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1.
Nucleic Acids Res ; 49(1): 568-583, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33332555

RESUMO

Infection with kinetoplastid parasites, including Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania can cause serious disease in humans. Like other kinetoplastid species, mRNAs of these disease-causing parasites must undergo posttranscriptional editing in order to be functional. mRNA editing is directed by gRNAs, a large group of small RNAs. Similar to mRNAs, gRNAs are also precisely regulated. In T. brucei, overexpression of RNase D ribonuclease (TbRND) leads to substantial reduction in the total gRNA population and subsequent inhibition of mRNA editing. However, the mechanisms regulating gRNA binding and cleavage by TbRND are not well defined. Here, we report a thorough structural study of TbRND. Besides Apo- and NMP-bound structures, we also solved one TbRND structure in complexed with single-stranded RNA. In combination with mutagenesis and in vitro cleavage assays, our structures indicated that TbRND follows the conserved two-cation-assisted mechanism in catalysis. TbRND is a unique RND member, as it contains a ZFD domain at its C-terminus. In addition to T. brucei, our studies also advanced our understanding on the potential gRNA degradation pathway in T. cruzi, Leishmania, as well for as other disease-associated parasites expressing ZFD-containing RNDs.


Assuntos
Proteínas de Protozoários/química , Estabilidade de RNA/fisiologia , RNA Guia/metabolismo , RNA de Protozoário/metabolismo , Ribonuclease III/química , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , Regulação da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Domínios Proteicos , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonuclease III/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Dedos de Zinco
2.
Proc Natl Acad Sci U S A ; 117(48): 30391-30399, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-33199628

RESUMO

Nucleosome Assembly Protein 1 (NAP1) family proteins are evolutionarily conserved histone chaperones that play important roles in diverse biological processes. In this study, we determined the crystal structure of Arabidopsis NAP1-Related Protein 1 (NRP1) complexed with H2A-H2B and uncovered a previously unknown interaction mechanism in histone chaperoning. Both in vitro binding and in vivo plant rescue assays proved that interaction mediated by the N-terminal α-helix (αN) domain is essential for NRP1 function. In addition, the C-terminal acidic domain (CTAD) of NRP1 binds to H2A-H2B through a conserved mode similar to other histone chaperones. We further extended previous knowledge of the NAP1-conserved earmuff domain by mapping the amino acids of NRP1 involved in association with H2A-H2B. Finally, we showed that H2A-H2B interactions mediated by αN, earmuff, and CTAD domains are all required for the effective chaperone activity of NRP1. Collectively, our results reveal multiple interaction modes of a NAP1 family histone chaperone and shed light on how histone chaperones shield H2A-H2B from nonspecific interaction with DNA.

3.
Nat Plants ; 6(9): 1194, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32724167

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

4.
Nat Plants ; 6(7): 823-837, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32572214

RESUMO

Histone demethylation is crucial for proper chromatin structure and to ensure normal development, and requires the large family of Jumonji C (JmjC)-containing demethylases; however, the molecular mechanisms that regulate the substrate specificity of these JmjC-containing demethylases remain largely unknown. Here, we show that the substrate specificity of the Arabidopsis histone demethylase JMJ16 is broadened from Lys 4 of histone H3 (H3K4) alone in somatic cells to both H3K4 and H3K9 when it binds to the meiocyte-specific histone reader MMD1. Consistent with this, the JMJ16 catalytic domain exhibits both H3K4 and H3K9 demethylation activities. Moreover, the JMJ16 C-terminal FYR domain interacts with the JMJ16 catalytic domain and probably restricts its substrate specificity. By contrast, MMD1 can compete with the N-terminal catalytic domain of JMJ16 for binding to the FYR-C domain, thereby expanding the substrate specificity of JMJ16 by preventing the FYR domain from binding to the catalytic domain. We propose that MMD1 and JMJ16 together in male meiocytes promote gene expression in an H3K9me3-dependent manner and thereby contribute to meiotic chromosome condensation.

