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1.
Gene ; 764: 145098, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-32861881

RESUMO

Neocaridina denticulate sinensis is a small freshwater economic shrimp, as well as excellent laboratory model for their short life cycle and easy availability. However, the response of N. denticulate sinensis to pervasive copper pollution in aquatic environments has not been deeply investigated yet. Herein, we preformed Illumina sequencing technology to mine the alterations of cephalothorax transcriptome under 2.5 µmol/L of Cu2+ after 48 h. 122,512 unigenes were assembled and 219 unigenes were identified as significantly differentially expressed genes (DEGs) between control and Cu2+ treatment groups. Functional enrichment analysis revealed that DEGs were mostly associated with immune responses and molting, such as endocytosis, Fc gamma R-mediated phagocytosis and chitin metabolic process. Seven genes were chosen for qPCR verification, and the results showed that the transcriptome sequencing data were consistent with the qPCR results. This is the first report of transcriptome information about N. denticulate sinensis. These results provided a direction for the future research of resistance to Cu2+ in this shrimp, and simultaneously enriched gene information of N. denticulate sinensis.


Assuntos
Cobre/toxicidade , Decápodes/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Frutos do Mar , Poluentes Químicos da Água/toxicidade , Animais , Quitina/metabolismo , Decápodes/efeitos dos fármacos , Decápodes/imunologia , Endocitose/efeitos dos fármacos , Endocitose/genética , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular , Muda/efeitos dos fármacos , Muda/genética , Fagocitose/efeitos dos fármacos , Fagocitose/genética , RNA-Seq , Transcriptoma/efeitos dos fármacos
2.
Proc Natl Acad Sci U S A ; 117(34): 20932-20942, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32778594

RESUMO

Many pathogenic fungi exploit stomata as invasion routes, causing destructive diseases of major cereal crops. Intensive interaction is expected to occur between guard cells and fungi. In the present study, we took advantage of well-conserved molecules derived from the fungal cell wall, chitin oligosaccharide (CTOS), and chitosan oligosaccharide (CSOS) to study how guard cells respond to fungal invasion. In Arabidopsis, CTOS induced stomatal closure through a signaling mediated by its receptor CERK1, Ca2+, and a major S-type anion channel, SLAC1. CSOS, which is converted from CTOS by chitin deacetylases from invading fungi, did not induce stomatal closure, suggesting that this conversion is a fungal strategy to evade stomatal closure. At higher concentrations, CSOS but not CTOS induced guard cell death in a manner dependent on Ca2+ but not CERK1. These results suggest that stomatal immunity against fungal invasion comprises not only CTOS-induced stomatal closure but also CSOS-induced guard cell death.


Assuntos
Quitina/metabolismo , Estômatos de Plantas/imunologia , Estômatos de Plantas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Quitina/fisiologia , Quitosana/metabolismo , Fungos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais/efeitos dos fármacos
3.
Nat Commun ; 11(1): 3897, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753587

RESUMO

Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by rhizobial bacteria that trigger the nodulation process in legumes, and by some fungi that also establish symbiotic relationships with plants, notably the arbuscular and ecto mycorrhizal fungi. Here, we show that many other fungi also produce LCOs. We tested 59 species representing most fungal phyla, and found that 53 species produce LCOs that can be detected by functional assays and/or by mass spectroscopy. LCO treatment affects spore germination, branching of hyphae, pseudohyphal growth, and transcription in non-symbiotic fungi from the Ascomycete and Basidiomycete phyla. Our findings suggest that LCO production is common among fungi, and LCOs may function as signals regulating fungal growth and development.


Assuntos
Quitina/análogos & derivados , Quitina/metabolismo , Fungos/crescimento & desenvolvimento , Fungos/metabolismo , Transdução de Sinais/fisiologia , Ascomicetos/crescimento & desenvolvimento , Basidiomycota/crescimento & desenvolvimento , Ecologia , Ácidos Graxos/metabolismo , Micorrizas/fisiologia , Rhizobium/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Simbiose/fisiologia
4.
Proc Natl Acad Sci U S A ; 117(32): 19178-19189, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723819

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO-substrate interaction. Using NMR spectroscopy, we have elucidated the solution-phase structure of apo-BlLPMO10A from Bacillus licheniformis, along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and 15N-HYSCORE spectroscopy study on the uniformly isotope-labeled 63Cu(II)-bound 15N-BlLPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal-ligand covalency and an attendant increase in the d(x2-y2) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu-O2 intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O2 activation in chitin-active AA10 LPMOs.


