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Leaves of the carnivorous sundew plants (Drosera spp.) secrete mucilage that hosts microorganisms, but whether this microbiota contributes to prey digestion is unclear. We identified the acidophilic fungus Acrodontium crateriforme as the dominant species in the mucilage microbial communities, thriving in multiple sundew species across the global range. The fungus grows and sporulates on sundew glands as its preferred acidic environment, and its presence in traps increased the prey digestion process. A. crateriforme has a reduced genome similar to other symbiotic fungi. During A. crateriforme-Drosera spatulata coexistence and digestion of prey insects, transcriptomes revealed significant gene co-option in both partners. Holobiont expression patterns during prey digestion further revealed synergistic effects in several gene families including fungal aspartic and sedolisin peptidases, facilitating prey digestion in leaves, as well as nutrient assimilation and jasmonate signalling pathway expression. This study establishes that botanical carnivory is defined by adaptations involving microbial partners and interspecies interactions.
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Since its emergence, SARS-CoV-2 has been continuously evolving, hampering the effectiveness of current vaccines against COVID-19. mAbs can be used to treat patients at risk of severe COVID-19. Thus, the development of broadly protective mAbs and an understanding of the underlying protective mechanisms are of great importance. Here, we isolated mAbs from donors with breakthrough infection with Omicron subvariants using a single-B cell screening platform. We identified a mAb, O5C2, which possesses broad-spectrum neutralization and antibody-dependent cell-mediated cytotoxic activities against SARS-CoV-2 variants, including EG.5.1. Single-particle analysis by cryo-electron microscopy revealed that O5C2 targeted an unusually large epitope within the receptor-binding domain of spike protein that overlapped with the angiotensin-converting enzyme 2 binding interface. Furthermore, O5C2 effectively protected against BA.5 Omicron infection in vivo by mediating changes in transcriptomes enriched in genes involved in apoptosis and interferon responses. Our findings provide insights into the development of pan-protective mAbs against SARS-CoV-2.
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Anticuerpos Antivirales , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , SARS-CoV-2/inmunología , Humanos , COVID-19/inmunología , COVID-19/virología , Anticuerpos Antivirales/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Animales , Ratones , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Microscopía por Crioelectrón , Epítopos/inmunología , Anticuerpos ampliamente neutralizantes/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , FemeninoRESUMEN
The miR390-derived TAS3 trans-acting short-interfering RNAs (tasiRNAs) module represents a conserved RNA silencing pathway in the plant kingdom; however, its characterization in the bryophyte Marchantia polymorpha is limited. This study elucidated that MpDCL4 processes MpTAS3 double-stranded RNA (dsRNA) to generate tasiRNAs, primarily from the 5'- and 3'-ends of dsRNA. Notably, we discovered a novel tasiRNA, tasi78A, which can negatively regulate a cytochrome P450 gene, MpCYP78A101. Additionally, tasi78A was abundant in MpAGO1, and transient expression assays underscored the role of tasi78A in repressing MpCYP78A101. A microRNA, miR11700, also regulates MpCYP78A101 expression. This coordinate regulation suggests a role in modulating auxin signaling at apical notches of gemma, influencing the growth and sexual organ development of M. polymorpha and emphasizing the significance of RNA silencing in MpCYP78A101 regulation. However, phylogenetic analysis identified another paralog of the CYP78 family, Mp1g14150, which may have a redundant role with MpCYP78A101, explaining the absence of noticeable morphological changes in loss-of-function plants. Taken together, our findings provide new insights into the combined regulatory roles of miR390/MpTAS3/miR11700 in controlling MpCYP78A101 and expand our knowledge about the biogenesis and regulation of tasiRNAs in M. polymorpha.
