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1.
Front Cell Infect Microbiol ; 14: 1369301, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38774630

RESUMO

Dual-specificity LAMMER kinases are highly evolutionarily conserved in eukaryotes and play pivotal roles in diverse physiological processes, such as growth, differentiation, and stress responses. Although the functions of LAMMER kinase in fungal pathogens in pathogenicity and stress responses have been characterized, its role in Cryptococcus neoformans, a human fungal pathogen and a model yeast of basidiomycetes, remains elusive. In this study, we identified a LKH1 homologous gene and constructed a strain with a deleted LKH1 and a complemented strain. Similar to other fungi, the lkh1Δ mutant showed intrinsic growth defects. We observed that C. neoformans Lkh1 was involved in diverse stress responses, including oxidative stress and cell wall stress. Particularly, Lkh1 regulates DNA damage responses in Rad53-dependent and -independent manners. Furthermore, the absence of LKH1 reduced basidiospore formation. Our observations indicate that Lkh1 becomes hyperphosphorylated upon treatment with rapamycin, a TOR protein inhibitor. Notably, LKH1 deletion led to defects in melanin synthesis and capsule formation. Furthermore, we found that the deletion of LKH1 led to the avirulence of C. neoformans in a systemic cryptococcosis murine model. Taken together, Lkh1 is required for the stress response, sexual differentiation, and virulence of C. neoformans.


Assuntos
Criptococose , Cryptococcus neoformans , Melaninas , Estresse Oxidativo , Estresse Fisiológico , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/genética , Cryptococcus neoformans/enzimologia , Virulência , Animais , Criptococose/microbiologia , Camundongos , Melaninas/metabolismo , Modelos Animais de Doenças , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Fosforilação , Dano ao DNA , Parede Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Cápsulas Fúngicas/metabolismo , Cápsulas Fúngicas/genética , Sirolimo/farmacologia , Camundongos Endogâmicos BALB C , Feminino , Esporos Fúngicos/crescimento & desenvolvimento
2.
Int J Biol Macromol ; 268(Pt 1): 131619, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38692998

RESUMO

The plant cell wall is a complex, heterogeneous structure primarily composed of cellulose, hemicelluloses, and lignin. Exploring the variations in these three macromolecules over time is crucial for understanding wood formation to enhance chemical processing and utilization. Here, we comprehensively analyzed the chemical composition of cell walls in the trunks of Pinus tabulaeformis using multiple techniques. In situ analysis showed that macromolecules accumulated gradually in the cell wall as the plant aged, and the distribution pattern of lignin was opposite that of polysaccharides, and both showed heterogenous distribution patterns. In addition, gel permeation chromatography (GPC) results revealed that the molecular weights of hemicelluloses decreased while that of lignin increased with age. Two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance (2D-HSQC NMR) analysis indicated that hemicelluloses mainly comprised galactoglucomannan and arabinoglucuronoxylan, and the lignin types were mainly comprised guaiacyl (G) and p-hydroxyphenyl (H) units with three main linkage types: ß-O-4, ß-ß, and ß-5. Furthermore, the C-O bond (ß-O-4) signals of lignin decreased while the C-C bonds (ß-ß and ß-5) signals increased over time. Taken together, these findings shed light on wood formation in P. tabulaeformis and lay the foundation for enhancing the processing and use of wood and timber products.


Assuntos
Parede Celular , Celulose , Lignina , Pinus , Polissacarídeos , Lignina/química , Pinus/química , Parede Celular/química , Polissacarídeos/química , Celulose/química , Peso Molecular , Árvores/química , Espectroscopia de Ressonância Magnética/métodos , Madeira/química
3.
Carbohydr Polym ; 337: 122149, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38710571

