Structural prediction of LPMOs unravels the information of the LPMO's binding site. Although the LPMO has been explored for its oxidative mechanism, a little light has been shed on its gene structure. This study provides insights into the LPMO gene structure in P. dendritiformis CRN18 and its potential in lignocellulose hydrolysis., Competing Interests: Declaration of competing interest The authors declare no financial or commercial conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Post-fermentation fungal biomass waste provides a viable source for chitin. Cell wall chitin of filamentous fungi, and in particular its de-N-acetylated derivative chitosan, has a wide range of commercial applications. Although the cell wall of filamentous fungi comprises 10-30% chitin, these yields are too low for cost-effective production. Therefore, we aimed to identify the genes involved in increased chitin deposition by screening a collection of UV-derived cell wall mutants in Aspergillus niger. This screen revealed a mutant strain (RD15.4#55) that showed a 30-40% increase in cell wall chitin compared to the wild type. In addition to the cell wall chitin phenotype, this strain also exhibited sensitivity to SDS and produces an unknown yellow pigment. Genome sequencing combined with classical genetic linkage analysis identified two mutated genes on chromosome VII that were linked with the mutant phenotype. Single gene knockouts and subsequent complementation analysis revealed that an 8 bp deletion in NRRL3_09595 is solely responsible for the associated phenotypes of RD15.4#55. The mutated gene, which was named cwcA (cell wall chitin A), encodes an orthologue of Saccharomyces cerevisiae Bypass of ESS1 (BYE1), a negative regulator of transcription elongation. We propose that this conserved fungal protein is involved in preventing cell wall integrity signaling under non-inducing conditions, where loss of function results in constitutive activation of the cell wall stress response pathway, and consequently leads to increased chitin content in the mutant cell wall., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Abirami Sasi et al.)

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., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

DrChit is class I chitinase involved in the digestion of insect prey of Drosera rotundifolia plants. Herein, we cloned the DrChit -S open reading frame lacking the 5'- sequence coding signal peptide into the pET32a vector and its derivate lacking the thioredoxin tag. After DrChit -S  + Trx and DrChit -S -Trx overexpression in Escherichia coli cells and purification on Ni-NTA agarose, both enzymes exhibited maximum activity at pH 6.0 and 38 °C. Surprisingly, the DrChit -S  + Trx exerted double enzyme activity and improved all kinetic parameters for FITC-chitin substrate degradation resulting in catalytic efficiency three times higher (46.2 mM -1 . s -1 ) than DrChit -S -Trx (13.63 mM -1 . s -1 ). The 3D-structure of DrChit -S  + Trx revealed different spatial arrangement of the three tyrosine residues in chitin-binding site, while their aromatic rings showed better stacking geometry for CH/π interactions with the carbohydrate substrate. In contrast, there were no significant differences between both enzymes when the effect of metal ions and their antifungal potential were tested. Quantitative in vitro assays showed growth suppression of Fusarium poae (40%), Trichoderma viride (43.8%), and Alternaria solani (52.6%) but not Rhizoctonia solani (sp.). Our study indicates that sundew chitinase has potential in biotechnology either for degradation of chitin to oligomers applicable in medicine or for plant defense fortification., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 35S Met- 35S Cys 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.

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, Vc ChiP, from the cell envelope of the V. cholerae type strain O1. We modeled the structure of Vc ChiP, revealing a trimeric cylinder that forms single channels in phospholipid bilayers. The membrane-reconstituted Vc ChiP channel was highly dynamic and voltage induced. Substate openings O 1 ', O 2 ', and O 3 ', between the fully open states O 1 , O 2 , and O 3 , were polarity selective, with nonohmic conductance profiles. Results of liposome-swelling assays suggested that Vc ChiP 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 Vc ChiP 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., Competing Interests: Conflict of interest—All authors declare that they have no competing financial interest., (© 2020 Soysa et al.)

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., Competing Interests: The authors declare no conflict of interest exists.

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., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Drechslera teres ( D. teres ) is an ascomycete, responsible for net blotch, the most serious barley disease causing an important economic impact. The cell wall is a crucial structure for the growth and development of fungi. Thus, understanding cell wall structure, composition and biosynthesis can help in designing new strategies for pest management. Despite the severity and economic impact of net blotch, this is the first study analyzing the cell wall-related genes in D. teres . We have identified key genes involved in the synthesis/remodeling of cell wall polysaccharides, namely chitin, β-(1,3)-glucan and mixed-linkage glucan synthases, as well as endo/exoglucanases and a mitogen-activated protein kinase. We have also analyzed the differential expression of these genes in D. teres spores and in the mycelium after cultivation on different media, as well as in the presence of Paraburkholderia phytofirmans strain PsJN, a plant growth-promoting bacterium (PGPB). The targeted gene expression analysis shows higher gene expression in the spores and in the mycelium with the application of PGPB. Besides analyzing key cell-wall-related genes, this study also identifies the most suitable reference genes to normalize qPCR results in D. teres , thus serving as a basis for future molecular studies on this ascomycete.