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Sistema Enzimático del Citocromo P-450 , Regulación de la Expresión Génica de las Plantas , Marchantia , MicroARNs , ARN Interferente Pequeño , Marchantia/genética , MicroARNs/genética , MicroARNs/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Filogenia , ARN de Planta/genética , ARN de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Our study presents a 319-gene panel targeting inherited retinal dystrophy (IRD) genes. Through a multi-center retrospective cohort study, we validated the assay's effectiveness and clinical utility and characterized the mutation spectrum of Taiwanese IRD patients. Between January 2018 and May 2022, 493 patients in 425 unrelated families, all initially suspected of having IRD without prior genetic diagnoses, underwent detailed ophthalmic and physical examinations (with extra-ocular features recorded) and genetic testing with our customized panel. Disease-causing variants were identified by segregation analysis and clinical interpretation, with validation via Sanger sequencing. We achieved a read depth of >200× for 94.2% of the targeted 1.2 Mb region. 68.5% (291/425) of the probands received molecular diagnoses, with 53.9% (229/425) resolved cases. Retinitis pigmentosa (RP) is the most prevalent initial clinical impression (64.2%), and 90.8% of the cohort have the five most prevalent phenotypes (RP, cone-rod syndrome, Usher's syndrome, Leber's congenital amaurosis, Bietti crystalline dystrophy). The most commonly mutated genes of probands that received molecular diagnosis are USH2A (13.7% of the cohort), EYS (11.3%), CYP4V2 (4.8%), ABCA4 (4.5%), RPGR (3.4%), and RP1 (3.1%), collectively accounted for 40.8% of diagnoses. We identify 87 unique unreported variants previously not associated with IRD and refine clinical diagnoses for 21 patients (7.22% of positive cases). We developed a customized gene panel and tested it on the largest Taiwanese cohort, showing that it provides excellent coverage for diverse IRD phenotypes.
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The superior photosynthetic efficiency of C4 leaves over C3 leaves is owing to their unique Kranz anatomy, in which the vein is surrounded by one layer of bundle sheath (BS) cells and one layer of mesophyll (M) cells. Kranz anatomy development starts from three contiguous ground meristem (GM) cells, but its regulators and underlying molecular mechanism are largely unknown. To identify the regulators, we obtained the transcriptomes of 11 maize embryonic leaf cell types from five stages of pre-Kranz cells starting from median GM cells and six stages of pre-M cells starting from undifferentiated cells. Principal component and clustering analyses of transcriptomic data revealed rapid pre-Kranz cell differentiation in the first two stages but slow differentiation in the last three stages, suggesting early Kranz cell fate determination. In contrast, pre-M cells exhibit a more prolonged transcriptional differentiation process. Differential gene expression and coexpression analyses identified gene coexpression modules, one of which included 3 auxin transporter and 18 transcription factor (TF) genes, including known regulators of Kranz anatomy and/or vascular development. In situ hybridization of 11 TF genes validated their expression in early Kranz development. We determined the binding motifs of 15 TFs, predicted TF target gene relationships among the 18 TF and 3 auxin transporter genes, and validated 67 predictions by electrophoresis mobility shift assay. From these data, we constructed a gene regulatory network for Kranz development. Our study sheds light on the regulation of early maize leaf development and provides candidate leaf development regulators for future study.