Assuntos
Bacillus licheniformis/enzimologia , Proteínas de Bactérias/química , Quitina/metabolismo , Oxigenases de Função Mista/química , Oxigênio/metabolismo , Bacillus licheniformis/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Quitina/química , Cobre/química , Cobre/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Imagem por Ressonância Magnética , Oxigenases de Função Mista/metabolismo , Oxigênio/química , Especificidade por Substrato
5.
Proc Natl Acad Sci U S A ; 117(33): 20180-20189, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32719134

RESUMO

Two-component signal transduction systems (TCSs) represent a major mechanism that bacteria use to sense and respond to their environment. Prototypical TCSs are composed of a membrane-embedded histidine kinase, which senses an environmental stimulus and subsequently phosphorylates a cognate partner protein called a response regulator that regulates gene expression in a phosphorylation-dependent manner. Vibrio cholerae uses the hybrid histidine kinase ChiS to activate the expression of the chitin utilization program, which is critical for the survival of this facultative pathogen in its aquatic reservoir. A cognate response regulator for ChiS has not been identified and the mechanism of ChiS-dependent signal transduction remains unclear. Here, we show that ChiS is a noncanonical membrane-embedded one-component system that can both sense chitin and directly regulate gene expression via a cryptic DNA binding domain. Unlike prototypical TCSs, we find that ChiS DNA binding is diminished, rather than stimulated, by phosphorylation. Finally, we provide evidence that ChiS likely activates gene expression by directly recruiting RNA polymerase. This work addresses the mechanism of action for a major transcription factor in V. cholerae and highlights the versatility of signal transduction systems in bacterial species.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , Vibrio cholerae/enzimologia , Proteínas de Bactérias/genética , Quitina/metabolismo , Polinucleotídeo 5'-Hidroxiquinase/genética , Ligação Proteica , Vibrio cholerae/metabolismo
6.
Chem Biol Interact ; 328: 109201, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32717190

RESUMO

The caseinate and glycated caseinate generated from the transglutaminase-catalyzed reaction of caseinate and oligochitosan were digested using pepsin and trypsin, and the activity of the resultant digests was measured in rat intestinal epithelial cell line (IEC-6) using several biological responses as indicators. Compared with the caseinate digest, the glycated caseinate digest had similar contents in 17 amino acids but less reactable -NH2 contents, and 6.57 g glucosamine per kg protein; moreover, it showed higher activity in the cells (P < 0.05) to promote cell growth, accumulate the cell-cycle progression at the S-phase, and prevent the camptothecin-induced cell apoptosis. The glycated caseinate digest also showed higher differentiation activity in the cells than the caseinate digest, resulting in enhanced activities of the three brush-border membrane enzymes (P < 0.05) and increased microvilli on the cell surfaces. The real-time reverse transcription-polymerase chain reaction, Western-blot assay, and Dickkopf-1 (a receptor inhibitor of the Wnt/ß-catenin signaling pathway) were used to determine both gene and protein expression changes in the cells. A Wnt/ß-catenin signaling pathway responsible for these enhanced effects was proposed because the five genes (glycogen synthase kinase 3ß, Wnt3a, ß-catenin, c-Myc, and cyclin D1) and three proteins (nuclear and cytosolic ß-catenin, cyclin D1, and c-Myc) as part of this signaling pathway were regulated in the treated cells. The oligochitosan glycation of caseinate induced by transglutaminase is thus suggested endowing the peptic-tryptic caseinate digest with higher activity in the cells through its effects on the Wnt/ß-catenin signaling pathway.


Assuntos
Caseínas/metabolismo , Quitina/análogos & derivados , Enterócitos/metabolismo , Pepsina A/metabolismo , Tripsina/metabolismo , Via de Sinalização Wnt , Animais , Apoptose , Bovinos , Pontos de Checagem do Ciclo Celular , Diferenciação Celular/genética , Proliferação de Células , Sobrevivência Celular , Quitina/metabolismo , Enterócitos/citologia , Enterócitos/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética
7.
PLoS Pathog ; 16(6): e1008652, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32574207

RESUMO

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.


