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1.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068366

RESUMO

Magnaporthe oryzae (M. oryzae) is a typical cause of rice blast in agricultural production. Isobavachalcone (IBC), an active ingredient of Psoralea corylifolia L. extract, is an effective fungicide against rice blast. To determine the mechanism of IBC against M. oryzae, the effect of IBC on the metabolic pathway of M. oryzae was explored by transcriptome profiling. In M. oryzae, the expression of pyruvate dehydrogenase E1 (PDHE1), part of the tricarboxylic acid (TCA cycle), was significantly decreased in response to treatment with IBC, which was verified by qPCR and testing of enzyme activity. To further elucidate the interactions between IBC and PDHE1, the 3D structure model of the PDHE1 from M. oryzae was established based on homology modeling. The model was utilized to analyze the molecular interactions through molecular docking and molecular dynamics simulation, revealing that IBC has π-π stacking interactions with residue TYR139 and undergoes hydrogen bonding with residue ASP217 of PDHE1. Additionally, the nonpolar residues PHE111, MET174, ILE 187, VAL188, and MET250 form strong hydrophobic interactions with IBC. The above results reveal that PDHE1 is a potential target for antifungal agents, which will be of great significance for guiding the design of new fungicides. This research clarified the mechanism of IBC against M. oryzae at the molecular level, which will underpin further studies of the inhibitory mechanism of flavonoids and the discovery of new targets. It also provides theoretical guidance for the field application of IBC.


Assuntos
Chalconas/farmacologia , Proteínas Fúngicas/metabolismo , Magnaporthe/efeitos dos fármacos , Oryza/enzimologia , Doenças das Plantas/imunologia , Piruvato Desidrogenase (Lipoamida)/antagonistas & inibidores , Transcriptoma/efeitos dos fármacos , Proteínas Fúngicas/genética , Fungicidas Industriais/farmacologia , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Magnaporthe/fisiologia , Simulação de Acoplamento Molecular , Oryza/efeitos dos fármacos , Oryza/microbiologia , Doenças das Plantas/microbiologia , Conformação Proteica , Piruvato Desidrogenase (Lipoamida)/genética , Piruvato Desidrogenase (Lipoamida)/metabolismo
2.
BMC Genomics ; 22(1): 324, 2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947322

RESUMO

BACKGROUND: Cyclocybe aegerita (syn. Agrocybe aegerita) is a commercially cultivated mushroom. Its archetypal agaric morphology and its ability to undergo its whole life cycle under laboratory conditions makes this fungus a well-suited model for studying fruiting body (basidiome, basidiocarp) development. To elucidate the so far barely understood biosynthesis of fungal volatiles, alterations in the transcriptome during different developmental stages of C. aegerita were analyzed and combined with changes in the volatile profile during its different fruiting stages. RESULTS: A transcriptomic study at seven points in time during fruiting body development of C. aegerita with seven mycelial and five fruiting body stages was conducted. Differential gene expression was observed for genes involved in fungal fruiting body formation showing interesting transcriptional patterns and correlations of these fruiting-related genes with the developmental stages. Combining transcriptome and volatilome data, enzymes putatively involved in the biosynthesis of C8 oxylipins in C. aegerita including lipoxygenases (LOXs), dioxygenases (DOXs), hydroperoxide lyases (HPLs), alcohol dehydrogenases (ADHs) and ene-reductases could be identified. Furthermore, we were able to localize the mycelium as the main source for sesquiterpenes predominant during sporulation in the headspace of C. aegerita cultures. In contrast, changes in the C8 profile detected in late stages of development are probably due to the activity of enzymes located in the fruiting bodies. CONCLUSIONS: In this study, the combination of volatilome and transcriptome data of C. aegerita revealed interesting candidates both for functional genetics-based analysis of fruiting-related genes and for prospective enzyme characterization studies to further elucidate the so far barely understood biosynthesis of fungal C8 oxylipins.