5.
Front Microbiol ; 11: 191, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32226412

RESUMO

We report for the first time the isolation of endophytic actinobacteria associated with wild populations of the Chinese medicinal herb Thymus roseus Schipcz obtained from the arid land in Ili and Tacheng of the Xinjiang Province, China. Strains were isolated by special pretreatment of plant tissues and identified based on their 16S rRNA gene sequences, and their antimicrobial activities in vitro were evaluated. A total of 126 endophytic actinobacteria belonging to two classes, eight orders, 14 families, and 24 genera were isolated from different organs at the Ili and Tacheng sites. In addition, the diversity of culturable endophytic actinobacteria genera was higher at Tacheng site (n = 71, 56.35%) than the Ili site (n = 55, 43.65%). A neighbor-joining tree of 126 isolated actinobacteria showing the phylogenetic relationships based on 16S rRNA gene sequences and the genus Streptomyces was the most dominant isolate. The number of endophytic actinobacteria genera obtained from root tissues (n = 54, 42.86%) was higher compared to stem (n = 35, 27.78%) and leaf tissue (n = 37, 29.36%). Among 126 endophytic actinobacteria, 54 strains were antagonistic against at least one or more indicator organisms in vitro. Notably, most strains of Streptomyces proved antagonistic activities. For example, strain T4SB028, namely Streptomyces polyantibioticus, showed the highest inhibition ratio reached 67.06, 64.20, and 70.55% against Alternaria solani, Valsa malicola, and Valsa mali, respectively. The results demonstrate that about 30.95%, 23.01% of the tested endophytic actinobacteria were capable of producing siderophores and chitinase, respectively. Additionally, the results of the amplification of biosynthetic genes polyketide synthetase (PKS-I) and non-ribosomal peptide synthetase (NRPS) indicated that at least one antibiotic biosynthetic gene was detected in 27 (50%) of the tested strains. Our result emphasizes that the endophytic actinobacteria communities are different based on the plant tissues and the geographical environment of the sampled area. Thus, we conclude that T. roseus Schipcz. provided a rich source of endophytic actinobacteria that exhibited a broad-spectrum antimicrobial agent.

6.
Front Plant Sci ; 11: 47, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32117385

RESUMO

As a result of climate change, salinity has become a major abiotic stress that reduces plant growth and crop productivity worldwide. A variety of endophytic bacteria alleviate salt stress; however, their ecology and biotechnological potential has not been fully realized. To address this gap, a collection of 117 endophytic bacteria were isolated from wild populations of the herb Thymus vulgaris in Sheikh Zuweid and Rafah of North Sinai Province, Egypt, and identified based on their 16S rRNA gene sequences. The endophytes were highly diverse, including 17 genera and 30 species. The number of bacterial species obtained from root tissues was higher (n = 18) compared to stem (n = 14) and leaf (n = 11) tissue. The endophytic bacteria exhibited several plant growth-promoting activities in vitro, including auxin synthesis, diazotrophy, phosphate solubilization, siderophore production, and production of lytic enzymes (i.e., chitinase, cellulase, protease, and lipase). Three endophytes representing Bacillus species associated with T. vulgaris such as EGY05, EGY21, and EGY25 were selected based on their ex-situ activities for growth chamber assays to test for their ability to promote the growth of tomato (Solanum lycopersicum L.) under various NaCl concentrations (50-200 mM). All three strains significantly (P < 0.05) promoted the growth of tomato plants under salt stress, compared to uninoculated controls. In addition, inoculated tomato plants by all tested strains decreased (P < 0.05) the activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidase). Six strains, representing Bacillus and Enterobacter species EGY01, EGY05, EGY16, EGY21, EGY25, and EGY31 were selected based on in vitro antagonistic activity to F. oxysporum for pot experiments under salt stress. All tested strains reduced the disease severity index (DSI) of tomato plants at all tested salt concentrations. Gas-chromatography/mass-spectrometry analysis of cell-free extracts of B. subtilis (EGY16) showed at least ten compounds were known to have antimicrobial activity, with the major peaks being benzene, 1,3-dimethyl-, p-xylene, dibutyl phthalate, bis (2-ethylhexyl) phthalate, and tetracosane. This study demonstrates that diverse endophytes grow in wild thyme populations and that some are able to alleviate salinity stress and inhibit F. oxysporum pathogenesis, making them promising candidates for biofertilizers and biocontrol agents.