RESUMO

Phytopathogen cell wall polysaccharides have important physiological functions. In this study, we isolated and characterized the alkali-insoluble residue on the inner layers of the Rhizoctonia solani AG1 IA cell wall (RsCW-AIR). Through chemical composition and structural analysis, RsCW-AIR was mainly identified as a complex of chitin/chitosan and glucan (ChCsGC), with glucose and glucosamine were present in a molar ratio of 2.7:1.0. The predominant glycosidic bond linkage of glucan in ChCsGC was ß-1,3-linked Glcp, both the α and ß-polymorphic forms of chitin were presented in it by IR, XRD, and solid-state NMR, and the ChCsGC exhibited a degree of deacetylation measuring 67.08 %. RsCW-AIR pretreatment effectively reduced the incidence of rice sheath blight, and its induced resistance activity in rice was evaluated, such as inducing a reactive oxygen species (ROS) burst, leading to the accumulation of salicylic acid (SA) and the up-regulation of SA-related gene expression. The recognition of RsCW-AIR in rice is partially dependent on CERK1.


Assuntos
Parede Celular , Quitina , Quitosana , Glucanos , Oryza , Doenças das Plantas , Rhizoctonia , Rhizoctonia/efeitos dos fármacos , Oryza/microbiologia , Oryza/química , Parede Celular/química , Quitosana/química , Quitosana/farmacologia , Quitina/química , Quitina/farmacologia , Glucanos/química , Glucanos/farmacologia , Doenças das Plantas/microbiologia , Resistência à Doença , Espécies Reativas de Oxigênio/metabolismo
4.
Nat Commun ; 15(1): 3792, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38710711

RESUMO

Infection with the apicomplexan protozoan Toxoplasma gondii can be life-threatening in immunocompromised hosts. Transmission frequently occurs through the oral ingestion of T. gondii bradyzoite cysts, which transition to tachyzoites, disseminate, and then form cysts containing bradyzoites in the central nervous system, resulting in latent infection. Encapsulation of bradyzoites by a cyst wall is critical for immune evasion, survival, and transmission. O-glycosylation of the protein CST1 by the mucin-type O-glycosyltransferase T. gondii (Txg) GalNAc-T3 influences cyst wall rigidity and stability. Here, we report X-ray crystal structures of TxgGalNAc-T3, revealing multiple features that are strictly conserved among its apicomplexan homologues. This includes a unique 2nd metal that is coupled to substrate binding and enzymatic activity in vitro and cyst wall O-glycosylation in T. gondii. The study illustrates the divergence of pathogenic protozoan GalNAc-Ts from their host homologues and lays the groundwork for studying apicomplexan GalNAc-Ts as therapeutic targets in disease.


Assuntos
Proteínas de Protozoários , Toxoplasma , Toxoplasma/enzimologia , Toxoplasma/genética , Glicosilação , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/química , Humanos , Cristalografia por Raios X , Glicosiltransferases/metabolismo , Glicosiltransferases/genética , Parede Celular/metabolismo , Animais
5.
Appl Microbiol Biotechnol ; 108(1): 324, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38713211

RESUMO

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system. KEY POINTS: • ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome. • Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces. • High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter.


Assuntos
Quitinases , Inativação Gênica , Lacase , Quitinases/genética , Quitinases/metabolismo , Quitinases/biossíntese , Lacase/genética , Lacase/metabolismo , Lacase/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Agaricales/genética , Agaricales/enzimologia , Fermentação , Interferência de RNA , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/enzimologia , Parede Celular/metabolismo , Parede Celular/genética
6.
Glycobiology ; 34(6)2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38690785

RESUMO

Cellulose is an abundant component of plant cell wall matrices, and this para-crystalline polysaccharide is synthesized at the plasma membrane by motile Cellulose Synthase Complexes (CSCs). However, the factors that control CSC activity and motility are not fully resolved. In a targeted chemical screen, we identified the alkylated nojirimycin analog N-Dodecyl Deoxynojirimycin (ND-DNJ) as a small molecule that severely impacts Arabidopsis seedling growth. Previous work suggests that ND-DNJ-related compounds inhibit the biosynthesis of glucosylceramides (GlcCers), a class of glycosphingolipid associated with plant membranes. Our work uncovered major changes in the sphingolipidome of plants treated with ND-DNJ, including reductions in GlcCer abundance and altered acyl chain length distributions. Crystalline cellulose content was also reduced in ND-DNJ-treated plants as well as plants treated with the known GlcCer biosynthesis inhibitor N-[2-hydroxy-1-(4-morpholinylmethyl)-2-phenyl ethyl]-decanamide (PDMP) or plants containing a genetic disruption in GLUCOSYLCERAMIDE SYNTHASE (GCS), the enzyme responsible for sphingolipid glucosylation that results in GlcCer synthesis. Live-cell imaging revealed that CSC speed distributions were reduced upon treatment with ND-DNJ or PDMP, further suggesting an important relationship between glycosylated sphingolipid composition and CSC motility across the plasma membrane. These results indicate that multiple interventions compromising GlcCer biosynthesis disrupt cellulose deposition and CSC motility, suggesting that GlcCers regulate cellulose biosynthesis in plants.