Assuntos
Agaricales , Transcriptoma , Agaricales/genética , Agrocybe , Carpóforos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Oxilipinas , Estudos Prospectivos
3.
Pestic Biochem Physiol ; 175: 104835, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33993960

RESUMO

Rice blast (Magnaporthe oryzae), a major fungal disease in rice producing areas all over the world as well as in China, seriously affects the safety of rice production. Citral, a mixture of Z/E and trans isomers, is a natural acycloid monoterpene compound with good bacteriostatic effect on rice blast. To further investigate the underlying molecular mechanism, a comparative proteomics analysis was conducted between citral-treated and non-treated M. oryzae spores through two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. Our analysis identified 1600-1800 proteins from M. oryzae ZB15, of which 147 were differentially expressed in 100 µg/mL citral-treated samples relative to the control group. Among these differentially expressed proteins (DEPs), 40 proteins showed significantly different expression. GO enrichment and NCBI conserved domains database analysis showed that the main groups of the cellular component were cytoplasm (23.33%), and the major molecular function categories were ion binding (31.37%), and the major categories of biological processes included small molecule metabolic process (22.22%) and transport (13.89%). Further analysis found that down-regulated proteins included the tubulin α chain, ATP synthase subunit ß and malate dehydrogenase, while the tubulin ß, enolase were upregulated. These DEPs could possibly limit the availability of energy required for many cellular processes and result in various physiological adaptions of M. oryzae. This study represents the first proteomic analysis of M. oryzae treated by citral and will help to uncover the mode-of-action of this biologically active compound against M. oryzae. These findings have practical implications with respect to the use of citral for fungal disease control.


Assuntos
Monoterpenos Acíclicos , Magnaporthe , Ascomicetos , China , Proteínas Fúngicas/genética , Oryza , Doenças das Plantas , Proteômica
4.
Nat Commun ; 12(1): 2661, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976182

RESUMO

Precursor messenger RNA (pre-mRNA) splicing is an essential and tightly regulated process in eukaryotic cells; however, the regulatory mechanisms for the splicing are not well understood. Here, we characterize a RNA binding protein named FgRbp1 in Fusarium graminearum, a fungal pathogen of cereal crops worldwide. Deletion of FgRbp1 leads to reduced splicing efficiency in 47% of the F. graminearum intron-containing gene transcripts that are involved in various cellular processes including vegetative growth, development, and virulence. The human ortholog RBM42 is able to fully rescue the growth defects of ΔFgRbp1. FgRbp1 binds to the motif CAAGR in its target mRNAs, and interacts with the splicing factor FgU2AF23, a highly conserved protein involved in 3' splice site recognition, leading to enhanced recruitment of FgU2AF23 to the target mRNAs. This study demonstrates that FgRbp1 is a splicing regulator and regulates the pre-mRNA splicing in a sequence-dependent manner in F. graminearum.


Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Fator de Processamento U2AF/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Grão Comestível/microbiologia , Proteínas Fúngicas/genética , Fusarium/genética , Fusarium/patogenicidade , Humanos , Íntrons/genética , Ligação Proteica , Precursores de RNA/genética , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Virulência
5.
Nat Commun ; 12(1): 2261, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859194

RESUMO

Expanding the portfolio of products that can be made from lignin will be critical to enabling a viable bio-based economy. Here, we engineer Pseudomonas putida for high-yield production of the tricarboxylic acid cycle-derived building block chemical, itaconic acid, from model aromatic compounds and aromatics derived from lignin. We develop a nitrogen starvation-detecting biosensor for dynamic two-stage bioproduction in which itaconic acid is produced during a non-growth associated production phase. Through the use of two distinct itaconic acid production pathways, the tuning of TCA cycle gene expression, deletion of competing pathways, and dynamic regulation, we achieve an overall maximum yield of 56% (mol/mol) and titer of 1.3 g/L from p-coumarate, and 1.4 g/L titer from monomeric aromatic compounds produced from alkali-treated lignin. This work illustrates a proof-of-principle that using dynamic metabolic control to reroute carbon after it enters central metabolism enables production of valuable chemicals from lignin at high yields by relieving the burden of constitutively expressing toxic heterologous pathways.


Assuntos
Lignina/metabolismo , Engenharia Metabólica/métodos , Pseudomonas putida/metabolismo , Succinatos/metabolismo , Álcalis/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Basidiomycota/enzimologia , Basidiomycota/genética , Técnicas Biossensoriais , Burkholderia/enzimologia , Burkholderia/genética , Carbono/metabolismo , Ciclo do Ácido Cítrico/genética , Ácidos Cumáricos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Microbiologia Industrial/métodos , Lignina/química , Estudo de Prova de Conceito , Pseudomonas putida/genética
6.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33800857

