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1.
J Nanobiotechnology ; 19(1): 273, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34496881

RESUMO

The control of contagious or refractory diseases requires early, rapid diagnostic assays that are simple, fast, and easy-to-use. Here, easy-to-implement CRISPR/Cas12a-based diagnostic platform through Raman transducer generated by Raman enhancement effect, term as SERS-CRISPR (S-CRISPR), are described. The S-CRISPR uses high-activity noble metallic nanoscopic materials to increase the sensitivity in the detection of nucleic acids, without amplification. This amplification-free platform, which can be performed within 30-40 min of incubation time, is then used for detection of SARS-CoV-2 derived nucleic acids in RNA extracts obtained from nasopharyngeal swab specimens (n = 112). Compared with the quantitative reverse transcription polymerase chain reaction (RT-qPCR), the sensitivity and specificity of S-CRISPR reaches 87.50% and 100%, respectively. In general, the S-CRISPR can rapidly identify the RNA of SARS-CoV-2 RNA without amplification and is a potential strategy for nucleic acid point of care test (POCT).


Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Amplificação de Ácido Nucleico , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Análise Espectral Raman , COVID-19/diagnóstico , COVID-19/virologia , Regulação Fúngica da Expressão Gênica , Genes Virais , Humanos , RNA Viral/análise , Sensibilidade e Especificidade
2.
FASEB J ; 35(9): e21778, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383971

RESUMO

As a result of the relatively few available antifungals and the increasing frequency of resistance to them, the development of novel antifungals is increasingly important. The plant natural product poacic acid (PA) inhibits ß-1,3-glucan synthesis in Saccharomyces cerevisiae and has antifungal activity against a wide range of plant pathogens. However, the mode of action of PA is unclear. Here, we reveal that PA specifically binds to ß-1,3-glucan, its affinity for which is ~30-fold that for chitin. Besides its effect on ß-1,3-glucan synthase activity, PA inhibited the yeast glucan-elongating activity of Gas1 and Gas2 and the chitin-glucan transglycosylase activity of Crh1. Regarding the cellular response to PA, transcriptional co-regulation was mediated by parallel activation of the cell-wall integrity (CWI) and high-osmolarity glycerol signaling pathways. Despite targeting ß-1,3-glucan remodeling, the transcriptional profiles and regulatory circuits activated by caspofungin, zymolyase, and PA differed, indicating that their effects on CWI have different mechanisms. The effects of PA on the growth of yeast strains indicated that it has a mode of action distinct from that of echinocandins, suggesting it is a unique antifungal agent.


Assuntos
Antifúngicos/farmacologia , Parede Celular/efeitos dos fármacos , Ácidos Cumáricos/farmacologia , Glicerol/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Estilbenos/farmacologia , Transcrição Genética/efeitos dos fármacos , beta-Glucanas/farmacologia , Caspofungina/farmacologia , Parede Celular/genética , Parede Celular/metabolismo , Quitina/farmacologia , Equinocandinas/farmacologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/genética , Concentração Osmolar , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcrição Genética/genética
4.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360997

RESUMO

Poly(dA:dT) tracts cause nucleosome depletion in many species, e.g., at promoters and replication origins. Their intrinsic biophysical sequence properties make them stiff and unfavorable for nucleosome assembly, as probed by in vitro nucleosome reconstitution. The mere correlation between nucleosome depletion over poly(dA:dT) tracts in in vitro reconstituted and in in vivo chromatin inspired an intrinsic nucleosome exclusion mechanism in vivo that is based only on DNA and histone properties. However, we compile here published and new evidence that this correlation does not reflect mechanistic causation. (1) Nucleosome depletion over poly(dA:dT) in vivo is not universal, e.g., very weak in S. pombe. (2) The energy penalty for incorporating poly(dA:dT) tracts into nucleosomes is modest (<10%) relative to ATP hydrolysis energy abundantly invested by chromatin remodelers. (3) Nucleosome depletion over poly(dA:dT) is much stronger in vivo than in vitro if monitored without MNase and (4) actively maintained in vivo. (5) S. cerevisiae promoters evolved a strand-biased poly(dA) versus poly(dT) distribution. (6) Nucleosome depletion over poly(dA) is directional in vivo. (7) The ATP dependent chromatin remodeler RSC preferentially and directionally displaces nucleosomes towards 5' of poly(dA). Especially distribution strand bias and displacement directionality would not be expected for an intrinsic mechanism. Together, this argues for an in vivo mechanism where active and species-specific read out of intrinsic sequence properties, e.g., by remodelers, shapes nucleosome organization.