Assuntos
Arabidopsis , Celulose , Glucosilceramidas , Glucosiltransferases , Arabidopsis/metabolismo , Glucosiltransferases/metabolismo , Glucosiltransferases/genética , Celulose/metabolismo , Celulose/biossíntese , Glucosilceramidas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , 1-Desoxinojirimicina/farmacologia , 1-Desoxinojirimicina/análogos & derivados , Parede Celular/metabolismo
7.
J R Soc Interface ; 21(214): 20240008, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38715319

RESUMO

Multicellular organisms grow and acquire their shapes through the differential expansion and deformation of their cells. Recent research has addressed the role of cell and tissue mechanical properties in these processes. In plants, it is believed that growth rate is a function of the mechanical stress exerted on the cell wall, the thin polymeric layer surrounding cells, involving an effective viscosity. Nevertheless, recent studies have questioned this view, suggesting that cell wall elasticity sets the growth rate or that uptake of water is limiting for plant growth. To assess these issues, we developed a microfluidic device to quantify the growth rates, elastic properties and hydraulic conductivity of individual Marchantia polymorpha plants in a controlled environment with a high throughput. We characterized the effect of osmotic treatment and abscisic acid on growth and hydromechanical properties. Overall, the instantaneous growth rate of individuals is correlated with both bulk elastic modulus and hydraulic conductivity. Our results are consistent with a framework in which the growth rate is determined primarily by the elasticity of the wall and its remodelling, and secondarily by hydraulic conductivity. Accordingly, the coupling between the chemistry of the cell wall and the hydromechanics of the cell appears as key to set growth patterns during morphogenesis.


Assuntos
Parede Celular , Parede Celular/fisiologia , Marchantia/crescimento & desenvolvimento , Marchantia/fisiologia , Ácido Abscísico/metabolismo , Modelos Biológicos , Fenômenos Biomecânicos , Desenvolvimento Vegetal/fisiologia
8.
Sci Rep ; 14(1): 10237, 2024 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-38702505

RESUMO

Enzymatic degradation of algae cell wall carbohydrates by microorganisms is under increasing investigation as marine organic matter gains more value as a sustainable resource. The fate of carbon in the marine ecosystem is in part driven by these degradation processes. In this study, we observe the microbiome dynamics of the macroalga Fucus vesiculosus in 25-day-enrichment cultures resulting in partial degradation of the brown algae. Microbial community analyses revealed the phylum Pseudomonadota as the main bacterial fraction dominated by the genera Marinomonas and Vibrio. More importantly, a metagenome-based Hidden Markov model for specific glycosyl hydrolyses and sulphatases identified Bacteroidota as the phylum with the highest potential for cell wall degradation, contrary to their low abundance. For experimental verification, we cloned, expressed, and biochemically characterised two α-L-fucosidases, FUJM18 and FUJM20. While protein structure predictions suggest the highest similarity to a Bacillota origin, protein-protein blasts solely showed weak similarities to defined Bacteroidota proteins. Both enzymes were remarkably active at elevated temperatures and are the basis for a potential synthetic enzyme cocktail for large-scale algal destruction.