RESUMO

Fusarium wilt of flax is an aggressive disease caused by the soil-borne fungal pathogen Fusarium oxysporum f. sp. lini. It is a challenging pathogen presenting a constant threat to flax production industry worldwide. Previously, we reported chromosome-level assemblies of 5 highly pathogenic F. oxysporum f. sp. lini strains. We sought to characterize the genomic architecture of the fungus and outline evolutionary mechanisms shaping the pathogen genome. Here, we reveal the complex multi-compartmentalized genome organization and uncover its diverse evolutionary dynamics, which boosts genetic diversity and facilitates host adaptation. In addition, our results suggest that host of functions implicated in the life cycle of mobile genetic elements are main contributors to dissimilarity between proteomes of different Fusaria. Finally, our experiments demonstrate that mobile genetics elements are expressed in planta upon infection, alluding to their role in pathogenicity. On the whole, these results pave the way for further in-depth studies of evolutionary forces shaping the host-pathogen interaction.


Assuntos
Linho/microbiologia , Fusarium/genética , Genoma Fúngico , Doenças das Plantas/microbiologia , Cromossomos Fúngicos/genética , Evolução Molecular , Proteínas Fúngicas/genética , Interações Hospedeiro-Patógeno/genética , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular , Filogenia , Proteoma , Especificidade da Espécie , Virulência/genética
7.
Int J Mol Sci ; 22(6)2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33808705

RESUMO

Fungal LysM effector proteins can dampen plant host-defence responses, protecting hyphae from plant chitinases, but little is known on these effectors from nonpathogenic fungal endophytes. We found four putative LysM effectors in the genome of the endophytic nematophagous fungus Pochonia chlamydosporia (Pc123). All four genes encoding putative LysM effectors are expressed constitutively by the fungus. Additionally, the gene encoding Lys1-the smallest one-is the most expressed in banana roots colonised by the fungus. Pc123 Lys1, 2 and 4 display high homology with those of other strains of the fungus and phylogenetically close entomopathogenic fungi. However, Pc123 Lys3 displays low homology with other fungi, but some similarities are found in saprophytes. This suggests evolutionary divergence in Pc123 LysM effectors. Additionally, molecular docking shows that the NAcGl binding sites of Pc123 Lys 2, 3 and 4 are adjacent to an alpha helix. Putative LysM effectors from fungal endophytes, such as Pc123, differ from those of plant pathogenic fungi. LysM motifs from endophytic fungi show clear conservation of cysteines in Positions 13, 51 and 63, unlike those of plant pathogens. LysM effectors could therefore be associated with the lifestyle of a fungus and give us a clue of how organisms could behave in different environments.


Assuntos
Proteínas Fúngicas/metabolismo , Fungos/fisiologia , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Fungos/classificação , Regulação Fúngica da Expressão Gênica , Interações Hospedeiro-Patógeno , Hifas , Hypocreales/fisiologia , Modelos Moleculares , Plantas/metabolismo , Plantas/microbiologia , Conformação Proteica , Relação Estrutura-Atividade
8.
Int J Mol Sci ; 22(8)2021 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-33921693

RESUMO

Plants lack a circulating adaptive immune system to protect themselves against pathogens. Therefore, they have evolved an innate immune system based upon complicated and efficient defense mechanisms, either constitutive or inducible. Plant defense responses are triggered by elicitors such as microbe-associated molecular patterns (MAMPs). These components are recognized by pattern recognition receptors (PRRs) which include plant cell surface receptors. Upon recognition, PRRs trigger pattern-triggered immunity (PTI). Ethylene Inducing Xylanase (EIX) is a fungal MAMP protein from the plant-growth-promoting fungi (PGPF)-Trichoderma. It elicits plant defense responses in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum), making it an excellent tool in the studies of plant immunity. Xylanases such as EIX are hydrolytic enzymes that act on xylan in hemicellulose. There are two types of xylanases: the endo-1, 4-ß-xylanases that hydrolyze within the xylan structure, and the ß-d-xylosidases that hydrolyze the ends of the xylan chain. Xylanases are mainly synthesized by fungi and bacteria. Filamentous fungi produce xylanases in high amounts and secrete them in liquid cultures, making them an ideal system for xylanase purification. Here, we describe a method for cost- and yield-effective xylanase production from Trichoderma using wheat bran as a growth substrate. Xylanase produced by this method possessed xylanase activity and immunogenic activity, effectively inducing a hypersensitive response, ethylene biosynthesis, and ROS burst.