Assuntos
Sequência Rica em At , Montagem e Desmontagem da Cromatina , Nucleossomos/genética , Regulação Fúngica da Expressão Gênica , Nucleossomos/química , Nucleossomos/metabolismo , Saccharomyces cerevisiae , Schizosaccharomyces
5.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360643

RESUMO

Filamentous fungi are able to synthesise a remarkable range of secondary metabolites, which play various key roles in the interaction between fungi and the rest of the biosphere, determining their ecological fitness. Many of them can have a beneficial activity to be exploited, as well as negative impact on human and animal health, as in the case of mycotoxins contaminating large quantities of food, feed, and agricultural products worldwide and posing serious health and economic risks. The elucidation of the molecular aspects of mycotoxin biosynthesis has been greatly sped up over the past decade due to the advent of next-generation sequencing technologies, which greatly reduced the cost of genome sequencing and related omic analyses. Here, we briefly highlight the recent progress in the use and integration of omic approaches for the study of mycotoxins biosynthesis. Particular attention has been paid to genomics and transcriptomic approaches for the identification and characterisation of biosynthetic gene clusters of mycotoxins and the understanding of the regulatory pathways activated in response to physiological and environmental factors leading to their production. The latest innovations in genome-editing technology have also provided a more powerful tool for the complete explanation of regulatory and biosynthesis pathways. Finally, we address the crucial issue of the interpretation of the combined omics data on the biology of the mycotoxigenic fungi. They are rapidly expanding and require the development of resources for more efficient integration, as well as the completeness and the availability of intertwined data for the research community.


Assuntos
Fungos/fisiologia , Regulação Fúngica da Expressão Gênica , Micotoxinas/biossíntese , Animais , Vias Biossintéticas , Genômica , Humanos , Micotoxinas/genética
6.
Gene ; 802: 145863, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34358628

RESUMO

Hydrophobins are small, secreted proteins with important physiological functions in mycelial growth and fungal development. Here, 1 nucleus-specific and 35 allelic hydrophobin genes were identified in the genome of a white rot fungus, Coriolopsis trogii. Among these, 22 were eight-cysteine class I hydrophobin genes and the other 14 were uncommon six-cysteine hydrophobin genes. The six-cysteine hydrophobins were speculated to have originated from a common ancestor. The hydrophobin genes favored a clustering distribution and two recent duplication pairs were identified. The genes had conserved gene structures with three exons and two introns. Cthyd18, Cthyd19, and Cthyd32 were constitutively highly expressed in all developmental stages. Cthyd20, Cthyd21, Cthyd22, Cthyd28, Cthyd30, Cthyd31, and Cthyd33 were highly expressed in mycelia, and Cthyd12 and Cthyd35 in the reproductive stages. Sixteen hydrophobin genes were regulated differently in the transition from mycelia to primordia; Cthyd35 showed maximal upregulation of 1922-fold, and Cthyd23 showed maximal downregulation of 552-fold. Most (32) hydrophobin genes showed significant differential expression between mycelia cultured in different media (potato dextrose agar or broth). Weighted gene co-expression network analysis and promoter analysis revealed that C2H2 zinc finger proteins may regulate hydrophobin genes. These results may support further research into the function and evolution of hydrophobins.