7.
Cell Discov ; 6: 13, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32194979

RESUMO

African swine fever virus (ASFV) is highly contagious and can cause lethal disease in pigs. ASFV is primarily replicated in the cytoplasm of pig macrophages, which is oxidative and caused constant damage to ASFV genome. ASFV AP endonuclease (AsfvAP) catalyzes DNA cleavage reaction at the abasic site and is a key enzyme of ASFV base excision repair (BER) system. Although it plays an essential role in ASFV survival in host cells, the basis underlying substrate binding and cleavage by AsfvAP remains unclear. Here, we reported the structural and functional studies of AsfvAP, showing that AsfvAP adopts a novel DNA-binding mode distinct from other APs. AsfvAP possesses many unique structural features, including one narrower nucleotide-binding pocket at the active site, the C16-C20 disulfide bond-containing region, and histidine-rich loop. As indicated by our mutagenesis, in vitro binding and cleavage assays, these features are important for AsfvAP to suit the acidic and oxidative environment. Owing to their functional importance, these unique features could serve as targets for designing small molecule inhibitors that could disrupt the repair process of ASFV genome and help fight against this deadly virus in the future.

8.
Plants (Basel) ; 9(2)2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-32079337

RESUMO

Salinization of agricultural land is a devastating phenomenon which will affect future food security. Understanding how plants survive and thrive in response to salinity is therefore critical to potentiate tolerance traits in crop species. The halophyte Salicornia europaea has been used as model system for this purpose. High salinity causes NH4+ accumulation in plant tissues and consequent toxicity symptoms that may further exacerbate those caused by NaCl. In this experiment we exposed Salicornia plants to five concentrations of NaCl (0, 1, 10, 50 and 200 mM) in combination with two concentrations of NH4Cl (1 and 50 mM). We confirmed the euhalophytic behavior of Salicornia that grew better at 200 vs. 0 mM NaCl in terms of both fresh (+34%) and dry (+46%) weights. Addition of 50 mM NH4Cl to the growth medium caused a general growth reduction, which was likely caused by NH4+ accumulation and toxicity in roots and shoots. When plants were exposed to high NH4Cl, high salinity reduced roots NH4+ concentration (-50%) compared to 0 mM NaCl. This correlates with the activation of the NH4+ assimilation enzymes, glutamine synthetase and glutamate dehydrogenase, and the growth inhibition was partially recovered. We argue that NH4+ detoxification is an important trait under high salinity that may differentiate halophytes from glycophytes and we present a possible model for NH4+ detoxification in response to salinity.

9.
Nucleic Acids Res ; 48(6): 3343-3355, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32016421

RESUMO

NrS-1 is the first known phage that can infect Epsilonproteobacteria, one of the predominant primary producers in the deep-sea hydrothermal vent ecosystems. NrS-1 polymerase is a multidomain enzyme and is one key component of the phage replisome. The N-terminal Prim/Pol and HBD domains are responsible for DNA polymerization and de novo primer synthesis activities of NrS-1 polymerase. However, the structure and function of the C-terminus (CTR) of NrS-1 polymerase are poorly understood. Here, we report two crystal structures, showing that NrS-1 CTR adopts one unique hexameric ring-shaped conformation. Although the central helicase domain of NrS-1 CTR shares structural similarity with the superfamily III helicases, the folds of the Head and Tail domains are completely novel. Via mutagenesis and in vitro biochemical analysis, we identified many residues important for the helicase and polymerization activities of NrS-1 polymerase. In addition to NrS-1 polymerase, our study may also help us identify and understand the functions of multidomain polymerases expressed by many NrS-1 related phages.