Assuntos
Proteínas Fúngicas/metabolismo , Trichoderma/enzimologia , Trichoderma/metabolismo , Xilosidases/metabolismo , Etilenos/metabolismo , Proteínas Fúngicas/genética , Lycopersicon esculentum/imunologia , Lycopersicon esculentum/metabolismo , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tabaco/imunologia , Tabaco/metabolismo , Xilosidases/genética , Xilosidases/isolamento & purificação
9.
Int J Mol Sci ; 22(9)2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33921959

RESUMO

Chloroxylenol (PCMX) is applied as a preservative and disinfectant in personal care products, currently recommended for use to inactivate the SARS-CoV-2 virus. Its intensive application leads to the release of PCMX into the environment, which can have a harmful impact on aquatic and soil biotas. The aim of this study was to assess the mechanism of chloroxylenol biodegradation by the fungal strains Cunninghamella elegans IM 1785/21GP and Trametes versicolor IM 373, and investigate the ecotoxicity of emerging by-products. The residues of PCMX and formed metabolites were analysed using GC-MS. The elimination of PCMX in the cultures of tested microorganisms was above 70%. Five fungal by-products were detected for the first time. Identified intermediates were performed by dechlorination, hydroxylation, and oxidation reactions catalysed by cytochrome P450 enzymes and laccase. A real-time quantitative PCR analysis confirmed an increase in CYP450 genes expression in C. elegans cells. In the case of T. versicolor, spectrophotometric measurement of the oxidation of 2,20-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) showed a significant rise in laccase activity during PCMX elimination. Furthermore, with the use of bioindicators from different ecosystems (Daphtoxkit F and Phytotoxkit), it was revealed that the biodegradation process of PCMX had a detoxifying nature.


Assuntos
Cunninghamella/metabolismo , Trametes/metabolismo , Xilenos/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Daphnia/efeitos dos fármacos , Daphnia/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica , Lacase/metabolismo , Oxirredução , Testes de Toxicidade , Xilenos/análise , Xilenos/farmacologia
10.
New Phytol ; 231(1): 416-431, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33843063

RESUMO

The biotrophic basidiomycete fungus Ustilago maydis causes smut disease in maize. Hallmarks of the disease are characteristic large tumors in which dark pigmented spores are formed. Here, we functionally characterized a novel core effector lep1 (late effector protein 1) which is highly expressed during tumor formation and contributes to virulence. We characterize lep1 mutants, localize the protein, determine phenotypic consequences upon deletion as well as constitutive expression, and analyze relationships with the repellent protein Rep1 and hydrophobins. In tumors, lep1 mutants show attenuated hyphal aggregation, fail to undergo massive late proliferation and produce only a few spores. Upon constitutive expression, cell aggregation is induced and the surface of filamentous colonies displays enhanced hydrophobicity. Lep1 is bound to the cell wall of biotrophic hyphae and associates with Rep1 when constitutively expressed in hyphae. We conclude that Lep1 acts as a novel kind of cell adhesin which functions together with other surface-active proteins to allow proliferation of diploid hyphae as well as for induction of the morphological changes associated with spore formation.


Assuntos
Hifas , Ustilago , Basidiomycota , Proteínas Fúngicas/genética , Doenças das Plantas , Tumores de Planta , Ustilago/genética , Zea mays
11.
Nat Commun ; 12(1): 2178, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846336

RESUMO

Many phytopathogens secrete cell wall degradation enzymes (CWDEs) to damage host cells and facilitate colonization. As the major components of the plant cell wall, cellulose and hemicellulose are the targets of CWDEs. Damaged plant cells often release damage-associated molecular patterns (DAMPs) to trigger plant immune responses. Here, we establish that the fungal pathogen Magnaporthe oryzae secretes the endoglucanases MoCel12A and MoCel12B during infection of rice (Oryza sativa). These endoglucanases target hemicellulose of the rice cell wall and release two specific oligosaccharides, namely the trisaccharide 31-ß-D-Cellobiosyl-glucose and the tetrasaccharide 31-ß-D-Cellotriosyl-glucose. 31-ß-D-Cellobiosyl-glucose and 31-ß-D-Cellotriosyl-glucose bind the immune receptor OsCERK1 but not the chitin binding protein OsCEBiP. However, they induce the dimerization of OsCERK1 and OsCEBiP. In addition, these Poaceae cell wall-specific oligosaccharides trigger a burst of reactive oxygen species (ROS) that is largely compromised in oscerk1 and oscebip mutants. We conclude that 31-ß-D-Cellobiosyl-glucose and 31-ß-D-Cellotriosyl-glucose are specific DAMPs released from the hemicellulose of rice cell wall, which are perceived by an OsCERK1 and OsCEBiP immune complex during M. oryzae infection in rice.