Assuntos
Parede Celular , Fucus , Metagenômica , Parede Celular/metabolismo , Fucus/metabolismo , Fucus/genética , Fucus/microbiologia , Metagenômica/métodos , Bacteroidetes/genética , Bacteroidetes/enzimologia , Metagenoma , Microbiota , Filogenia
9.
Curr Genet ; 70(1): 6, 2024 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-38733432

RESUMO

The gene products of PRS1-PRS5 in Saccharomyces cerevisiae are responsible for the production of PRPP (5-phospho-D-ribosyl-α-1-pyrophosphate). However, it has been demonstrated that they are also involved in the cell wall integrity (CWI) signalling pathway as shown by protein-protein interactions (PPIs) with, for example Slt2, the MAP kinase of the CWI pathway. The following databases: SGD, BioGRID and Hit Predict, which collate PPIs from various research papers, have been scrutinized for evidence of PPIs between Prs1-Prs5 and components of the CWI pathway. The level of certainty in PPIs was verified by interaction scores available in the Hit Predict database revealing that well-documented interactions correspond with higher interaction scores and can be graded as high confidence interactions based on a score > 0.28, an annotation score ≥ 0.5 and a method-based high confidence score level of ≥ 0.485. Each of the Prs1-Prs5 polypeptides shows some degree of interaction with the CWI pathway. However, Prs5 has a vital role in the expression of FKS2 and Rlm1, previously only documented by reporter assay studies. This report emphasizes the importance of investigating interactions using more than one approach since every method has its limitations and the use of different methods, as described herein, provides complementary experimental and statistical data, thereby corroborating PPIs. Since the experimental data described so far are consistent with a link between PRPP synthetase and the CWI pathway, our aim was to demonstrate that these data are also supported by high-throughput bioinformatic analyses promoting our hypothesis that two of the five PRS-encoding genes contain information required for the maintenance of CWI by combining data from our targeted approach with relevant, unbiased data from high-throughput analyses.


Assuntos
Parede Celular , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Parede Celular/metabolismo , Parede Celular/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Mapas de Interação de Proteínas , Mapeamento de Interação de Proteínas
10.
Proc Natl Acad Sci U S A ; 121(21): e2314570121, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38739804

RESUMO

Lipid polymers such as cutin and suberin strengthen the diffusion barrier properties of the cell wall in specific cell types and are essential for water relations, mineral nutrition, and stress protection in plants. Land plant-specific glycerol-3-phosphate acyltransferases (GPATs) of different clades are central players in cutin and suberin monomer biosynthesis. Here, we show that the GPAT4/6/8 clade in Arabidopsis thaliana, which is known to mediate cutin formation, is also required for developmentally regulated root suberization, in addition to the established roles of GPAT5/7 in suberization. The GPAT5/7 clade is mainly required for abscisic acid-regulated suberization. In addition, the GPAT5/7 clade is crucial for the formation of the typical lamellated suberin ultrastructure observed by transmission electron microscopy, as distinct amorphous globular polyester structures were deposited in the apoplast of the gpat5 gpat7 double mutant, in contrast to the thinner but still lamellated suberin deposition in the gpat4 gpat6 gpat8 triple mutant. Site-directed mutagenesis revealed that the intrinsic phosphatase activity of GPAT4, GPAT6, and GPAT8, which leads to monoacylglycerol biosynthesis, contributes to suberin formation. GPAT5/7 lack an active phosphatase domain and the amorphous globular polyester structure observed in the gpat5 gpat7 double mutant was partially reverted by treatment with a phosphatase inhibitor or the expression of phosphatase-dead variants of GPAT4/6/8. Thus, GPATs that lack an active phosphatase domain synthetize lysophosphatidic acids that might play a role in the formation of the lamellated structure of suberin. GPATs with active and nonactive phosphatase domains appear to have nonredundant functions and must cooperate to achieve the efficient biosynthesis of correctly structured suberin.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Lipídeos , Raízes de Plantas , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Lipídeos/química , Regulação da Expressão Gênica de Plantas , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Glicerol-3-Fosfato O-Aciltransferase/genética , Lipídeos de Membrana/metabolismo , Ácido Abscísico/metabolismo , Parede Celular/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase
11.
Sci Rep ; 14(1): 11454, 2024 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-38769105