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., (© 2019 The Royal Entomological Society.)

Toxicities of the marine algae Alexandrium minutum and its excreted gonyautoxins (GTXs) to the marine crustacean Artemia salina were investigated. Mortality was observed for neither larvae nor adult A. salina exposed to A. minutum at a density of 5000 cells/mL or 0.5 μM GTX2/3. After exposure, the full transcriptome of adult A. salina was assembled and functionally annotated. A total of 599,286 transcripts were obtained, which were clustered into 515,196 unigenes. Results of the transcriptional effect level index revealed that direct exposure to the toxic algae A. minutum caused greater alterations in the transcriptome than did exposure to the extracellular product GTX2/3. Mechanisms of effects were different between exposure of A. salina to A. minutum cells or GTX2/3. Exposure to A. minutum modulated formation of the ribonucleoprotein complex and metabolism of amino acids and lipids in A. salina. Exposure to GTX2/3 exposure inhibited expression of genes related to metabolism of chitin, which might result in disruption of molting process or disturbed sheath morphogenesis. Overall, effects on transcription observed in this study represent the first report based on application of next generation sequencing techniques to investigate the transcriptomic response of A. salina exposed to an environmentally realistic level of A. minutum or GTX2/3., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The cell wall of Aspergillus fumigatus is predominantly composed of polysaccharides. The central fibrillar core of the cell wall is composed of a branched β(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., (© 2019 John Wiley & Sons Ltd.)

Phytophthora capsici has been the most destructive pathogen of pepper plants (Capsicum annuum L.), possessing the ability to quickly overcome the host defense system. In this context, the chitin-binding protein (CBP) family member CaChiIV1 regulates the response to P. capsici and abiotic stresses. The relevance of functional characterization and regulation of CaChiIV1 has not been explored in horticultural crops, especially pepper plants. The target gene (CaChiIV1) was isolated from pepper plants and cloned; the encoded protein carries a chitin-binding domain (CBD) that is rich in cysteine residues and has a hinge region with an abundance of proline and glycine residues. Additionally, the conserved regions in the promoter have a remarkable motif, "TTGACC". The expression of CaChiIV1 was markedly regulated by methyl-jasmonate (MeJA), hydrogen peroxide (H 2 O 2 ), melatonin, mannitol and P. capsici (PC and HX-9) infection. Knockdown of CaChiIV1 in pepper plants increased sensitivity to P. capsici (PC strain). Higher malondialdehyde (MDA) content and relative electrolyte leakage (REL) but lower antioxidant enzyme activities, chlorophyll content, root activity, and proline content were observed in CaChiIV1-silenced plants than in control plants. In conclusion, CaChiIV1-silenced pepper plants displayed increased susceptibility to P. capsici infection due to changes in expression of defense-related genes, thus showing its coregulation affect in particular conditions. Furthermore, antioxidant enzymes and proline content were largely diminished in CaChiIV1-silenced plants. Therefore, this evidence suggests that the CaChiIV1 gene plays a prominent role in the defense mechanism of pepper plants against P. capsici infection. In the future, the potential role of the CaChiIV1 gene in defense regulatory pathways and its coregulation with other pathogen-related genes should be identified.

Rhizoctonia solani is a fungal species that belongs to the fungal division Basidiomycota. It is a soil-borne pathogen that attacks a broad range of plant species and crops. Disease symptoms are commonly seen as damping off of seedlings and root rot, although it can infect plants at any developmental stage. Despite the severity of this disease, many aspects in R. solani infection biology remain unclear. Here we investigated the role of a LysM effector, previously predicted from the genome of a R. solani AG2-2IIIB strain that has sugar beet as a host. Gene expression analysis showed that RsLysM was highly induced upon sugar beet infection. When RsLysM was heterologously expressed in Cercospora beticola, necrotic lesion size and fungal colonization ability were increased, indicating a role in virulence. RsLysM displayed chitin-binding affinity and suppression of chitin-triggered immunity but could not protect hyphae from hydrolysis. Overall, this study is the first characterization of a LysM effector from Basidiomycota, suggesting that this necrotrophic fungal species relies on perturbation of chitin-triggered immunity to establish a successful infection.