Assuntos
Ascomicetos/fisiologia , Parede Celular/metabolismo , Oligossacarídeos/metabolismo , Oryza/imunologia , Oryza/microbiologia , Imunidade Vegetal , Proteínas de Plantas/metabolismo , Celulase/metabolismo , Resistência à Doença , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Modelos Biológicos , Fenótipo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Especificidade da Espécie , Transcrição Genética
12.
Nat Commun ; 12(1): 2132, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33837197

RESUMO

Oxidative plant cell-wall processing enzymes are of great importance in biology and biotechnology. Yet, our insight into the functional interplay amongst such oxidative enzymes remains limited. Here, a phylogenetic analysis of the auxiliary activity 7 family (AA7), currently harbouring oligosaccharide flavo-oxidases, reveals a striking abundance of AA7-genes in phytopathogenic fungi and Oomycetes. Expression of five fungal enzymes, including three from unexplored clades, expands the AA7-substrate range and unveils a cellooligosaccharide dehydrogenase activity, previously unknown within AA7. Sequence and structural analyses identify unique signatures distinguishing the strict dehydrogenase clade from canonical AA7 oxidases. The discovered dehydrogenase directly is able to transfer electrons to an AA9 lytic polysaccharide monooxygenase (LPMO) and fuel cellulose degradation by LPMOs without exogenous reductants. The expansion of redox-profiles and substrate range highlights the functional diversity within AA7 and sets the stage for harnessing AA7 dehydrogenases to fine-tune LPMO activity in biotechnological conversion of plant feedstocks.


Assuntos
Celulose/metabolismo , Proteínas Fúngicas/metabolismo , Oomicetos/enzimologia , Oxirredutases/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Cristalografia por Raios X , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Flavoproteínas Transferidoras de Elétrons/metabolismo , Ensaios Enzimáticos , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/ultraestrutura , Microbiologia Industrial/métodos , Espectroscopia de Ressonância Magnética , Oomicetos/genética , Oxirredução , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Oxirredutases/ultraestrutura , Filogenia , Análise de Sequência de DNA , Especificidade por Substrato
13.
Nat Commun ; 12(1): 2451, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33907187

RESUMO

Many pathogens infect hosts through specific organs, such as Ustilaginoidea virens, which infects rice panicles. Here, we show that a microbe-associated molecular pattern (MAMP), Ser-Thr-rich Glycosyl-phosphatidyl-inositol-anchored protein (SGP1) from U. virens, induces immune responses in rice leaves but not panicles. SGP1 is widely distributed among fungi and acts as a proteinaceous, thermostable elicitor of BAK1-dependent defense responses in N. benthamiana. Plants specifically recognize a 22 amino acid peptide (SGP1 N terminus peptide 22, SNP22) in its N-terminus that induces cell death, oxidative burst, and defense-related gene expression. Exposure to SNP22 enhances rice immunity signaling and resistance to infection by multiple fungal and bacterial pathogens. Interestingly, while SGP1 can activate immune responses in leaves, SGP1 is required for U. virens infection of rice panicles in vivo, showing it contributes to the virulence of a panicle adapted pathogen.


Assuntos
Proteínas Fúngicas/imunologia , Hypocreales/patogenicidade , Oryza/imunologia , Doenças das Plantas/imunologia , Folhas de Planta/imunologia , Proteínas de Plantas/imunologia , Sequência de Aminoácidos , Morte Celular/genética , Morte Celular/imunologia , Proteínas Fúngicas/genética , Regulação da Expressão Gênica , Glicosilfosfatidilinositóis/química , Glicosilfosfatidilinositóis/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Hypocreales/genética , Hypocreales/crescimento & desenvolvimento , Hypocreales/imunologia , Inflorescência/genética , Inflorescência/imunologia , Inflorescência/microbiologia , Oryza/genética , Oryza/microbiologia , Padrões Moleculares Associados a Patógenos/imunologia , Padrões Moleculares Associados a Patógenos/metabolismo , Peptídeos/genética , Peptídeos/imunologia , Células Vegetais/imunologia , Células Vegetais/patologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Folhas de Planta/genética , Folhas de Planta/microbiologia , Proteínas de Plantas/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Virulência
14.
Int J Mol Sci ; 22(7)2021 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-33917505