Assuntos
Proteínas Fúngicas/genética , Polyporaceae/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Micélio/genética , Micélio/crescimento & desenvolvimento , Polyporaceae/crescimento & desenvolvimento , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real
7.
Nat Commun ; 12(1): 4790, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373465

RESUMO

Eukaryotic organisms play an important role in industrial biotechnology, from the production of fuels and commodity chemicals to therapeutic proteins. To optimize these industrial systems, a mathematical approach can be used to integrate the description of multiple biological networks into a single model for cell analysis and engineering. One of the most accurate models of biological systems include Expression and Thermodynamics FLux (ETFL), which efficiently integrates RNA and protein synthesis with traditional genome-scale metabolic models. However, ETFL is so far only applicable for E. coli. To adapt this model for Saccharomyces cerevisiae, we developed yETFL, in which we augmented the original formulation with additional considerations for biomass composition, the compartmentalized cellular expression system, and the energetic costs of biological processes. We demonstrated the ability of yETFL to predict maximum growth rate, essential genes, and the phenotype of overflow metabolism. We envision that the presented formulation can be extended to a wide range of eukaryotic organisms to the benefit of academic and industrial research.


Assuntos
Genoma , Engenharia Metabólica , Redes e Vias Metabólicas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Biomassa , Biotecnologia , Simulação por Computador , Escherichia coli/genética , Regulação Fúngica da Expressão Gênica , Glucose , Modelos Biológicos , Fenótipo , Termodinâmica
8.
Curr Microbiol ; 78(8): 3201-3211, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34213616

RESUMO

Cellulase plays an important role in addressing the issue of the energy crisis. However, the yield and degradation efficiency of cellulase remain a major challenge. In the present study, we aimed to verify whether ammonium ion (NH4+) could induce cellulase synthesis from T. koningii AS3.2774 and to explore new functional genes related to the cellulase production. Our results indicated that NH4+ induces cellulase production in a way different from nitrogen sources. NH4+-mediated mycelia displayed a significant increase in transport vesicles. Under NH4+ mediation, CBHI, CBHII, glycoside hydrolase family 5 proteins, Hap2/3/5 complexes, "ribosome biogenesis", and "heme binding" were significantly up-regulated, and differentially expressed genes (DEGs) were mainly involved in "Metabolism". Collectively, our findings illustrated that NH4+ induced the cellulase production at morphological and gene expression levels, which might be related to the Hap2/3/5 complex, ribosomes, and genes involved in various amino acid metabolism, pyruvate metabolism, and glycolysis/gluconeogenesis. Taken together, our results provided valuable insights into the regulatory network of cellulase gene expression in filamentous fungi.


Assuntos
Compostos de Amônio , Celulase , Trichoderma , Celulase/genética , Celulase/metabolismo , Regulação Fúngica da Expressão Gênica , Hypocreales , Íons , Trichoderma/genética , Trichoderma/metabolismo
9.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298948

RESUMO

Verticillium wilt, caused by Verticillium dahliae, is a devastating disease for many important crops, including cotton. Kiwellins (KWLs), a group of cysteine-rich proteins synthesized in many plants, have been shown to be involved in response to various phytopathogens. To evaluate genes for their function in resistance to Verticillium wilt, we investigated KWL homologs in cotton. Thirty-five KWL genes (GhKWLs) were identified from the genome of upland cotton (Gossypium hirsutum). Among them, GhKWL1 was shown to be localized in nucleus and cytosol, and its gene expression is induced by the infection of V. dahliae. We revealed that GhKWL1 was a positive regulator of GhERF105. Silencing of GhKWL1 resulted in a decrease, whereas overexpression led to an increase in resistance of transgenic plants to Verticillium wilt. Interestingly, through binding to GhKWL1, the pathogenic effector protein VdISC1 produced by V. dahliae could impair the defense response mediated by GhKWL1. Therefore, our study suggests there is a GhKWL1-mediated defense response in cotton, which can be hijacked by V. dahliae through the interaction of VdISC1 with GhKWL1.