Coccidioides is the causative agent of San Joaquin Valley fever, a fungal disease prevalent in the semiarid regions of the Americas. Efforts to develop a fungal vaccine over the last 2 decades were unsuccessful. A candidate antigen, Antigen 2 (Ag2), is notoriously difficult to express in Escherichia coli, and this study sought to accumulate the antigen at high levels in maize. Transformed maize lines accumulated recombinant Ag2 at levels >1 g/kg. Mice immunized with this antigen and challenged with live Coccidioides arthroconidia showed a reduction in the fungal load when Ag2 derived from either E. coli or maize was loaded into glucan chitin particles. A fusion of Ag2 to dendritic cell carrier peptide (DCpep) induced a T-helper type 17 response in the spleen when orally delivered, indicative of a protective immune response. The maize production platform and the glucan chitin particle adjuvant system show promise for development of a Coccidioides vaccine, but further testing is needed to fully assess the optimal method of administration., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The availability of different host chassis will greatly expand the range of applications in synthetic biology. Members of the Acetobacteraceae family of Gram-negative bacteria form an attractive class of nonmodel microorganisms that can be exploited to produce industrial chemicals, food and beverage, and biomaterials. One such biomaterial is bacterial cellulose, which is a strong and ultrapure natural polymer used in tissue engineering scaffolds, wound dressings, electronics, food additives, and other products. However, despite the potential of Acetobacteraceae in biotechnology, there has been considerably little effort to fundamentally reprogram the bacteria for enhanced performance. One limiting factor is the lack of a well-characterized, comprehensive toolkit to control expression of genes in biosynthetic pathways and regulatory networks to optimize production and cell viability. Here, we address this shortcoming by building an expanded genetic toolkit for synthetic biology applications in Acetobacteraceae. We characterized the performance of multiple natural and synthetic promoters, ribosome binding sites, terminators, and degradation tags in three different strains, namely, Gluconacetobacter xylinus ATCC 700178, Gluconacetobacter hansenii ATCC 53582, and Komagataeibacter rhaeticus iGEM. Our quantitative data revealed strain-specific and common design rules for the precise control of gene expression in these industrially relevant bacterial species. We further applied our tools to synthesize a biodegradable cellulose-chitin copolymer, adjust the structure of the cellulose film produced, and implement CRISPR interference for ready down-regulation of gene expression. Collectively, our genetic parts will enable the efficient engineering of Acetobacteraceae bacteria for the biomanufacturing of cellulose-based materials and other commercially valuable products.