RESUMO

This study focuses on pectin covalently linked in cell walls from two sources, apples and carrots, that was extracted using diluted alkali, and it describes changes in the rheological properties of diluted alkali-soluble pectin (DASP) due to enzymatic treatment. Given DASP's richness of rhamnogalacturonan I (RG-I), RG-I acetyl esterase (RGAE), rhamnogalacturonan endolyase (RGL), and arabinofuranosidase (ABF) were employed in various combinations for targeted degradation of RG-I pectin chains. Enzymatic degradations were followed by structural studies of pectin molecules using atomic force microscopy (AFM) as well as measurements of rheological and spectral properties. AFM imaging revealed a significant increase in the length of branched molecules after incubation with ABF, suggesting that arabinose side chains limit RG-I aggregation. Structural modifications were confirmed by changes in the intensity of bands in the pectin fingerprint and anomeric region on Fourier transform infrared spectra. ABF treatment led to a decrease in the stability of pectic gels, while the simultaneous use of ABF, RGAE, and RGL enzymes did not increase the degree of aggregation compared to the control sample. These findings suggest that the association of pectin chains within the DASP fraction may rely significantly on intermolecular interactions. Two mechanisms are proposed, which involve side chains as short-range attachment points or an extended linear homogalacturonan conformation favoring inter-chain interactions over self-association.


Assuntos
Pectinas , Reologia , Pectinas/química , Pectinas/metabolismo , Microscopia de Força Atômica , Álcalis/química , Glicosídeo Hidrolases/metabolismo , Glicosídeo Hidrolases/química , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier , Daucus carota/química , Polissacarídeo-Liases/metabolismo , Polissacarídeo-Liases/química , Parede Celular/química , Parede Celular/metabolismo
12.
Proc Natl Acad Sci U S A ; 121(21): e2319707121, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38743622

RESUMO

Glycogen is a glucose storage molecule composed of branched α-1,4-glucan chains, best known as an energy reserve that can be broken down to fuel central metabolism. Because fungal cells have a specialized need for glucose in building cell wall glucans, we investigated whether glycogen is used for this process. For these studies, we focused on the pathogenic yeast Cryptococcus neoformans, which causes ~150,000 deaths per year worldwide. We identified two proteins that influence formation of both glycogen and the cell wall: glycogenin (Glg1), which initiates glycogen synthesis, and a protein that we call Glucan organizing enzyme 1 (Goe1). We found that cells missing Glg1 lack α-1,4-glucan in their walls, indicating that this material is derived from glycogen. Without Goe1, glycogen rosettes are mislocalized and ß-1,3-glucan in the cell wall is reduced. Altogether, our results provide mechanisms for a close association between glycogen and cell wall.


Assuntos
Parede Celular , Cryptococcus neoformans , Proteínas Fúngicas , Glucanos , Glicogênio , Parede Celular/metabolismo , Glicogênio/metabolismo , Glucanos/metabolismo , Proteínas Fúngicas/metabolismo , Cryptococcus neoformans/metabolismo , Glucosiltransferases/metabolismo , beta-Glucanas/metabolismo
13.
Methods Mol Biol ; 2775: 225-237, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38758321

RESUMO

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size varies widely between strains, has the ability to grow rapidly when introduced to stressful or low-nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. Inducing the growth of the C. neoformans capsule is used during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here, we describe one of the standard methods of capsule induction and detail two accepted methods of staining: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we outline how to measure capsule diameter manually and offer a protocol for automated diameter measurement of India ink-stained samples using computational image analysis.