Assuntos
Ascomicetos , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Gossypium , Doenças das Plantas , Fatores de Transcrição , Regulação para Cima , Fatores de Virulência , Ascomicetos/genética , Ascomicetos/metabolismo , Ascomicetos/patogenicidade , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Gossypium/genética , Gossypium/metabolismo , Gossypium/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Virulência/biossíntese , Fatores de Virulência/genética
10.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298922

RESUMO

The coordinated transcription of the genome is the fundamental mechanism in molecular biology. Transcription in eukaryotes is carried out by three main RNA polymerases: Pol I, II, and III. One basic problem is how a decrease in tRNA levels, by downregulating Pol III efficiency, influences the expression pattern of protein-coding genes. The purpose of this study was to determine the mRNA levels in the yeast mutant rpc128-1007 and its overdose suppressors, RBS1 and PRT1. The rpc128-1007 mutant prevents assembly of the Pol III complex and functionally mimics similar mutations in human Pol III, which cause hypomyelinating leukodystrophies. We applied RNAseq followed by the hierarchical clustering of our complete RNA-seq transcriptome and functional analysis of genes from the clusters. mRNA upregulation in rpc128-1007 cells was generally stronger than downregulation. The observed induction of mRNA expression was mostly indirect and resulted from the derepression of general transcription factor Gcn4, differently modulated by suppressor genes. rpc128-1007 mutation, regardless of the presence of suppressors, also resulted in a weak increase in the expression of ribosome biogenesis genes. mRNA genes that were downregulated by the reduction of Pol III assembly comprise the proteasome complex. In summary, our results provide the regulatory links affected by Pol III assembly that contribute differently to cellular fitness.


Assuntos
RNA Polimerase III/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação para Baixo/genética , Regulação Fúngica da Expressão Gênica/genética , Humanos , RNA Polimerase II/genética , RNA de Transferência/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição Genética/genética , Ativação Transcricional/genética , Transcriptoma/genética , Regulação para Cima/genética
11.
Appl Microbiol Biotechnol ; 105(13): 5553-5564, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34236481

RESUMO

Aspergillus niger is a filamentous fungus well known for its ability to produce a wide variety of pectinolytic enzymes, which have many applications in the industry. The transcriptional activator GaaR is induced by 2-keto-3-deoxy-L-galactonate, a compound derived from D-galacturonic acid, and plays a major role in the regulation of pectinolytic genes. The requirement for inducer molecules can be a limiting factor for the production of enzymes. Therefore, the generation of chimeric transcription factors able to activate the expression of pectinolytic genes by using underutilized agricultural residues would be highly valuable for industrial applications. In this study, we used the CRISPR/Cas9 system to generate three chimeric GaaR-XlnR transcription factors expressed by the xlnR promoter by swapping the N-terminal region of the xylanolytic regulator XlnR to that of the GaaR in A. niger. As a test case, we constructed a PpgaX-hph reporter strain to evaluate the alteration of transcription factor specificity in the chimeric mutants. Our results showed that the chimeric GaaR-XlnR transcription factor was induced in the presence of D-xylose. Additionally, we generated a constitutively active GaaR-XlnR V756F version of the most efficient chimeric transcription factor to better assess its activity. Proteomics analysis confirmed the production of several pectinolytic enzymes by ΔgaaR mutants carrying the chimeric transcription factor. This correlates with the improved release of D-galacturonic acid from pectin by the GaaR-XlnR V756F mutant, as well as by the increased L-arabinose release from the pectin side chains by both chimeric mutants under inducing condition, which is required for efficient degradation of pectin. KEY POINTS: • Chimeric transcription factors were generated by on-site mutations using CRISPR/Cas9. • PpgaX-hph reporter strain allowed for the screening of functional GaaR-XlnR mutants. • Chimeric GaaR-XlnR induced pectinolytic activities in the presence of D-xylose.


Assuntos
Aspergillus niger , Fatores de Transcrição , Aspergillus niger/genética , Aspergillus niger/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Xilose
12.
BMC Plant Biol ; 21(1): 332, 2021 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253186