Assuntos
Ascomicetos/química , Quitina/química , Proteínas Fúngicas/química , Hifas/química , Multimerização Proteica , Ascomicetos/genética , Ascomicetos/metabolismo , Quitina/genética , Quitina/metabolismo , Cladosporium/química , Cladosporium/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hifas/genética , Hifas/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Estrutura Quaternária de Proteína , Triticum/genética , Triticum/metabolismo , Triticum/microbiologia
8.
Food Chem ; 330: 127230, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32526651

RESUMO

Chitooligosaccharides are oligosaccharides with many biological activities that can be used in food production for sweeteners, preservatives and humectants, among other products. Chitin, a long-chain polymer of N-acetylglucosamine and a derivative of glucose, can be hydrolyzed by applying chitinase to break down glycosidic bonds to form chitooligosaccharides. Chitinases arising from heterologous gene expression are usually linked to a 6 × His-tag to facilitate easy purification. Heterologously expressed chitinase linked to a 6 × His-tag is a transgenic element, but enzyme activity tests cannot be used to distinguish transgenic elements from natural elements. In this study, we established a rapid and easy method to detect His-tag-containing chitinase using gold nanoparticles (AuNPs) and ssDNA aptamers. Using this method, His-tag-containing chitinase could be detected at concentrations as low as 0.136 nM within 5 min. Color changes of AuNPs showed a positive correlation with His-tag-containing chitinase concentrations.


Assuntos
Aptâmeros de Nucleotídeos/química , Quitinases/metabolismo , DNA de Cadeia Simples/química , Ouro/química , Nanopartículas Metálicas/química , Quitina/análogos & derivados , Quitina/metabolismo , Cor , Hidrólise , Fatores de Tempo
9.
PLoS Pathog ; 16(5): e1008342, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365117

RESUMO

Chitinases are important enzymes that contribute to the generation of carbon and nitrogen from chitin, a long chain polymer of N-acetylglucosamine that is abundant in insects, fungi, invertebrates and fish. Although mammals do not produce chitin, chitinases have been identified in bacteria that are key virulence factors in severe respiratory, gastrointestinal and urinary diseases. However, it is unclear how these enzymes are able to carry out this dual function. Legionella pneumophila is the causative agent of Legionnaires' disease, an often-fatal pneumonia and its chitinase ChiA is essential for the survival of L. pneumophila in the lung. Here we report the first atomic resolution insight into the pathogenic mechanism of a bacterial chitinase. We derive an experimental model of intact ChiA and show how its N-terminal region targets ChiA to the bacterial surface after its secretion. We provide the first evidence that L. pneumophila can bind mucins on its surface, but this is not dependent on ChiA. This demonstrates that additional peripheral mucin binding proteins are also expressed in L. pneumophila. We also show that the ChiA C-terminal chitinase domain has novel Zn2+-dependent peptidase activity against mammalian mucin-like proteins, namely MUC5AC and the C1-esterase inhibitor, and that ChiA promotes bacterial penetration of mucin gels. Our findings suggest that ChiA can facilitate passage of L. pneumophila through the alveolar mucosa, can modulate the host complement system and that ChiA may be a promising target for vaccine development.


Assuntos
Quitinases/metabolismo , Legionella pneumophila/metabolismo , Acetilglucosamina/metabolismo , Proteínas de Bactérias/metabolismo , Quitina/metabolismo , Quitinases/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Doença dos Legionários/metabolismo , Metais , Mucina-1/metabolismo , Mucinas/metabolismo , Proteólise , Relação Estrutura-Atividade , Fatores de Virulência/metabolismo
10.
Proc Natl Acad Sci U S A ; 117(17): 9621-9629, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32284410