Assuntos
Bacteriófagos/enzimologia , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/ultraestrutura , Conformação Proteica , Sequência de Aminoácidos/genética , Bacteriófagos/genética , Bacteriófagos/ultraestrutura , Cristalografia por Raios X , DNA Polimerase Dirigida por DNA/química , Ecossistema , Epsilonproteobacteria/genética , Epsilonproteobacteria/virologia , Fontes Hidrotermais/química
10.
Sci Rep ; 10(1): 2388, 2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-32024923

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

11.
BMC Plant Biol ; 19(1): 473, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694537

RESUMO

BACKGROUND: Soil salinization and alkalization are among the major agricultural threats that affect crop productivity worldwide, which are increasing day by day with an alarming rate. In recent years, several halophytes have been investigated for their utilization in soil remediation and to decipher the mechanism of salt-tolerance in these high salt tolerant genetic repositories. Suaeda salsa is an annual halophytic herb in the family Amaranthaceae, displaying high salt and alkali-resistance and having nutritive value. However, the fundamental biological characteristics of this valuable plant remain to be elucidated until today. RESULTS: In this study, we observed the morphology and development of Suaeda salsa, including seed morphology, seed germination, plant morphology, and flower development. Using microscopy, we observed the male and female gametophyte developments of Suaeda salsa. Also, chromosome behaviour during the meiosis of male gametophyte was studied. Eventually, the genome size of Suaeda salsa was estimated through flow cytometry using Arabidopsis as reference. CONCLUSIONS: Our findings suggest that the male and female gametophyte developments of Suaeda salsa are similar to those of the model plant Arabidopsis, and the diploid Suaeda salsa contains nine pairs of chromosomes. The findings also indicate that the haploid genome of Suaeda salsa is approximately 437.5 MB. The observations and results discussed in this study will provide an insight into future research on Suaeda salsa.


Assuntos
Amaranthaceae/genética , Cromossomos de Plantas , Genoma de Planta , Plantas Tolerantes a Sal/genética , Amaranthaceae/anatomia & histologia , Flores/anatomia & histologia , Tamanho do Genoma , Células Germinativas Vegetais/crescimento & desenvolvimento , Tolerância ao Sal/genética , Plantas Tolerantes a Sal/anatomia & histologia
12.
Nat Cell Biol ; 21(10): 1261-1272, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570835

RESUMO

The repression of transposons by the Piwi-interacting RNA (piRNA) pathway is essential to protect animal germ cells. In Drosophila, Panoramix enforces transcriptional silencing by binding to the target-engaged Piwi-piRNA complex, although the precise mechanisms by which this occurs remain elusive. Here, we show that Panoramix functions together with a germline-specific paralogue of a nuclear export factor, dNxf2, and its cofactor dNxt1 (p15), to suppress transposon expression. The transposon RNA-binding protein dNxf2 is required for animal fertility and Panoramix-mediated silencing. Transient tethering of dNxf2 to nascent transcripts leads to their nuclear retention. The NTF2 domain of dNxf2 competes dNxf1 (TAP) off nucleoporins, a process required for proper RNA export. Thus, dNxf2 functions in a Panoramix-dNxf2-dependent TAP/p15 silencing (Pandas) complex that counteracts the canonical RNA exporting machinery and restricts transposons to the nuclear peripheries. Our findings may have broader implications for understanding how RNA metabolism modulates heterochromatin formation.