RESUMO

The APSES family proteins are transcription factors (TFs) with a basic helix-loop-helix domain, known to regulate growth, development, secondary metabolism, and other biological processes in Aspergillus species. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus, five genes predicted to encode APSES TFs are present. Here, we report the characterization of one of these genes, called mbsA (Afu7g05620). The deletion (Δ) of mbsA resulted in significantly decreased hyphal growth and asexual sporulation (conidiation), and lowered mRNA levels of the key conidiation genes abaA, brlA, and wetA. Moreover, ΔmbsA resulted in reduced spore germination rates, elevated sensitivity toward Nikkomycin Z, and significantly lowered transcripts levels of genes associated with chitin synthesis. The mbsA deletion also resulted in significantly reduced levels of proteins and transcripts of genes associated with the SakA MAP kinase pathway. Importantly, the cell wall hydrophobicity and architecture of the ΔmbsA asexual spores (conidia) were altered, notably lacking the rodlet layer on the surface of the ΔmbsA conidium. Comparative transcriptomic analyses revealed that the ΔmbsA mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes, which was corroborated by elevated levels of GT production in the mutant. While the ΔmbsA mutant produced higher amount of GT, ΔmbsA strains showed reduced virulence in the murine model, likely due to the defective spore integrity. In summary, the putative APSES TF MbsA plays a multiple role in governing growth, development, spore wall architecture, GT production, and virulence, which may be associated with the attenuated SakA signaling pathway.


Assuntos
Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Esporos Fúngicos/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Aspergillus fumigatus/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Esporos Fúngicos/genética , Fatores de Transcrição/genética
15.
Int J Mol Sci ; 22(5)2021 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-33801529

RESUMO

During the infection of a host, plant pathogenic fungi secrete small proteins called effectors, which then modulate the defence response of the host. In the Fusarium oxysporum species complex (FOSC), the secreted in xylem (SIX) gene effectors are important for host-specific pathogenicity, and are also useful markers for identifying the various host-specific lineages. While the presence and diversity of the SIX genes has been explored in many of the pathogenic lineages of F. oxysporum, there is a limited understanding of these genes in non-pathogenic, endophytic isolates of F. oxysporum. In this study, universal primers for each of the known SIX genes are designed and used to screen a panel of endophytically-associated Fusarium species isolated from healthy, asymptomatic banana tissue. SIX gene orthologues are identified in the majority of the Fusarium isolates screened in this study. Furthermore, the SIX gene profiles of these endophytic isolates do not overlap with the SIX genes present in the pathogenic lineages of F. oxysporum that are assessed in this study. SIX gene orthologues have not been commonly identified in Fusarium species outside of the FOSC nor in non-pathogenic isolates of F. oxysporum. The results of this study indicate that the SIX gene effectors may be more broadly distributed throughout the Fusarium genus than previously thought. This has important implications for understanding the evolution of pathogenicity in the FOSC.


Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/genética , Regulação Fúngica da Expressão Gênica , Especificidade de Hospedeiro , Musa/microbiologia , Doenças das Plantas/microbiologia , Transcriptoma , Proteínas Fúngicas/genética , Fusarium/patogenicidade , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Filogenia , Virulência/genética
16.
Molecules ; 26(6)2021 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-33806828

RESUMO

Cyanide is a hazardous and detrimental chemical that causes the inactivation of the respiration system through the inactivation of cytochrome c oxidase. Because of the limitation in the number of cyanide-degrading enzymes, there is a great demand to design and introduce new enzymes with better functionality. This study developed an integrated method of protein-homology-modelling and ligand-docking protein-design approaches that reconstructs a better active site from cyanide hydratase (CHT) structure. Designing a mutant CHT (mCHT) can improve the CHT performance. A computational design procedure that focuses on mutation for constructing a new model of cyanide hydratase with better activity was used. In fact, this study predicted the three-dimensional (3D) structure of CHT for subsequent analysis. Inducing mutation on CHT of Trichoderma harzianum was performed and molecular docking was used to compare protein interaction with cyanide as a ligand in both CHT and mCHT. By combining multiple designed mutations, a significant improvement in docking for CHT was obtained. The results demonstrate computational capabilities for enhancing and accelerating enzyme activity. The result of sequence alignment and homology modeling show that catalytic triad (Cys-Glu-Lys) was conserved in CHT of Trichoderma harzianum. By inducing mutation in CHT structure, MolDock score enhanced from -18.1752 to -23.8575, thus the nucleophilic attack can occur rapidly by adding Cys in the catalytic cavity and the total charge of protein in pH 6.5 is increased from -6.0004 to -5.0004. Also, molecular dynamic simulation shows a stable protein-ligand complex model. These changes would help in the cyanide degradation process by mCHT.