Assuntos
Cryptococcus neoformans , Coloração e Rotulagem , Cryptococcus neoformans/citologia , Coloração e Rotulagem/métodos , Microscopia Confocal/métodos , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Cápsulas Fúngicas/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Corantes Fluorescentes/química , Carbono
14.
Methods Mol Biol ; 2775: 329-347, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38758327

RESUMO

The cell wall of the fungal pathogens Cryptococcus neoformans and C. gattii is critical for cell wall integrity and signaling external threats to the cell, allowing it to adapt and grow in a variety of changing environments. Chitin is a polysaccharide found in the cell walls of fungi that is considered to be essential for fungal survival. Chitosan is a polysaccharide derived from chitin via deacetylation that is also essential for cryptococcal cell wall integrity, fungal pathogenicity, and virulence. Cryptococcus has evolved mechanisms to regulate the amount of chitin and chitosan during growth under laboratory conditions or during mammalian infection. Therefore, levels of chitin and chitosan have been useful phenotypes to define mutant Cryptococcus strains. As a result, we have developed and/or refined various qualitative and quantitative methods for measuring chitin and chitosan. These techniques include those that use fluorescent probes that are known to bind to chitin (e.g., calcofluor white and wheat germ agglutinin), as well as those that preferentially bind to chitosan (e.g., eosin Y and cibacron brilliant red 3B-A). Techniques that enhance the localization and quantification of chitin and chitosan in the cell wall include (i) fluorescence microscopy, (ii) flow cytometry, (iii) and spectrofluorometry. We have also modified two highly selective biochemical methods to measure cellular chitin and chitosan content: the Morgan-Elson and the 3-methyl-2-benzothiazolone hydrazine hydrochloride (MBTH) assays, respectively.


Assuntos
Parede Celular , Quitina , Quitosana , Quitina/metabolismo , Quitina/química , Quitina/análise , Quitosana/química , Quitosana/metabolismo , Parede Celular/metabolismo , Parede Celular/química , Cryptococcus neoformans/metabolismo , Corantes Fluorescentes/química , Cryptococcus/metabolismo , Microscopia de Fluorescência/métodos
15.
Artigo em Inglês | MEDLINE | ID: mdl-38752995

RESUMO

A novel actinobacterium, strain ZYX-F-186T, was isolated from marine sediment sampled on Yongxing Island, Hainan Province, PR China. Based on the results of 16S rRNA gene sequence analysis, strain ZYX-F-186T belongs to the genus Phytohabitans, with high similarity to Phytohabitans kaempferiae KK1-3T (98.3 %), Phytohabitans rumicis K11-0047T (98.1 %), Phytohabitans flavus K09-0627T (98.1 %), Phytohabitans houttuyneae K11-0057T (97.9 %), Phytohabitans suffuscus K07-0523T (97.7 %), and Phytohabitans aurantiacus RD004123T (97.7 %). Phylogenetic analysis of 16S rRNA gene sequences showed that the strain formed a single subclade in the genus Phytohabitans. The novel isolate contained meso-diaminopimelic acid, d-glutamic acid, glycine, d-alanine, and l-lysine in the cell wall. The whole-cell sugars were xylose, arabinose, ribose, and rhamnose. The predominant menaquinones were MK-9(H8), MK-9(H6), and MK-9(H4). The characteristic phospholipids were phosphatidylethanolamine, phosphatidylinositol, phosphatidylmethylethanolamine, phosphatidylglycerol, and an unknown phospholipid. The major fatty acids (>5 %) were iso-C16 : 0, anteiso-C17 : 0, and iso-C18 : 0. Genome sequencing showed a DNA G+C content of 71.9 mol%. Low average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity values demonstrated that strain ZYX-F-186T could be readily distinguished from its closely related species. Based on its phylogenetic, chemotaxonomic, and physiological characteristics, strain ZYX-F-186T represents a novel species of the genus Phytohabitans, for which the name Phytohabitans maris sp. nov. is proposed. The type strain is ZYX-F-186T (=CGMCC 4.8025T=CCTCC AA 2023025T=JCM 36507T).