RESUMO

BACKGROUND: Pogoniopsis schenckii Cogn. is a mycoheterotrophic orchid that can be used as a model to understand the influence of mycoheterotrophy at different stages of the reproductive cycle. We aimed to verify the presence of endophytic and epiphytic fungi at each stage of the reproductive process and investigated how the breeding system may relate to genetic structure and diversity of populations. In this study we performed anatomical and ultrastructural analyses of the reproductive organs, field tests to confirm the breeding system, and molecular analysis to assess genetic diversity and structure of populations. RESULTS: During the development of the pollen grain, embryo sac and embryogenesis, no fungal infestation was observed. The presence of endophytic fungal hyphae was observed just within floral stems and indehiscent fruit. Beyond assuring the presence of fungus that promote seed germination, specific fungi hyphae in the fruit may affect other process, such as fruit ripening. As other mycoheterotrophic orchids, P. schenckii is autogamous, which may explain the low genetic diversity and high genetic structure in populations. CONCLUSIONS: We discuss an interesting interaction: fungal hyphae in the indehiscent fruit. These fungal hyphae seem to play different roles inside fruit tissues, such as acting in the fruit maturation process and increasing the proximity between fungi and plant seeds even before dispersion occurs. As other mycoheterotrophic orchids, P. schenckii is autogamous, which may explain the low genetic diversity and high genetic structure in populations. Altogether, our findings provide important novel information about the mechanisms shaping ecology and evolution of fragmented populations of mycoheterotrophic plant.


Assuntos
Micorrizas/genética , Orchidaceae/crescimento & desenvolvimento , Orchidaceae/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , Reprodução/genética , Simbiose/genética , Brasil , DNA Fúngico , Regulação Fúngica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Orchidaceae/microbiologia , Raízes de Plantas/microbiologia
13.
Commun Biol ; 4(1): 822, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193958

RESUMO

Stochastic gene expression leads to inherent variability in expression outcomes even in isogenic single-celled organisms grown in the same environment. The Drop-Seq technology facilitates transcriptomic studies of individual mammalian cells, and it has had transformative effects on the characterization of cell identity and function based on single-cell transcript counts. However, application of this technology to organisms with different cell size and morphology characteristics has been challenging. Here we present yeastDrop-Seq, a yeast-optimized platform for quantifying the number of distinct mRNA molecules in a cell-specific manner in individual yeast cells. Using yeastDrop-Seq, we measured the transcriptomic impact of the lifespan-extending compound mycophenolic acid and its epistatic agent guanine. Each treatment condition had a distinct transcriptomic footprint on isogenic yeast cells as indicated by distinct clustering with clear separations among the different groups. The yeastDrop-Seq platform facilitates transcriptomic profiling of yeast cells for basic science and biotechnology applications.


Assuntos
Perfilação da Expressão Gênica/métodos , Regulação Fúngica da Expressão Gênica/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Análise de Célula Única/métodos , Transcriptoma/genética , Análise por Conglomerados , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Guanina/metabolismo , Guanina/farmacologia , Ácido Micofenólico/metabolismo , Ácido Micofenólico/farmacologia , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/citologia , Análise de Sequência de RNA/métodos , Transcriptoma/efeitos dos fármacos
14.
Mol Cell ; 81(16): 3294-3309.e12, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34293321

RESUMO

Temperature is a variable component of the environment, and all organisms must deal with or adapt to temperature change. Acute temperature change activates cellular stress responses, resulting in refolding or removal of damaged proteins. However, how organisms adapt to long-term temperature change remains largely unexplored. Here we report that budding yeast responds to long-term high temperature challenge by switching from chaperone induction to reduction of temperature-sensitive proteins and re-localizing a portion of its proteome. Surprisingly, we also find that many proteins adopt an alternative conformation. Using Fet3p as an example, we find that the temperature-dependent conformational difference is accompanied by distinct thermostability, subcellular localization, and, importantly, cellular functions. We postulate that, in addition to the known mechanisms of adaptation, conformational plasticity allows some polypeptides to acquire new biophysical properties and functions when environmental change endures.


Assuntos
Adaptação Fisiológica/genética , Proteoma/genética , Estresse Fisiológico/genética , Transcriptoma/genética , Aclimatação/genética , Animais , Exposição Ambiental/efeitos adversos , Regulação Fúngica da Expressão Gênica/genética , Temperatura Alta/efeitos adversos , Saccharomycetales/genética
15.
Int J Mol Sci ; 22(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299357