Assuntos
Proteínas Argonauta/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Inativação Gênica , Heterocromatina/metabolismo , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , Montagem e Desmontagem da Cromatina , Elementos de DNA Transponíveis , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/ultraestrutura , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Oócitos/metabolismo , Oócitos/ultraestrutura , Ovário/citologia , Ovário/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
13.
Mol Cell ; 75(6): 1188-1202.e11, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31399345

RESUMO

The maternal-to-zygotic transition (MZT) is a conserved and fundamental process during which the maternal environment is converted to an environment of embryonic-driven development through dramatic reprogramming. However, how maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein, through genome-wide profiling of RNA 5-methylcytosine (m5C) modification in zebrafish early embryos, we found that m5C-modified maternal mRNAs display higher stability than non-m5C-modified mRNAs during MZT. We discovered that Y-box binding protein 1 (Ybx1) preferentially recognizes m5C-modified mRNAs through π-π interactions with a key residue, Trp45, in Ybx1's cold shock domain (CSD), which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Together with the mRNA stabilizer Pabpc1a, Ybx1 promotes the stability of its target mRNAs in an m5C-dependent manner. Our study demonstrates an unexpected mechanism of RNA m5C-regulated maternal mRNA stabilization during zebrafish MZT, highlighting the critical role of m5C mRNA modification in early development.


Assuntos
5-Metilcitosina/metabolismo , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/fisiologia , Estabilidade de RNA/fisiologia , RNA Mensageiro Estocado/metabolismo , Peixe-Zebra/embriologia , Animais , Células HeLa , Humanos , Camundongos , RNA Mensageiro Estocado/genética , Peixe-Zebra/genética
14.
Biochem Biophys Res Commun ; 517(2): 390-397, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31358319

RESUMO

Although core xylose on glycoproteins has been implicated in allergy, infection and other biological processes, research on core xylose modification is rare. The lack of a ß-d-xylosidase that can catalytically remove the core xylose directly from glycoproteins is a reason for this. Through functional genomic analysis, we identified a glycoprotein core xylosidase and named it gpcXase I. gpcXase I is located immediately downstream of glycoprotein core fucosidase cFase I in Elizabethkingia meningoseptica. These two genes form a functional operon for glycoprotein core modifications. Three acidic residues (Asp-200, Asp-304 and Glu-649) were identified as key catalytic sites for gpcXase I activity, suggesting a unique triacdic mechanize for its activity. Asp-200 was identified a novel and essential base catalysts in the catalytic process, Asp-304 and Glu-649 was function as catalytic nucleophiles and acid catalysts, respectively. In addition, IgE-specific reactions were detected in 55% of serum samples collected from 40 allergic patients, and the reactions were significantly attenuated by removal of the core xylose of the allergen by treatment with gpcXase I. gpcXase I is a novel tool for basic and clinical glycomics.


Assuntos
Proteínas de Bactérias/metabolismo , Flavobacteriaceae/metabolismo , Glicoproteínas/metabolismo , Xilose/metabolismo , Xilosidases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Flavobacteriaceae/química , Flavobacteriaceae/genética , Infecções por Flavobacteriaceae/microbiologia , Humanos , Cinética , Modelos Moleculares , Filogenia , Alinhamento de Sequência , Especificidade por Substrato , Xilosidases/química , Xilosidases/genética
15.
Nat Cell Biol ; 21(8): 978-990, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31358969

RESUMO

Although 5-methylcytosine (m5C) is a widespread modification in RNAs, its regulation and biological role in pathological conditions (such as cancer) remain unknown. Here, we provide the single-nucleotide resolution landscape of messenger RNA m5C modifications in human urothelial carcinoma of the bladder (UCB). We identify numerous oncogene RNAs with hypermethylated m5C sites causally linked to their upregulation in UCBs and further demonstrate YBX1 as an m5C 'reader' recognizing m5C-modified mRNAs through the indole ring of W65 in its cold-shock domain. YBX1 maintains the stability of its target mRNA by recruiting ELAVL1. Moreover, NSUN2 and YBX1 are demonstrated to drive UCB pathogenesis by targeting the m5C methylation site in the HDGF 3' untranslated region. Clinically, a high coexpression of NUSN2, YBX1 and HDGF predicts the poorest survival. Our findings reveal an unprecedented mechanism of RNA m5C-regulated oncogene activation, providing a potential therapeutic strategy for UCB.