Assuntos
Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano , Ácidos Graxos , Sedimentos Geológicos , Filogenia , RNA Ribossômico 16S , Análise de Sequência de DNA , Sedimentos Geológicos/microbiologia , RNA Ribossômico 16S/genética , China , DNA Bacteriano/genética , Ácidos Graxos/química , Fosfolipídeos , Vitamina K 2/análogos & derivados , Vitamina K 2/análise , Vitamina K 2/química , Hibridização de Ácido Nucleico , Parede Celular/química
16.
Int J Mol Sci ; 25(9)2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38732136

RESUMO

In the context of sustainable agriculture and biomaterial development, understanding and enhancing plant secondary cell wall formation are crucial for improving crop fiber quality and biomass conversion efficiency. This is especially critical for economically important crops like upland cotton (Gossypium hirsutum L.), for which fiber quality and its processing properties are essential. Through comprehensive genome-wide screening and analysis of expression patterns, we identified a particularly high expression of an R2R3 MYB transcription factor, GhMYB52 Like, in the development of the secondary cell wall in cotton fiber cells. Utilizing gene-editing technology to generate a loss-of-function mutant to clarify the role of GhMYB52 Like, we revealed that GhMYB52 Like does not directly contribute to cellulose synthesis in cotton fibers but instead represses a subset of lignin biosynthesis genes, establishing it as a lignin biosynthesis inhibitor. Concurrently, a substantial decrease in the lint index, a critical measure of cotton yield, was noted in parallel with an elevation in lignin levels. This study not only deepens our understanding of the molecular mechanisms underlying cotton fiber development but also offers new perspectives for the molecular improvement of other economically important crops and the enhancement of biomass energy utilization.


Assuntos
Fibra de Algodão , Regulação da Expressão Gênica de Plantas , Gossypium , Lignina , Proteínas de Plantas , Lignina/biossíntese , Gossypium/genética , Gossypium/metabolismo , Gossypium/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Parede Celular/metabolismo , Parede Celular/genética , Celulose/biossíntese , Celulose/metabolismo , Vias Biossintéticas
17.
Toxins (Basel) ; 16(5)2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38787069

RESUMO

The fungal cell wall serves as the primary interface between fungi and their external environment, providing protection and facilitating interactions with the surroundings. Chitin is a vital structural element in fungal cell wall. Chitin deacetylase (CDA) can transform chitin into chitosan through deacetylation, providing various biological functions across fungal species. Although this modification is widespread in fungi, the biological functions of CDA enzymes in Aspergillus flavus remain largely unexplored. In this study, we aimed to investigate the biofunctions of the CDA family in A. flavus. The A. flavus genome contains six annotated putative chitin deacetylases. We constructed knockout strains targeting each member of the CDA family, including Δcda1, Δcda2, Δcda3, Δcda4, Δcda5, and Δcda6. Functional analyses revealed that the deletion of CDA family members neither significantly affects the chitin content nor exhibits the expected chitin deacetylation function in A. flavus. However, the Δcda6 strain displayed distinct phenotypic characteristics compared to the wild-type (WT), including an increased conidia count, decreased mycelium production, heightened aflatoxin production, and impaired seed colonization. Subcellular localization experiments indicated the cellular localization of CDA6 protein within the cell wall of A. flavus filaments. Moreover, our findings highlight the significance of the CBD1 and CBD2 structural domains in mediating the functional role of the CDA6 protein. Overall, we analyzed the gene functions of CDA family in A. flavus, which contribute to a deeper understanding of the mechanisms underlying aflatoxin contamination and lay the groundwork for potential biocontrol strategies targeting A. flavus.


Assuntos
Aflatoxinas , Amidoidrolases , Aspergillus flavus , Aspergillus flavus/genética , Aspergillus flavus/enzimologia , Aspergillus flavus/metabolismo , Amidoidrolases/genética , Amidoidrolases/metabolismo , Aflatoxinas/biossíntese , Aflatoxinas/metabolismo , Aflatoxinas/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Quitina/metabolismo , Parede Celular/metabolismo
18.
Physiol Plant ; 176(3): e14330, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38698648