Insect cuticle is a composite made of chitin filaments embedded in a proteinaceous matrix, consisting mainly of structural cuticular proteins. In the present study, an endocuticle structural glycoprotein gene, LmAbd-9, was characterized based on the Locusta migratoria transcriptome. LmAbd-9 encodes a glycoprotein with a chitin binding domain 4, belonging to RR-1 subclass of the CPR family, which has two potential O-linked glycosylation sites (S115 and T137) at which glycosylation modification may occur. LmAbd-9 was highly expressed in the integument and showed periodic expression during molting. The expression levels of LmAbd-9 were significantly down-regulated after injection with 20-hydroxyecdysone (20E) for 6, 12, and 24 h, whereas it was upregulated after double-stranded RNA-mediated RNA interference of the 20E receptor gene LmEcR and LmFTZ-F1beta at day 2 of fifth instar nymphs for 48 h. After injection of dsLmAbd-9 on day 2 of fifth instar nymphs, the insects could normally molt to adults and showed no macroscopic phenotype; however, the cuticle of the adults was thinner, and there were significantly fewer endocuticular lamellae than in the control. Thus, LmAbd-9 that negatively regulated by the 20E signaling pathway was involved in the formation of the endocuticle in L. migratoria., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The fungal cell wall is very important for cell growth and survival during stress, and the target of rapamycin (TOR) pathway plays a major role in regulating cell growth in response to environmental cues. Ganoderma lucidum is an important edible and medicinal fungus, and the function of TOR in this organism remains unclear. As shown in the present study, the TOR pathway regulates cell wall integrity (CWI) in G. lucidum. Inhibition of TOR signaling by RNA interference (RNAi) or rapamycin treatment reduced the growth of G. lucidum mycelia, increased contents of the cell wall components chitin and β-1,3-glucan, and increased cell wall thickness. Furthermore, inhibition of TOR signaling enhanced the relative level of phosphorylated Slt2, a member of the MAPK cascade involved in CWI signaling. Moreover, when treated with rapamycin, significantly lower chitin and β-1,3-glucan contents were observed in Slt2-silenced strains than in WT strains, indicating that TOR regulates the synthesis of these cell wall components through the Slt2-MAPK pathway. These results indicate a potential relationship between TOR signaling and CWI signaling. Additionally, participation of Slt2-MAPK in TOR-mediated regulation of cell wall component production has not previously been reported in a microorganism., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The formation of a glucan/chitin/glycoprotein cell wall matrix is vital for fungal survival, growth, and morphogenesis. The cell wall proteins are important cell wall components and function in adhesion, signal transduction, and as cell wall structural elements. In this report we demonstrate that Neurospora crassa GH72 glucan transferases function to crosslink cell wall glycoproteins into the cell wall. With an in vitro assay, we show that the glucan transferases are able to attach lichenin, a cell wall glucan with a repeating β-1,4-glucose-β-1,4-glucose-β-1,3-glucose structure, to cell wall glycoproteins. We propose that the pathway for attachment of lichenin to the glycoprotein has four steps. First, N-linked oligosaccharides present on the glycoproteins are modified by the addition of a galactomannan. As part of our report we have characterized the structure of the galactomannan, which consists of an α-1,6-mannose backbone with galactofuranose side chains. In the second step, the galactomannan is processed by members of the GH76 α-1,6-mannanases. In the third step, the glucan transferases cleave the lichenin and create substrate-enzyme intermediates. In the final step, the transferases transfer the lichenin to the processed galactomannan. We demonstrate that the N. crassa glucan transferases have demonstrate specificity for the processed galactomannan and for lichenin. The energy from the cleaved glycosidic bond in lichenin is retained in the substrate-enzyme intermediate and used to create a new glycosidic bond between the lichenin and the processed galactomannan. The pathway effectively crosslinks glycoproteins into the fungal cell wall., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

An expression strategy is presented in order to produce nanobodies modified with a clickable alkyne functionality at their C-terminus via the intein-mediated protein ligation (IPL) technique. The protocol focuses on the cytoplasmic expression and extraction of a nanobody-intein-chitin binding domain (CBD) fusion protein in E. coli SHuffle ® T7 cells, in the commonly used Luria-Bertani (LB) medium. The combination of these factors results in a high yield and nearly complete alkynation of the nanobody at its C-terminus via IPL. The resulting alkynated nanobodies retain excellent binding capacity toward the nanobody targeted antigen. The presented protocol benefits from time- and cost-effectiveness and allows for a feasible upscaling of functionalized (here alkynated) nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to (1) novel biosurface applications that demand for homogeneously oriented nanobodies having their active site fully accessible for target (e.g., biomarker) binding, and (2) innovative applications such as localized drug delivery and image guided surgery by covalent "click" chemistry coupling of these alkynated nanobodies to a multitude of azide-containing counterparts as there are drug containing polymers and contrast labeling agents.

Chitinase is an important enzyme for many physiological processes. Mytichitin-1 is a chitinase-like protein in Mytilus coruscus, and its C-terminal 55-AA fragment (mytichitin-CB) is a novel antimicrobial peptide, suggesting a new immune process in which chitinase is involved; mytichtin-1 may have various forms in the different biological processes of M. coruscus. Thus, the study of mytichitin-1 will be helpful for understanding the mechanism of mussel immune biology and the functional diversity of chitinase. In this study, mytichitin-1 was recombinantly expressed with different lengths, full-length mytichtin-1 (rMchi-F) and the N-terminal region (rMchi-N) in Escherichia coli BL21 with codon optimization. The results of SDS-PAGE, Western blotting, and mass spectrometry confirmed that the two forms of mytichitin-1 had been successfully recombinant expressed with a yield of 40 mg purified enzyme per L culture. In addition, the 55-AA fragment of mytichitin-CB was chemically synthesized (sMchi-CB). After purification and oxidation, the functions of the three protein products were analysed, including chitin degradation, chitin binding, and antimicrobial activities. Both rMchi-F and rMchi-N displayed enzymatic activity with the optimum pH of 4.0 and optimum temperature of 40 °C, and rMchi-N showed a stronger activity than rMchi-F. Enzymatic activities of rMchi-F and rMchi-N were stimulated by the metal ions Fe 2+ , Ba 2+ , and Na + and partially inhibited by Cu 2+ , Ni 2+ and Zn 2+ . rMchi-F, rMchi-N, and sMchi-CB had the ability to combine with colloid chitin. The antimicrobial activities of these proteins were tested against bacteria and fungi, and the results indicated the strongest activity for sMchi-CB and the weakest activity for rMchi-N. Using a prepared anti-rMchi-F polyclonal antibody, immunohistochemistry and immunoprecipitation were performed and the results revealed the location of mytichitin-1 in mantle, digestive gland and blood cells. In addition, two forms of mytichitin-1, mytichitin-CB (6 kD) and full-length mytichitin-1 (48 kD), were detected, and a 35 kD protein was identified as the third form of mytichitin-1, existing in various tissues of M. coruscus. These findings suggest that mytichitin-1 may play different roles, with at least three forms, in different M. coruscus tissues., (Copyright © 2018. Published by Elsevier Ltd.)