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Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Hojas de la Planta , Transcriptoma , Zea mays , Ácidos Indolacéticos/metabolismo , Captura por Microdisección con Láser , Fotosíntesis/genética , Hojas de la Planta/embriología , Hojas de la Planta/genética , Zea mays/enzimología , Zea mays/genéticaRESUMEN
TRIM28 is a scaffold protein that interacts with DNA-binding proteins and recruits corepressor complexes to cause gene silencing. TRIM28 contributes to physiological functions such as cell growth and differentiation. In the chronic myeloid leukemia cell line K562, we edited TRIM28 using CRISPR/Cas9 technology, and the complete and partial knockout (KO) cell clones were obtained and confirmed using quantitative droplet digital PCR (ddPCR) technology. The amplicon sequencing demonstrated no off-target effects in our gene editing experiments. The TRIM28 KO cells grew slowly and appeared red, seeming to have a tendency towards erythroid differentiation. To understand how TRIM28 controls K562 cell proliferation and differentiation, transcriptome profiling analysis was performed in wild-type and KO cells to identify TRIM28-regulated genes. Some of the RNAs that encode the proteins regulating the cell cycle were increased (such as p21) or decreased (such as cyclin D2) in TRIM28 KO cell clones; a tumor marker, the MAGE (melanoma antigen) family, which is involved in cell proliferation was reduced. Moreover, we found that knockout of TRIM28 can induce miR-874 expression to downregulate MAGEC2 mRNA via post-transcriptional regulation. The embryonic epsilon-globin gene was significantly increased in TRIM28 KO cell clones through the downregulation of transcription repressor SOX6. Taken together, we provide evidence to demonstrate the regulatory network of TRIM28-mediated cell growth and erythroid differentiation in K562 leukemia cells.
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Edición Génica , MicroARNs , Sistemas CRISPR-Cas , Proliferación Celular/genética , Expresión Génica , Subunidades de Hemoglobina/genética , Subunidades de Hemoglobina/metabolismo , Humanos , Células K562 , Factores de Transcripción/metabolismo , Proteína 28 que Contiene Motivos Tripartito/metabolismoRESUMEN
The ecology and genetic diversity of the model yeast Saccharomyces cerevisiae before human domestication remain poorly understood. Taiwan is regarded as part of this yeast's geographic birthplace, where the most divergent natural lineage was discovered. Here, we extensively sampled the broadleaf forests across this continental island to probe the ancestral species' diversity. We found that S. cerevisiae is distributed ubiquitously at low abundance in the forests. Whole-genome sequencing of 121 isolates revealed nine distinct lineages that diverged from Asian lineages during the Pleistocene, when a transient continental shelf land bridge connected Taiwan to other major landmasses. Three lineages are endemic to Taiwan and six are widespread in Asia, making this region a focal biodiversity hotspot. Both ancient and recent admixture events were detected between the natural lineages, and a genetic ancestry component associated with isolates from fruits was detected in most admixed isolates. Collectively, Taiwanese isolates harbor genetic diversity comparable to that of the whole Asia continent, and different lineages have coexisted at a fine spatial scale even on the same tree. Patterns of variations within each lineage revealed that S. cerevisiae is highly clonal and predominantly reproduces asexually in nature. We identified different selection patterns shaping the coding sequences of natural lineages and found fewer gene family expansion and contractions that contrast with domesticated lineages. This study establishes that S. cerevisiae has rich natural diversity sheltered from human influences, making it a powerful model system in microbial ecology.
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Biodiversidad , Saccharomyces cerevisiae , Asia , Humanos , Filogenia , Saccharomyces cerevisiae/genética , Taiwán , Secuenciación Completa del GenomaRESUMEN
Colletotrichum scovillei causes anthracnose of chili pepper in many countries. Three strains of this pathogen, Coll-524, Coll-153, and Coll-365, show varied virulence on chili pepper. Among the three strains, Coll-365 showed significant defects in growth and virulence. To decipher the genetic variations among these strains and identify genes contributing to growth and virulence, comparative genomic analysis and gene transformation to show gene function were applied in this study. Compared to Coll-524, Coll-153, and Coll-365 had numerous gene losses including 32 candidate effector genes that are mainly exist in acutatum species complex. A cluster of 14 genes in a 34-kb genomic fragment was lost in Coll-365. Through gene transformation, three genes in the 34-kb fragment were identified to have functions in growth and/or virulence of C. scovillei. CsPLAA encoding a phospholipase A2-activating protein enhanced the growth of Coll-365. A combination of CsPLAA with one transcription factor CsBZTF and one C6 zinc finger domain-containing protein CsCZCP was found to enhance the pathogenicity of Coll-365. Introduction of CsGIP, which encodes a hypothetical protein, into Coll-365 caused a reduction in the germination rate of Coll-365. In conclusion, the highest virulent strain Coll-524 had more genes and encoded more pathogenicity related proteins and transposable elements than the other two strains, which may contribute to the high virulence of Coll-524. In addition, the absence of the 34-kb fragment plays a critical role in the defects of growth and virulence of strain Coll-365.