RESUMO

The airborne fungus Aspergillus fumigatus causes opportunistic infections in humans with high mortality rates in immunocompromised patients. Previous work established that the bZIP transcription factor HapX is essential for virulence via adaptation to iron limitation by repressing iron-consuming pathways and activating iron acquisition mechanisms. Moreover, HapX was shown to be essential for transcriptional activation of vacuolar iron storage and iron-dependent pathways in response to iron availability. Here, we demonstrate that HapX has a very short half-life during iron starvation, which is further decreased in response to iron, while siderophore biosynthetic enzymes are very stable. We identified Fbx22 and SumO as HapX interactors and, in agreement, HapX post-translational modifications including ubiquitination of lysine161, sumoylation of lysine242 and phosphorylation of threonine319. All three modifications were enriched in the immediate adaptation from iron-limiting to iron-replete conditions. Interfering with these post-translational modifications, either by point mutations or by inactivation, of Fbx22 or SumO, altered HapX degradation, heme biosynthesis and iron resistance to different extents. Consistent with the need to precisely regulate HapX protein levels, overexpression of hapX caused significant growth defects under iron sufficiency. Taken together, our results indicate that post-translational regulation of HapX is important to control iron homeostasis in A. fumigatus.


Assuntos
Aspergillus fumigatus/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Homeostase/genética , Ferro/metabolismo , Processamento de Proteína Pós-Traducional/genética , Adaptação Fisiológica/genética , Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Mutação Puntual/genética , Sideróforos/genética , Treonina/genética , Virulência/genética
16.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206573

RESUMO

Processing of the RNA polymerase I pre-rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs requires removing the "spacer" sequences. The canonical pathway for the removal of the ITS1 spacer involves cleavages at the 3' end of 18S rRNA and at two sites inside ITS1. The process can generate either a long or a short 5.8S rRNA that differs in the number of ITS1 nucleotides retained at the 5.8S 5' end. Here we document a novel pathway to the long 5.8S, which bypasses cleavage within ITS1. Instead, the entire ITS1 is degraded from its 5' end by exonuclease Xrn1. Mutations in RNase MRP increase the accumulation of long relative to short 5.8S rRNA. Traditionally this is attributed to a decreased rate of RNase MRP cleavage at its target in ITS1, called A3. However, results from this work show that the MRP-induced switch between long and short 5.8S rRNA formation occurs even when the A3 site is deleted. Based on this and our published data, we propose that the link between RNase MRP and 5.8S 5' end formation involves RNase MRP cleavage at unknown sites elsewhere in pre-rRNA or in RNA molecules other than pre-rRNA.


Assuntos
RNA Ribossômico 5,8S/genética , RNA Ribossômico 5,8S/metabolismo , DNA Espaçador Ribossômico , Endorribonucleases , Regulação Fúngica da Expressão Gênica , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA , RNA Fúngico , RNA Ribossômico 5,8S/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência
17.
Int J Mol Sci ; 22(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201004

RESUMO

Cadmium is a carcinogen that can induce ER stress, DNA damage, oxidative stress and cell death. The yeast mitogen-activated protein kinase (MAPK) signalling pathways paly crucial roles in response to various stresses. Here, we demonstrate that the unfolded protein response (UPR) pathway, the high osmolarity glycerol (HOG) pathway and the cell wall integrity (CWI) pathway are all essential for yeast cells to defend against the cadmium-induced toxicity, including the elevated ROS and cell death levels induced by cadmium. We show that the UPR pathway is required for the cadmium-induced phosphorylation of HOG_MAPK Hog1 but not for CWI_MAPK Slt2, while Slt2 but not Hog1 is required for the activation of the UPR pathway through the transcription factors of Swi6 and Rlm1. Moreover, deletion of HAC1 and IRE1 could promote the nuclear accumulation of Hog1, and increase the cytosolic and bud neck localisation of Slt2, indicating crucial roles of Hog1 and Slt2 in regulating the cellular process in the absence of UPR pathway. Altogether, our findings highlight the significance of these two MAPK pathways of HOG and CWI and their interrelationship with the UPR pathway in responding to cadmium-induced toxicity in budding yeast.