Understanding the evolution of the developmental programs active during dipteran embryogenesis depends on comparative studies. As a counterpoint to the intensively investigated and highly derived cyclorrhaphan flies that include the model organism Drosophila melanogaster, we are studying the basal Diptera Bradysia hygida, a member of the Sciaridae family that is amenable to laboratory cultivation. Here we describe the B. hygida embryogenesis, which lasts 9 days at 22 °C. The use of standard fixation D. melanogaster protocols resulted in embryos refractory to DAPI staining and to overcome this, a new enzyme-based method was developed. Calcofluor-White staining of enzimatically-treated embryos revealed that this method removes chitin from the serosal cuticle surrounding the B. hygida embryo. Chitin is one of the main components of serosal cuticles and searches in a B. hygida embryonic transcriptome database revealed conservation of the chitin synthesis pathway, further supporting the occurrence of chitin biosynthesis in B. hygida embryos. Combining the enzymatic treatment protocol with the use of both DIC and fluorescence microscopy allowed the first complete description of the B. hygida embryogenesis. Our results constitute an important step towards the understanding of early development of a basal Diptera and pave the way for future evo-devo studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Vicilins are 7S globulins which constitute the major seed storage proteins in leguminous species. Variant vicilins showing differential binding affinities for chitin have been implicated in the resistance and susceptibility of cowpea to the bruchid Callosobruchus maculatus. These proteins are members of the cupin superfamily, which includes a wide variety of enzymes and non-catalytic seed storage proteins. The cupin fold does not share similarity with any known chitin-biding domain. Therefore, it is poorly understood how these storage proteins bind to chitin. In this work, partial cDNA sequences encoding β-vignin, the major component of cowpea vicilins, were obtained from developing seeds. Three-dimensional molecular models of β-vignin showed the characteristic cupin fold and computational simulations revealed that each vicilin trimer contained 3 chitin-binding sites. Interaction models showed that chito-oligosaccharides bound to β-vignin were stabilized mainly by hydrogen bonds, a common structural feature of typical carbohydrate-binding proteins. Furthermore, many of the residues involved in the chitin-binding sites of β-vignin are conserved in other 7S globulins. These results support previous experimental evidences on the ability of vicilin-like proteins from cowpea and other leguminous species to bind in vitro to chitin as well as in vivo to chitinous structures of larval C. maculatus midgut., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Lytic polysaccharide monooxygenases (LPMOs) contribute to enzymatic conversion of recalcitrant polysaccharides such as chitin and cellulose and may also play a role in bacterial infections. Some LPMOs are multimodular, the implications of which remain only partly understood. We have studied the properties of a tetra-modular LPMO from the food poisoning bacterium Bacillus cereus (named BcLPMO10A). We show that BcLPMO10A, comprising an LPMO domain, two fibronectin-type III (FnIII)-like domains, and a carbohydrate-binding module (CBM5), is a powerful chitin-active LPMO. While the role of the FnIII domains remains unclear, we show that enzyme functionality strongly depends on the CBM5, which, by promoting substrate binding, protects the enzyme from inactivation. BcLPMO10A enhances the activity of chitinases during the degradation of α-chitin., (© 2018 Federation of European Biochemical Societies.)