RESUMO

The plasma membrane (PM) is composed of heterogeneous subdomains, characterized by differences in protein and lipid composition. PM receptors can be dynamically sorted into membrane domains to underpin signaling in response to extracellular stimuli. In plants, the plasmodesmal PM is a discrete microdomain that hosts specific receptors and responses. We exploited the independence of this PM domain to investigate how membrane domains can independently integrate a signal that triggers responses across the cell. Focusing on chitin signaling, we found that responses in the plasmodesmal PM require the LysM receptor kinases LYK4 and LYK5 in addition to LYM2. Chitin induces dynamic changes in the localization, association, or mobility of these receptors, but only LYM2 and LYK4 are detected in the plasmodesmal PM. We further uncovered that chitin-induced production of reactive oxygen species and callose depends on specific signaling events that lead to plasmodesmata closure. Our results demonstrate that distinct membrane domains can integrate a common signal with specific machinery that initiates discrete signaling cascades to produce a localized response.


Assuntos
Arabidopsis/fisiologia , Quitina/metabolismo , Plasmodesmos/fisiologia , Tabaco/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fenômenos Biomecânicos , Membrana Celular/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Mecanotransdução Celular/fisiologia , Folhas de Planta/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio
11.
PLoS Pathog ; 16(4): e1008518, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32324832

RESUMO

Competition is one of the fundamental driving forces of natural selection. Beauveria bassiana is a soil and plant phylloplane/root fungus capable of parasitizing insect hosts. Soil and plant environments are often enriched with other fungi against which B. bassiana competes for survival. Here, we report an antifungal peptide (BbAFP1), specifically expressed and localized to the conidial cell wall and is released into the surrounding microenvironment inhibiting growth of competing fungi. B. bassiana strains expressing BbAFP1, including overexpression strains, inhibited growth of Alternaria brassicae in co-cultured experiments, whereas targeted gene deletion of BbAFP1 significantly decreased (25%) this inhibitory effect. Recombinant BbAFP1 showed chitin and glucan binding abilities, and growth inhibition of a wide range of phytopathogenic fungi by disrupting membrane integrity and eliciting reactive oxygen species (ROS) production. A phenylalanine residue (F50) contributes to chitin binding and antifungal activity, but was not required for the latter. Expression of BbAFP1 in tomato resulted in transgenic plants with enhanced resistance to plant fungal pathogens. These results highlight the importance of fungal competition in shaping primitive competition strategies, with antimicrobial compounds that can be embedded in the spore cell wall to be released into the environment during the critical initial phases of germination for successful growth in its environmental niche. Furthermore, these peptides can be exploited to increase plant resistance to fungal pathogens.


Assuntos
Antifúngicos/metabolismo , Beauveria/metabolismo , Esporos Fúngicos/metabolismo , Animais , Antifúngicos/farmacologia , Beauveria/genética , Parede Celular/metabolismo , Quitina/metabolismo , Proteínas Fúngicas/metabolismo , Glucanos/metabolismo , Insetos/microbiologia , Peptídeos , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Espécies Reativas de Oxigênio , Estresse Fisiológico/efeitos dos fármacos , Virulência
12.
PLoS One ; 15(3): e0230431, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32191756

RESUMO

Mollusk shell is composed of two CaCO3 polymorphs (calcite and aragonite) and an organic matrix that consists of acetic acid- or ethylenediaminetetraacetic acid (EDTA)-soluble and insoluble proteins and other biomolecules (polysaccharides, ß-chitin). However, the shell matrix proteins involved in nacre formation are not fully known. Thus, the aim of this study was to identify and characterize a novel protein from the acetic acid-insoluble fraction from the shell of Pteria sterna, named in this study as Ps19, to have a better understanding of the biomineralization process. Ps19 biochemical characterization showed that it is a glycoprotein that exhibits calcium- and chitin-binding capabilities. Additionally, it is capable of inducing aragonite plate crystallization in vitro. Ps19 partial peptide sequence showed similarity with other known shell matrix proteins, but it displayed similarity with proteins from Crassostrea gigas, Mizuhopecten yessoensis, Biomphalaria glabrata, Alpysia californica, Lottia gigantea and Elysia chlorotica. The results obtained indicated that Ps19 might play an important role in nacre growth of mollusk shells.