Assuntos
Cianetos/química , Proteínas Fúngicas/química , Hidroliases/química , Hypocreales/enzimologia , Simulação de Acoplamento Molecular , Substituição de Aminoácidos , Proteínas Fúngicas/genética , Hidroliases/genética , Hypocreales/genética , Mutação de Sentido Incorreto
17.
Appl Microbiol Biotechnol ; 105(7): 2747-2758, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33686455

RESUMO

The E3 ubiquitin ligase Ubr1 is a core player in yeast ubiquitylation and protein quality control required for cellular events including proteasomal degradation and gene activity but has been rarely explored in filamentous fungi. We show here an essentiality of orthologous Ubr1-mediated ubiquitylation for the activation of central developmental pathway (CPD) and the CPD-controlled cellular events in Beauveria bassiana, a filamentous fungal insect pathogen that undergoes an asexual cycle in vitro or in vivo. As a result of ubr1 disruption, intracellular free ubiquitin accumulation increased by 1.4-fold, indicating an impaired ability for the disruptant to transfer ubiquitin to target proteins. Consequently, the disruptant was compromised in polar growth featured with curved or hook-like germ tubes and abnormally branched hyphae, leading to impeded propagation of aberrant hyphal bodies in infected insect hemocoel and attenuated virulence. In the mutant, sharply repressed expression of three CDP activator genes (brlA, abaA, and wetA) correlated well with severe defects in aerial conidiation and submerged blastospore (hyphal body) production in insect hemolymph or a mimicking medium. Moreover, the disruptant was sensitive to cell wall perturbation or lysing and showed increased catalase activity and resistance to hydrogen peroxide despite null response to high osmolarity or heat shock. Most of the examined genes involved in polar growth and cell wall integrity were down-regulated in the disruptant. These findings uncover that the Ubr1-mediated ubiquitylation orchestrates polar growth and the CDP-regulated asexual cycle in vitro and in vivo in B. bassiana. KEY POINTS: • Ubr1 is an E3 ubiquitin ligase essential for ubiquitylation in Beauveria bassiana. • Ubr1-mediated ubiquitylation is required for activation of central development pathway. • Ubr1 orchestrates polar growth and asexual cycle in vitro and in vivo.


Assuntos
Beauveria , Animais , Beauveria/genética , Beauveria/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Esporos Fúngicos/metabolismo , Estresse Fisiológico , Ubiquitinação , Virulência
18.
New Phytol ; 231(1): 399-415, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33786841

RESUMO

Ustilago maydis is a biotrophic fungus causing smut disease in corn. The infectious forms are dikaryotic hyphae. Here we analyze mutants lacking the nlt1 transcription factor and investigate why these mutants are unable to induce leaf tumors. The study involved reverse genetics, complementation, epistasis analysis, microscopy, gene expression analysis by quantitative reverse transcriptase PCR and virulence assays. We show that nlt1 mutants colonize maize leaves efficiently but fail to undergo karyogamy and are attenuated in late proliferation. Nlt1 activates transcription of ros1, a transcription factor controlling karyogamy, and represses see1, an effector previously shown to contribute to leaf tumor induction. In mononuclate solopathogenic strains, nlt1 mutants cause attenuated leaf tumor formation. In actively dividing maize organs, nlt1 mutants undergo karyogamy and induce tumor formation. Sporisorium reilianum, a smut fungus unable to induce leaf tumors, possesses an ortholog of nlt1 that controls the fusion of dikaryotic nuclei late in infection during cob colonization. Our results have established a regulatory connection between nlt1, ros1 and see1 and suggest the existence of two stages contributing to leaf tumor formation, one before nuclear fusion and involving nlt1 and one after karyogamy that is nlt1 independent.