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Here we report the 409.5 Mb chromosome-level assembly of the first bred semi-dwarf rice, the Taichung Native 1 (TN1), which served as the template for the development of the Green Revolution (GR) cultivar IR8 "miracle rice". We sequenced the TN1 genome utilizing multiple platforms and produced PacBio long reads, Illumina paired-end reads, Illumina mate-pair reads and 10x Genomics linked reads. We used a hybrid approach to assemble the 226× coverage of sequences by a combination of de novo and reference-guided approaches. The assembled TN1 genome has an N50 scaffold size of 33.1 Mb with the longest measuring 45.5 Mb. We annotated 37,526 genes, in which 24,102 (64.23%) were assigned Blast2GO annotations. The genome has 4672 or 95.4% complete BUSCOs and a repeat content of 51.52%. We developed our own method of creating a GR pangenome using the orthologous relationships of the proteins of TN1, IR8, MH63 and IR64, identifying 16,999 core orthologue groups of Green Revolution. From the pangenome, we identified a set of shared and unique gene ontology terms for the accessory clusters, characterizing TN1, IR8, MH63 and IR64. This TN1 genome assembly and GR pangenome will be a resource for new genomic discoveries about Green Revolution, and for improving the disease and insect resistances and the yield of rice.
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Oryza , Cromosomas , Genoma , Genómica , Oryza/genética , FitomejoramientoRESUMEN
Mutational profiling of patients' tumors has suggested that the development of oral cavity squamous cell carcinoma (OCSCC) is driven by multiple genes in multiple pathways. This study aimed to examine the association between genomic alterations and clinical outcomes in patients with advanced stages OCSCC to facilitate prognostic stratification. We re-analyzed our previous whole-exome sequencing data from 165 long-term follow-ups of stages III and IV patients with OCSCC. Their frequent mutations were mapped to 10 oncogenic signaling pathways. Clinicopathological risk factors, relapse, and survival were analyzed to identify the genetic factors associated with advanced OCSCC. Frequent genetic alterations included point mutations in TP53, FAT1, NOTCH1, CASP8, CDKN2A, HRAS, PIK3CA, KMT2B (also known as MLL4), and LINC00273; amplified segments in CCND1, EGFR, CTTN, and FGFR1; and lost segments in CDKN2A, ADAM3A, and CFHR1/CFHR4. Comprehensive analysis of genetic alterations revealed that subgroups based on mutational signatures had a significant negative impact on disease-free survival (p = 0.0005) and overall survival (p = 0.0024). Several important signaling pathways were identified to be frequently genetically altered in our cohort. A specific subgroup of patients with alterations in NOTCH, RTK/RAS/MAPK, and TGF-beta pathways that had a significantly negative impact on disease-free survival (p = 0.0009). Thirty percent of samples had multiple targetable mutations in multiple pathways, indicating opportunities for novel therapy.