Assuntos
Calcificação Fisiológica , Carbonato de Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Quitina/metabolismo , Pinctada/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Cristalização , Peptídeos/química , Peptídeos/metabolismo , Sais , Solubilidade , Análise Espectral Raman
13.
Syst Appl Microbiol ; 43(3): 126075, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32173136

RESUMO

Bacillus cereus s.l., Gram-positive endospore-forming bacilli, persist ubiquitously in different natural habitats and play various ecological roles. Nevertheless, although chitin is one of the most abundant polymer on Earth, the study of the ability of B. cereus s.l. to hydrolyze this polymer were limited to individual B. cereus and B. thuringiensis strains only. Thus, to fill this gap in this research we focused on (i) the linkage between the capability to chitin degradation and the phylogenetic relatedness of B. cereus s.l. strains, and (ii) the genetic background of chitinolytic properties of these bacilli. Our results showed that chitin degradation is common among the B. cereus group members, yet strains clustered into particular phylogenetic groups differ in their chitinolytic capacity. Separate clustering of chitinolytic and non-chitinolytic strains in the phylogenetic tree indicates the ecotypic structure of these isolates. Two proteins belonging to subfamily A (ChiA) and subfamily B (ChiB) of the glycoside hydrolase GH18 family exhibited simultaneous chitobiosidase and endochitinase activities, and are responsible for chitin utilization by environmental B. cereus s.l. isolates.


Assuntos
Bacillus cereus/classificação , Bacillus cereus/fisiologia , Quitina/metabolismo , Microbiologia Ambiental , Filogenia , Quitinases/genética , Quitinases/metabolismo , Ativação Enzimática , Evolução Molecular , Patrimônio Genético , Variação Genética , Hidrólise
14.
Carbohydr Polym ; 235: 115952, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32122487

RESUMO

Chitin is the second most abundant and renewable polysaccharide, next to cellulose. Hydrolysis of abundant and highly crystalline α-chitin, pretreated with KOH and KOH-urea aqueous solutions, by a single modular endo-chitinase from Enterobacter cloacae subsp. cloacae (EcChi1) was investigated. The hydrolysis of untreated α-chitin and colloidal chitin by EcChi1 produced N-acetylglucosamine and N, N'-diacetylchitobiose, whereas, hydrolysis of treated substrates generated N, N', N''-triacetylchitotriose, in addition to N-acetylglucosamine and N, N'-diacetylchitobiose. The total amount of chitooligosaccharides (COS) generated by EcChi1 from pretreated substrates was 10 to 25-fold higher compared to untreated α-chitin at 24 h (depending on the solvent type and state of substrate). EcChi1 released higher amount of DP1 and DP2 products on treated α-chitin, with a fold change of 45 and 18, respectively. Treatment of α-chitin with KOH/KOH-urea is, therefore, a promising approach for an efficient conversion of rich source of chitin to soluble COS by chitinases like EcChi1.


Assuntos
Quitina/química , Quitinases/química , Enterobacter cloacae/enzimologia , Hidróxidos/química , Compostos de Potássio/química , Ureia/química , Quitina/metabolismo , Quitinases/metabolismo , Hidrólise , Hidróxidos/metabolismo , Compostos de Potássio/metabolismo , Ureia/metabolismo
15.
Arch Insect Biochem Physiol ; 104(1): e21666, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32112466

RESUMO

Chitin deacetylase (CDA) is a hydrolytic enzyme that modifies chitin into chitosan in the body of insects. In this study, we obtained a full-length complementary DNA sequence (MsCDA1) from the oriental armyworm Mythimna separata by high-throughput sequencing. MsCDA1 is 1,952 bp long and includes 1,620 bp open reading frame encoding 539 amino acids. Analysis by quantitative real time polymerase chain reaction showed that MsCDA1 expression was higher at the adult stage than at earlier developmental stages. MsCDA1 was expressed in all larval tissues examined, in which the highest expression level was found in the midgut. The RNA interference (RNAi) suppressed MsCDA1 expression levels at 12, 24, and 48 hr after injection of double-stranded RNA (1-4 µg per larva) specific to MsCDA1. Under RNAi condition, CDA enzyme activity was significantly reduced and changes an ultramicroscopic structure of M. separata peritrophic matrix especially in its microfibrillar organization exhibiting loose network. In contrast, the surface of the peritrophic matrix was relatively smooth and well organized at control or low RNAi conditions. Moreover, RNAi of MsCDA1 expression impaired larval growth and development, occasionally leading to larval death. These results demonstrate that MsCDA1 plays a crucial role in maintaining peritrophic matrix integrity in M. separata.