The pancrustacean theory groups crustaceans and hexapods (once thought to comprise separate clades within the Arthropoda) into a single clade. A key feature common to all pancrustaceans is their chitinous exoskeleton, with a major contribution by cuticular proteins. Among these, are the CPAP3's, a family of cuticular proteins, first identified in the hexapod Drosophila melanogaster and characterized by an N-terminal signaling peptide and three chitin-binding domains. In this study, CPAP3 proteins were mined from a transcriptomic library of a decapod crustacean, the crayfish Cherax quadricarinatus. Phylogenetic analysis of other CPAP3 proteins from hexapods and other crustaceans showed a high degree of conservation. Characterization of the crayfish proteins, designated CqCPAP3's, suggested a major role for CPAP3'sin cuticle formation. Loss-of-function experiments using RNAi supported such a notion by demonstrating crucial roles for several CqCPAP3 proteins during molting. A putative mode of action for the CqCPAP3 proteins -theoretically binding three chitin strands- was suggested by the structural data obtained from a representative recombinant CqCPAP3. The similarities between the CqCPAP3 proteins and their hexapod homologues further demonstrated common genetic and proteinaceous features of cuticle formation in pancrustaceans, thereby reinforcing the linkage between these two highly important phylogenetic groups.

Dolichols are isoprenoid lipids of varying length that act as sugar carriers in glycosylation reactions in the endoplasmic reticulum. In Saccharomyces cerevisiae , there are two cis -prenyltransferases that synthesize polyprenol-an essential precursor to dolichol. These enzymes are heterodimers composed of Nus1 and either Rer2 or Srt1. Rer2-Nus1 and Srt1-Nus1 can both generate dolichol in vegetative cells, but srt1 ∆ cells grow normally while rer2 ∆ grows very slowly, indicating that Rer2-Nus1 is the primary enzyme used in mitotically dividing cells. In contrast, SRT1 performs an important function in sporulating cells, where the haploid genomes created by meiosis are packaged into spores. The spore wall is a multilaminar structure and SRT1 is required for the generation of the outer chitosan and dityrosine layers of the spore wall. Srt1 specifically localizes to lipid droplets associated with spore walls, and, during sporulation there is an SRT1 -dependent increase in long-chain polyprenols and dolichols in these lipid droplets. Synthesis of chitin by Chs3, the chitin synthase responsible for chitosan layer formation, is dependent on the cis -prenyltransferase activity of Srt1, indicating that polyprenols are necessary to coordinate assembly of the spore wall layers. This work shows that a developmentally regulated cis -prenyltransferase can produce polyprenols that function in cellular processes besides protein glycosylation., (Copyright © 2017 by the Genetics Society of America.)

Plants interpret their immediate environment through perception of small molecules. Microbe-associated molecular patterns (MAMPs) such as flagellin and chitin are likely to be more abundant in the rhizosphere than plant-derived damage-associated molecular patterns (DAMPs). We investigated how the Arabidopsis thaliana root interprets MAMPs and DAMPs as danger signals. We monitored root development during exposure to increasing concentrations of the MAMPs flg22 and the chitin heptamer as well as of the DAMP AtPep1. The tissue-specific expression of defence-related genes in roots was analysed using a toolkit of promoter::YFPN lines reporting jasmonic acid (JA)-, salicylic acid (SA)-, ethylene (ET)- and reactive oxygen species (ROS)- dependent signalling. Finally, marker responses were analysed during invasion by the root pathogen Fusarium oxysporum. The DAMP AtPep1 triggered a stronger activation of the defence markers compared to flg22 and the chitin heptamer. In contrast to the tested MAMPs, AtPep1 induced SA- and JA-signalling markers in the root and caused a severe inhibition of root growth. Fungal attack resulted in a strong activation of defence genes in tissues close to the invading fungal hyphae. The results collectively suggest that AtPep1 presents a stronger danger signal to the Arabidopsis root than the MAMPs flg22 and chitin heptamer.

Broad-Spectrum Resistance 1 (BSR1) encodes a rice receptor-like cytoplasmic kinase, and enhances disease resistance when overexpressed. Rice plants overexpressing BSR1 are highly resistant to diverse pathogens, including rice blast fungus. However, the mechanism responsible for this resistance has not been fully characterized. To analyze the BSR1 function, BSR1-knockout (BSR1-KO) plants were generated using a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Experiments using suspension-cultured cells revealed that defense responses including H 2 O 2 production (i.e. oxidative burst) and expression of defense-related genes induced by autoclaved conidia of the rice blast fungus significantly decreased in BSR1-KO cells. Furthermore, a treatment with chitin oligomers which function as microbe-associated molecular patterns (MAMPs) of the rice blast fungus resulted in considerably suppressed defense responses in BSR1-KO cells. These results suggest that BSR1 is important for the rice innate immunity triggered by the perception of chitin.