Assuntos
5-Metilcitosina/metabolismo , Regulação da Expressão Gênica/genética , Metiltransferases/genética , Neoplasias da Bexiga Urinária/genética , Proteína 1 de Ligação a Y-Box/genética , Animais , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/metabolismo , Humanos , Camundongos , RNA Mensageiro/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia
16.
Nucleic Acids Res ; 47(15): 7886-7900, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31216029

RESUMO

The intron-lariat spliceosome (ILS) complex is highly conserved among eukaryotes, and its disassembly marks the end of a canonical splicing cycle. In this study, we show that two conserved disassembly factors of the ILS complex, Increased Level of Polyploidy1-1D (ILP1) and NTC-Related protein 1 (NTR1), positively regulate microRNA (miRNA) biogenesis by facilitating transcriptional elongation of MIRNA (MIR) genes in Arabidopsis thaliana. ILP1 and NTR1 formed a stable complex and co-regulated alternative splicing of more than a hundred genes across the Arabidopsis genome, including some primary transcripts of miRNAs (pri-miRNAs). Intriguingly, pri-miRNAs, regardless of having introns or not, were globally down-regulated when the ILP1 or NTR1 function was compromised. ILP1 and NTR1 interacted with core miRNA processing proteins Dicer-like 1 and Serrate, and were required for proper RNA polymerase II occupancy at elongated regions of MIR chromatin, without affecting either MIR promoter activity or pri-miRNA decay. Our results provide further insights into the regulatory role of spliceosomal machineries in the biogenesis of miRNAs.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Genoma de Planta , MicroRNAs/genética , Proteínas Periplásmicas de Ligação/genética , Processamento de RNA , Proteínas Repressoras/metabolismo , Spliceossomos/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Éxons , Regulação da Expressão Gênica de Plantas , Íntrons , MicroRNAs/biossíntese , Proteínas Periplásmicas de Ligação/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Spliceossomos/metabolismo
17.
Nat Commun ; 10(1): 2063, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048693

RESUMO

RELATIVE OF EARLY FLOWERING 6 (REF6/JMJ12), a Jumonji C (JmjC)-domain-containing H3K27me3 histone demethylase, finds its target loci in Arabidopsis genome by directly recognizing the CTCTGYTY motif via its zinc-finger (ZnF) domains. REF6 tends to bind motifs located in active chromatin states that are depleted for heterochromatic modifications. However, the underlying mechanism remains unknown. Here, we show that REF6 preferentially bind to hypo-methylated CTCTGYTY motifs in vivo, and that CHG methylation decreases REF6 DNA binding affinity in vitro. In addition, crystal structures of ZnF-clusters in complex with DNA oligonucleotides reveal that 5-methylcytosine is unfavorable for REF6 binding. In drm1 drm2 cmt2 cmt3 (ddcc) quadruple mutants, in which non-CG methylation is significantly reduced, REF6 can ectopically bind a small number of new target loci, most of which are located in or neighbored with short TEs in euchromatic regions. Collectively, our findings reveal that DNA methylation, likely acting in combination with other epigenetic modifications, may partially explain why REF6 binding is depleted in heterochromatic loci.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Metilação de DNA/fisiologia , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fatores de Transcrição/metabolismo , 5-Metilcitosina/metabolismo , Epigênese Genética/fisiologia , Eucromatina/metabolismo , Heterocromatina/metabolismo , Mutação , Plantas Geneticamente Modificadas , Dedos de Zinco/fisiologia
18.
Front Microbiol ; 10: 968, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31134016

RESUMO

Eukaryotic ribosomes are essential for proliferation, differentiation, and cell growth. RPs26 is a ribosomal subunit structural protein involved in the growth and development process. Little is known about the function of PsRPs26 in pathogenic fungi. In this study, we isolated the RPs26 gene, PsRPs26, from Puccinia striiformis f. sp. tritici (Pst). PsRPs26 contains a eukaryotic-specific Y62-K70 motif and is more than 90% identical with its ortholog gene in other fungi. PsRPs26 was found to be localized in both the nucleus and cytoplasm. Expression of PsRPs26 increased when wheat seedlings were inoculated with the Pst CYR31 isolate. Moreover, knockdown of PsRPs26 by a host-induced gene silencing system inhibited growth and limited urediospore production in Pst. Our discovery that PsRPs26 may contribute to the pathogenicity of Pst and open a new way in the pathogenic function of PsRPs26 in cereal rust fungi.