Assuntos
Amidoidrolases/genética , Mariposas/enzimologia , Amidoidrolases/metabolismo , Sequência de Aminoácidos , Animais , Quitina/metabolismo , Trato Gastrointestinal/ultraestrutura , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Interferência de RNA , Análise de Sequência de DNA
16.
Mol Phylogenet Evol ; 148: 106805, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32217169

RESUMO

The anemone-crab mutualism is ubiquitous in temperate and tropical marine environments. In this symbiosis, one or more anemones live on a shell inhabited by a hermit crab and reciprocal phoretic, trophic, and defensive benefits are exchanged between the partners. Sea anemone-hermit crab symbionts belong to three families: Hormathiidae (Calliactis and Paracalliactis), Sagartiidae (Carcinactis and Verrillactis), and Actiniidae (Stylobates). Hermit crabs establish most partnerships by detaching anemones and placing them on their shell; sea anemones can also mount shells unaided, triggered by a mollusc-derived substance in the periostracum of the shell. At least partial cooperation by the anemones is necessary for successful establishment of the symbiosis. Here, we expand the evolutionary framework for hormathiid symbionts by generating a phylogeny with at least one member of each actiniarian symbiotic genus with hermit crabs using five molecular markers (16S, 12S, 18S, 28S, CO3). We not only corroborated the results from a previous study by finding two origins of hermit crab symbiosis within Hormathiidae, but also found additional origins for hermit crab symbiosis within Actiniaria. We provide for the first time evidence of a close relationship between symbionts Carcinactis dolosa and V. paguri. The ability to secrete chitin by the ectoderm of the column is inferred to be broadly convergent within Actiniaria whereas the secretion of a chitinous carcinoecium by the pedal disc is a distinct but convergent morphological adaptation of several lineages within Actiniaria. Our finding of multiple origins for both the hermit crab and gastropod symbioses suggests that the shell-mounting behavior might only have been the precursor of the hermit crab association among Calliactis spp.


Assuntos
Anomuros/fisiologia , Evolução Biológica , Gastrópodes/fisiologia , Anêmonas-do-Mar/fisiologia , Simbiose/fisiologia , Animais , Quitina/metabolismo , Filogenia , Anêmonas-do-Mar/classificação
17.
Gene ; 735: 144403, 2020 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-32004668

RESUMO

Melon (Cucumis melo L.), an economically beneficial crop widely cultivated around the world, is vulnerable to powdery mildew (PM). However, the studies on molecular mechanism of melon response to PM fungi is still limited. Long non coding RNAs (lncRNAs) have emerged as new regulators in plants response to biotic stresses. We predicted and identified the intricate regulatory roles of lncRNAs in melon response to PM fungi. A total of 539 lncRNAs were identified from PM-resistant (MR-1) and susceptible melon (Top Mark), in which 254 were significantly altered after PM fungi infection. Multiple target genes of lncRNAs were found to be involved in the hydrolysis of chitin, callose deposition and cell wall thickening, plant-pathogen interaction and plant hormone signal transduction pathway. Additionally, a total of 42 lncRNAs possess the various functions with microRNAs (miRNAs), including lncRNAs that are targeted by miRNAs and function as miRNA precursors or miRNA sponges. These findings provide a comprehensive view of potentially functional lncRNAs, corresponding target genes and related lncRNA-miRNA pairs, which will greatly increase our knowledge of the mechanism underlying susceptibility and resistance to PM in melon.