Assuntos
Tumores de Planta/microbiologia , Ustilago/patogenicidade , Zea mays/microbiologia , Basidiomycota , Proteínas Fúngicas/genética , Doenças das Plantas , Folhas de Planta , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas , Ustilago/genética , Zea mays/genética
19.
Methods Mol Biol ; 2280: 15-30, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33751426

RESUMO

The approaches used by the authors to design the Candida famata strains capable to overproduce riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) are described. The metabolic engineering approaches include overexpression of SEF1 gene encoding positive regulator of riboflavin biosynthesis, IMH3 (coding for IMP dehydrogenase) orthologs from another species of flavinogenic yeast Debaryomyces hansenii, and the homologous genes RIB1 and RIB7 encoding GTP cyclohydrolase II and riboflavin synthase, the first and the last enzymes of riboflavin biosynthesis pathway, respectively. Overexpression of the above mentioned genes in the genetically stable riboflavin overproducer AF-4 obtained by classical selection resulted in fourfold increase of riboflavin production in shake flask experiments.Overexpression of engineered enzymes phosphoribosyl pyrophosphate synthetase and phosphoribosyl pyrophosphate amidotransferase catalyzing the initial steps of purine nucleotide biosynthesis enhances riboflavin synthesis in the flavinogenic yeast C. famata even more.Recombinant strains of C. famata containing FMN1 gene from D. hansenii encoding riboflavin kinase under control of the strong constitutive TEF1 promoter were constructed. Overexpression of the FMN1 gene in the riboflavin-producing mutant led to the 30-fold increase of the riboflavin kinase activity and 400-fold increase of FMN production in the resulting recombinant strains which reached maximally 318.2 mg/L.FAD overproducing strains of C. famata were also constructed. This was achieved by overexpression of FAD1 gene from D. hansenii in C. famata FMN overproducing strain. The 7- to 15-fold increase in FAD synthetase activity as compared to the wild-type strain and FAD accumulation into cultural medium were observed. The maximal FAD titer 451.5 mg/L was achieved.


Assuntos
Candida/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Engenharia Metabólica/métodos , Técnicas de Cultura Celular por Lotes , Vias Biossintéticas , Candida/genética , Candida/metabolismo , Mononucleotídeo de Flavina/biossíntese , Mononucleotídeo de Flavina/genética , Flavina-Adenina Dinucleotídeo/biossíntese , Flavina-Adenina Dinucleotídeo/genética , Riboflavina/biossíntese , Riboflavina/genética
20.
Methods Mol Biol ; 2280: 31-42, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33751427

RESUMO

Many microorganisms are capable of riboflavin oversynthesis and accumulation in a medium, suggesting that they efficiently excrete riboflavin. The mechanisms of riboflavin efflux in microorganisms remain elusive. Candida famata are representatives of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B2) in response to iron limitation. The riboflavin overproducers of this yeast species have been obtained by classical mutagenesis and metabolic engineering. Overproduced riboflavin accumulates in the cultural medium rather than in the cells suggesting existence of the special mechanisms involved in riboflavin excretion. The appropriate protein and gene have not been identified in yeasts till recently. At the same time, the gene BCRP (breast cancer resistance protein) has been identified in mammal mammary glands. Several homologs of the mammal BCRP gene encoding putative riboflavin efflux protein (excretase) were identified in the flavinogenic yeasts Debaryomyces hansenii and C. famata. Here we evaluate the yeast homologs of BCRP with respect to improvement of a riboflavin production by C. famata. The closest homologs from D. hansenii or C. famata were expressed under the control of TEF1 promoter of these yeasts in the wild-type and riboflavin-overproducing strains of C. famata. Resulted transformants overexpressed the corresponding genes (designated as DhRFE and CfRFE) and produced 1.4- to 6-fold more riboflavin as compared to the corresponding parental strains. They also were characterized by overexpression of RIB1 and RIB6 genes which encode the first and the last structural enzymes of riboflavin synthesis and exhibited elevated specific activity of GTP cyclohydrolase II. Thus, overexpression of yeast homolog of mammal gene BCRP may be useful to increase the riboflavin yield in a riboflavin production process using a recombinant overproducing C. famata strain or other flavinogenic microorganisms.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Candida/crescimento & desenvolvimento , Engenharia Metabólica/métodos , Riboflavina/biossíntese , Candida/genética , Candida/metabolismo , Clonagem Molecular , Meios de Cultura/química , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Fator 1 de Elongação de Peptídeos/genética , Regiões Promotoras Genéticas , Regulação para Cima
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