RESUMO

Wall-associated kinases (WAKs) have been determined to recognize pathogenic signals and initiate plant immune responses. However, the roles of the family members in host resistance against Valsa canker, a serious fungal disease of apples and pears, are largely unknown. Here, we identified MbWAK1 in Malus baccata, a resistant germplasm differentially expressed during infection by Valsa mali (Vm). Over-expression of MbWAK1 enhanced the Valsa canker resistance of apple and pear fruits and 'Duli-G03' (Pyrus betulifolia) suspension cells. A large number of phloem, cell wall, and lipid metabolic process-related genes were differentially expressed in overexpressed suspension cell lines in response to Valsa pyri (Vp) signals. Among these, the expression of xyloglucan endotransglucosylase/hydrolase (XTH) gene PbeXTH1 and sieve element occlusion B-like (SEOB) gene PbeSEOB1 were significantly inhibited. Transient expression of PbeXTH1 or PbeSEOB1 compromised the expressional induction of MbWAK1 and the resistance contributed by MbWAK1. In addition, PbeXTH1 and PbeSEOB1 suppressed the immune response induced by MbWAK1. Our results enriched the molecular mechanisms for MbWAK1 against Valsa canker and resistant breeding.


Assuntos
Resistência à Doença , Regulação da Expressão Gênica de Plantas , Malus , Doenças das Plantas , Proteínas de Plantas , Pyrus , Doenças das Plantas/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Resistência à Doença/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pyrus/genética , Pyrus/microbiologia , Malus/genética , Malus/microbiologia , Malus/imunologia , Malus/enzimologia , Parede Celular/metabolismo
19.
Int J Biol Macromol ; 268(Pt 1): 131601, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38626833

RESUMO

This study investigates the impact of water and salinity stress on Aloe vera, focusing on the role of Aloe vera polysaccharides in mitigating these stresses. Pectins and acemannan were the most affected polymers. Low soil moisture and high salinity (NaCl 80 mM) increased pectic substances, altering rhamnogalacturonan type I in Aloe vera gel. Aloe vera pectins maintained a consistent 60 % methyl-esterification regardless of conditions. Interestingly, acemannan content rose with salinity, particularly under low moisture, accompanied by 90 to 150 % acetylation increase. These changes improved the functionality of Aloe vera polysaccharides: pectins increased cell wall reinforcement and interactions, while highly acetylated acemannan retained water for sustained plant functions. This study highlights the crucial role of Aloe vera polysaccharides in enhancing plant resilience to water and salinity stress, leading to improved functional properties.


Assuntos
Aloe , Mananas , Pectinas , Aloe/química , Mananas/química , Pectinas/química , Água/química , Parede Celular/química , Parede Celular/efeitos dos fármacos , Salinidade , Polissacarídeos/química , Polissacarídeos/farmacologia , Tolerância ao Sal/efeitos dos fármacos , Acetilação , Estresse Fisiológico/efeitos dos fármacos
20.
Int J Biol Macromol ; 268(Pt 1): 131559, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38631576

RESUMO

Expansins are important plant cell wall proteins. They can loosen and soften the cell walls and lead to wall extension and cell expansion. To investigate their role in wood formation and fiber elongation, the PagEXPA1 that highly expressed in cell differentiation and expansion tissues was cloned from 84K poplar (Populus alba × P. glandulosa). The subcellular localization showed that PagEXPA1 located in the cell wall and it was highly expressed in primary stems and young leaves. Compared with non-transgenic 84K poplar, overexpression of PagEXPA1 can promote plant-growth, lignification, and fiber cell elongation, while PagEXPA1 Cas9-editing mutant lines exhibited the opposite phenotype. Transcriptome analysis revealed that DEGs were mainly enriched in some important processes, which are associated with cell wall formation and cellulose synthesis. The protein interaction prediction and expression analysis showed that PagCDKB2:1 and PagEXPA1 might have an interaction relationship. The luciferase complementary assay and bimolecular fluorescence complementary assay validated that PagEXPA1 can combined with PagCDKB2;1. So they promoted the expansion of xylem vascular tissues and the development of poplar though participating in the regulation of cell division and differentiation by programming the cell-cycle. It provides good foundation for molecular breeding of fast-growing and high-quality poplar varieties.


Assuntos
Parede Celular , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Populus , Populus/genética , Populus/crescimento & desenvolvimento , Populus/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Parede Celular/metabolismo , Parede Celular/genética , Plantas Geneticamente Modificadas , Perfilação da Expressão Gênica , Xilema/metabolismo , Xilema/genética , Desenvolvimento Vegetal/genética , Madeira/genética , Madeira/crescimento & desenvolvimento
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