Assuntos
Ascomicetos/patogenicidade , Cucurbitaceae/genética , Resistência à Doença , Redes Reguladoras de Genes , RNA Longo não Codificante/genética , Quitina/metabolismo , Cucurbitaceae/microbiologia , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Transcriptoma
18.
PLoS Pathog ; 16(2): e1008320, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32078661

RESUMO

Fungal parasitism depends on the ability to invade host organisms and mandates adaptive cell wall remodeling to avoid detection and defense reactions by the host. All plant and human pathogens share invasive strategies, which aid to escape the chitin-triggered and chitin-targeted host immune system. Here we describe the full spectrum of the chitin/chitosan-modifying enzymes in the mycoparasite Trichoderma atroviride with a central role in cell wall remodeling. Rapid adaption to a variety of growth conditions, environmental stresses and host defense mechanisms such as oxidative stress depend on the concerted interplay of these enzymes and, ultimately, are necessary for the success of the mycoparasitic attack. To our knowledge, we provide the first in class description of chitin and associated glycopolymer synthesis in a mycoparasite and demonstrate that they are essential for biocontrol. Eight chitin synthases, six chitin deacetylases, additional chitinolytic enzymes, including six chitosanases, transglycosylases as well as accessory proteins are involved in this intricately regulated process. Systematic and biochemical classification, phenotypic characterization and mycoparasitic confrontation assays emphasize the importance of chitin and chitosan assembly in vegetative development and biocontrol in T. atroviride. Our findings critically contribute to understanding the molecular mechanism of chitin synthesis in filamentous fungi and mycoparasites with the overarching goal to selectively exploit the discovered biocontrol strategies.


Assuntos
Quitina/metabolismo , Quitosana/metabolismo , Trichoderma/metabolismo , Parede Celular/metabolismo , Quitina/fisiologia , Quitina Sintase/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Glicosídeo Hidrolases , Filogenia , Plantas/metabolismo , Trichoderma/crescimento & desenvolvimento , Trichoderma/patogenicidade
19.
Mar Drugs ; 18(2)2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32019265

RESUMO

: Chitin is one of the most abundant biomolecules on earth, occurring in crustacean shells and cell walls of fungi. While the polysaccharide is threatening to pollute coastal ecosystems in the form of accumulating shell-waste, it has the potential to be converted into highly profitable derivatives with applications in medicine, biotechnology, and wastewater treatment, among others. Traditionally this is still mostly done by the employment of aggressive chemicals, yielding low quality while producing toxic by-products. In the last decades, the enzymatic conversion of chitin has been on the rise, albeit still not on the same level of cost-effectiveness compared to the traditional methods due to its multi-step character. Another severe drawback of the biotechnological approach is the highly ordered structure of chitin, which renders it nigh impossible for most glycosidic hydrolases to act upon. So far, only the Auxiliary Activity 10 family (AA10), including lytic polysaccharide monooxygenases (LPMOs), is known to hydrolyse native recalcitrant chitin, which spares the expensive first step of chemical or mechanical pre-treatment to enlarge the substrate surface. The main advantages of enzymatic conversion of chitin over conventional chemical methods are the biocompability and, more strikingly, the higher product specificity, product quality, and yield of the process. Products with a higher Mw due to no unspecific depolymerisation besides an exactly defined degree and pattern of acetylation can be yielded. This provides a new toolset of thousands of new chitin and chitosan derivatives, as the physio-chemical properties can be modified according to the desired application. This review aims to provide an overview of the biotechnological tools currently at hand, as well as challenges and crucial steps to achieve the long-term goal of enzymatic conversion of native chitin into specialty chemical products.


Assuntos
Biotecnologia , Quitina/química , Quitosana/química , Animais , Quitina/isolamento & purificação , Quitina/metabolismo , Quitosana/metabolismo , Crustáceos/metabolismo , Ecossistema , Fungos/metabolismo
20.
Molecules ; 25(3)2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-32012651

RESUMO

Chitin biomass, a rich renewable resource, is the second most abundant natural polysaccharide after cellulose. Conversion of chitin biomass to high value-added chemicals can play a significant role in alleviating the global energy crisis and environmental pollution. In this review, the recent achievements in converting chitin biomass to high-value chemicals, such as 5-hydroxymethylfurfural (HMF), under different conditions using chitin, chitosan, glucosamine, and N-acetylglucosamine as raw materials are summarized. Related research on pretreatment technology of chitin biomass is also discussed. New approaches for transformation of chitin biomass to HMF are also proposed. This review promotes the development of industrial technologies for degradation of chitin biomass and preparation of HMF. It also provides insight into a sustainable future in terms of renewable resources.


Assuntos
Quitina/metabolismo , Poluição Ambiental/prevenção & controle , Furaldeído/análogos & derivados , Biomassa , Furaldeído/metabolismo
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