Assuntos
Cádmio/toxicidade , Parede Celular/química , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glicerol/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Parede Celular/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Concentração Osmolar , Fosforilação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transdução de Sinais
18.
Appl Microbiol Biotechnol ; 105(14-15): 5915-5929, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34292355

RESUMO

Arginine is an important amino acid involved in processes such as cell signal transduction, protein synthesis, and sexual reproduction. To understand the biological roles of arginine biosynthesis in pathogenic fungi, we used Cpa1, the carbamoyl phosphate synthase arginine-specific small chain subunit in Saccharomyces cerevisiae as a query to identify its ortholog in the Magnaporthe oryzae genome and named it MoCpa1. MoCpa1 is a 471-amino acid protein containing a CPSase_sm_chain domain and a GATase domain. MoCpa1 transcripts were highly expressed at the conidiation, early-infection, and late-infection stages of the fungus. Targeted deletion of the MoCPA1 gene resulted in a ΔMocpa1 mutant exhibiting arginine auxotrophy on minimum culture medium (MM), confirming its role in de novo arginine biosynthesis. The ΔMocpa1 mutant presented significantly decreased sporulation with some of its conidia being defective in morphology. Furthermore, the ΔMocpa1 mutant was nonpathogenic on rice and barley leaves, which was a result of defects in appressorium-mediated penetration and restricted invasive hyphal growth within host cells. Addition of exogenous arginine partially rescued conidiation and pathogenicity defects on the barley and rice leaves, while introduction of the MoCPA1 gene into the ΔMocpa1 mutant fully complemented the lost phenotype. Further confocal microscopy examination revealed that MoCpa1 is localized in the mitochondria. In summary, our results demonstrate that MoCpa1-mediated arginine biosynthesis is crucial for fungal development, conidiation, appressorium formation, and infection-related morphogenesis in M. oryzae, thus serving as an attractive target for mitigating obstinate fungal plant pathogens. KEY POINTS: • MoCpa1 is important for aerial hyphal growth and arginine biosynthesis. • MoCpa1 is pivotal for conidial morphogenesis and appressorium formation. • MoCpa1 is crucial for full virulence in M. oryzae.


Assuntos
Magnaporthe , Oryza , Arginina , Ascomicetos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Magnaporthe/genética , Magnaporthe/metabolismo , Oryza/metabolismo , Doenças das Plantas , Esporos Fúngicos/metabolismo
19.
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
20.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066497

RESUMO

Autophagy is an intracellular process in all eukaryotes which is responsible for the degradation of cytoplasmic constituents, recycling of organelles, and recycling of proteins. It is an important cellular process responsible for the effective virulence of several pathogenic plant fungal strains, having critical impacts on important crop plants including potatoes. However, the detailed physiological mechanisms of autophagy involved in the infection biology of soil-borne pathogens in the potato crop needs to be investigated further. In this study, the autophagy-related gene, FoATG12, in potato dry rot fungus Fusarium oxysporum was investigated by means of target gene replacement and overexpression. The deletion mutant ∆FoATG12 showed reduction in conidial formation and exhibited impaired aerial hyphae. The FoATG12 affected the expression of genes involved in pathogenicity and vegetative growth, as well as on morphology features of the colony under stressors. It was found that the disease symptoms were delayed upon being inoculated by the deletion mutant of FoATG12 compared to the wild-type (WT) and overexpression (OE), while the deletion mutant showed the disease symptoms on tomato plants. The results confirmed the significant role of the autophagy-related ATG12 gene in the production of aerial hyphae and the effective virulence of F. oxysporum in the potato crop. The current findings provid an enhanced gene-level understanding of the autophagy-related virulence of F. oxysporum, which could be helpful in pathogen control research and could have vital impacts on the potato crop.


Assuntos
Proteína 12 Relacionada à Autofagia/genética , Autofagia/genética , Proteínas Fúngicas/genética , Fusarium/citologia , Fusarium/genética , Genes Fúngicos , Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia , Proteína 12 Relacionada à Autofagia/metabolismo , Proteínas Fúngicas/metabolismo , Fusarium/patogenicidade , Regulação Fúngica da Expressão Gênica , Hifas/crescimento & desenvolvimento , Mutação/genética , Fenótipo , Doenças das Plantas/genética , Esporos Fúngicos/crescimento & desenvolvimento , Estresse Fisiológico/genética
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