19.
Proc Natl Acad Sci U S A ; 116(16): 8054-8059, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30926664

RESUMO

Phytophthora are eukaryotic pathogens that cause enormous losses in agriculture and forestry. Each Phytophthora species encodes hundreds of effector proteins that collectively have essential roles in manipulating host cellular processes and facilitating disease development. Here we report the crystal structure of the effector Phytophthora suppressor of RNA silencing 2 (PSR2). PSR2 produced by the soybean pathogen Phytophthora sojae (PsPSR2) consists of seven tandem repeat units, including one W-Y motif and six L-W-Y motifs. Each L-W-Y motif forms a highly conserved fold consisting of five α-helices. Adjacent units are connected through stable, directional linkages between an internal loop at the C terminus of one unit and a hydrophobic pocket at the N terminus of the following unit. This unique concatenation results in an overall stick-like structure of PsPSR2. Genome-wide analyses reveal 293 effectors from five Phytophthora species that have the PsPSR2-like arrangement, that is, containing a W-Y motif as the "start" unit, various numbers of L-W-Y motifs as the "middle" units, and a degenerate L-W-Y as the "end" unit. Residues involved in the interunit interactions show significant conservation, suggesting that these effectors also use the conserved concatenation mechanism. Furthermore, functional analysis demonstrates differential contributions of individual units to the virulence activity of PsPSR2. These findings suggest that the L-W-Y fold is a basic structural and functional module that may serve as a "building block" to accelerate effector evolution in Phytophthora.


Assuntos
Proteínas de Bactérias/química , Phytophthora/patogenicidade , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/fisiologia , Proteínas de Bactérias/genética , Modelos Moleculares , Phytophthora/química , Phytophthora/genética , Doenças das Plantas/microbiologia , Sequências de Repetição em Tandem/genética
20.
J Exp Bot ; 70(12): 3057-3073, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-30921464

RESUMO

Varieties of Gram-negative bacterial pathogens infect their eukaryotic hosts by deploying the type III translocon to deliver effector proteins into the cytosol of eukaryotic cells in which effectors execute their pathological functions. The translocon is hypothetically assembled by bacterial translocators in association with the assumed receptors situated on eukaryotic plasma membranes. This hypothesis is partially verified in the present study with genetic, biochemical, and pathological evidence for the role of a rice aquaporin, plasma membrane intrinsic protein PIP1;3, in the cytosolic import of the transcription activator-like effector PthXo1 from the bacterial blight pathogen. PIP1;3 interacts with the bacterial translocator Hpa1 at rice plasma membranes to control PthXo1 translocation from cells of a well-characterized strain of the bacterial blight pathogen into the cytosol of cells of a susceptible rice variety. An extracellular loop sequence of PIP1;3 and the α-helix motif of Hpa1 determine both the molecular interaction and its consequences with respect to the effector translocation and the bacterial virulence on the susceptible rice variety. Overall, these results provide multiple experimental avenues to support the hypothesis that interactions between bacterial translocators and their interactors at the target membrane are essential for bacterial effector translocation.


Assuntos
Aquaporinas/genética , Proteínas de Bactérias/genética , Glicosiltransferases/genética , Interações Hospedeiro-Patógeno , Oryza/genética , Proteínas de Plantas/genética , Xanthomonas/genética , Aquaporinas/metabolismo , Proteínas de Bactérias/metabolismo , Glicosiltransferases/metabolismo , Oryza/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Xanthomonas/metabolismo
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