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In higher plants, whole-genome duplication (WGD) is thought to facilitate the evolution of C4 photosynthesis from C3 photosynthesis. To understand this issue, we used new and existing leaf-development transcriptomes to construct two coding sequence databases for C4Gynandropsis gynandra and C3Tarenaya hassleriana, which shared a WGD before their divergence. We compared duplicated genes in the two species and found that the WGD contributed to four aspects of the evolution of C4 photosynthesis in G. gynandra. First, G. gynandra has retained the duplicates of ALAAT (alanine aminotransferase) and GOGAT (glutamine oxoglutarate aminotransferase) for nitrogen recycling to establish a photorespiratory CO2 pump in bundle sheath (BS) cells for increasing photosynthesis efficiency, suggesting that G. gynandra experienced a C3-C4 intermediate stage during the C4 evolution. Second, G. gynandra has retained almost all known vein-development-related paralogous genes derived from the WGD event, likely contributing to the high vein complexity of G. gynandra. Third, the WGD facilitated the evolution of C4 enzyme genes and their recruitment into the C4 pathway. Fourth, several genes encoding photosystem I proteins were derived from the WGD and are upregulated in G. gynandra, likely enabling the NADH dehydrogenase-like complex to produce extra ATPs for the C4 CO2 concentration mechanism. Thus, the WGD apparently played an enabler role in the evolution of C4 photosynthesis in G. gynandra. Importantly, an ALAAT duplicate became highly expressed in BS cells in G. gynandra, facilitating nitrogen recycling and transition to the C4 cycle. This study revealed how WDG may facilitate C4 photosynthesis evolution.
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Magnoliopsida , Hojas de la Planta , Duplicación de Gen , Magnoliopsida/genética , Fotosíntesis/genética , Hojas de la Planta/genética , TranscriptomaRESUMEN
Calonectria ilicicola (anamorph: Cylindrocladium parasiticum) is a soilborne plant-pathogenic fungus with a broad host range, and it can cause red crown rot of soybean and Cylindrocladium black rot of peanut, which has become an emerging threat to crop production worldwide. Limited molecular studies have focused on Calonectria ilicicola and one of the possible difficulties is the lack of genomic resources. This study presents the first high quality and near-completed genome of C. ilicicola, using the Oxford Nanopore GridION sequencing platform. A total of 16 contigs were assembled and the genome of C. ilicicola isolate F018 was estimated to have 11 chromosomes. Currently, the C. ilicicola F018 genome represents the most contiguous assembly, which has the lowest contig number and the highest contig N50 among all Calonectria genome resources. Putative protein-coding sequences and secretory proteins were estimated to be 17,308 and 1,930 in the C. ilicicola F018 genome, respectively; and the prediction was close to other plant-pathogenic fungi, such as Fusarium species, within the Nectriaceae family. The availability of this high-quality genome resource is expected to facilitate research on fungal biology and genetics of C. ilicicola and to support advanced understanding of pathogen virulence and disease management.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
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Fusarium , Hypocreales , Enfermedades de las Plantas , Glycine maxRESUMEN
Studies in various animals have shown that asymmetrically localized maternal transcripts play important roles in axial patterning and cell fate specification in early embryos. However, comprehensive analyses of the maternal transcriptomes with spatial information are scarce and limited to a handful of model organisms. In cephalochordates (amphioxus), an early branching chordate group, maternal transcripts of germline determinants form a compact granule that is inherited by a single blastomere during cleavage stages. Further blastomere separation experiments suggest that other transcripts associated with the granule are likely responsible for organizing the posterior structure in amphioxus; however, the identities of these determinants remain unknown. In this study, we used high-throughput RNA sequencing of separated blastomeres to examine asymmetrically localized transcripts in two-cell and eight-cell stage embryos of the amphioxus Branchiostoma floridae. We identified 111 and 391 differentially enriched transcripts at the 2-cell stage and the 8-cell stage, respectively, and used in situ hybridization to validate the spatial distribution patterns for a subset of these transcripts. The identified transcripts could be categorized into two major groups: (1) vegetal tier/germ granule-enriched and (2) animal tier/anterior-enriched transcripts. Using zebrafish as a surrogate model system, we showed that overexpression of one animal tier/anterior-localized amphioxus transcript, zfp665, causes a dorsalization/anteriorization phenotype in zebrafish embryos by downregulating the expression of the ventral gene, eve1, suggesting a potential function of zfp665 in early axial patterning. Our results provide a global transcriptomic blueprint for early-stage amphioxus embryos. This dataset represents a rich platform to guide future characterization of molecular players in early amphioxus development and to elucidate conservation and divergence of developmental programs during chordate evolution.
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Blastómeros/metabolismo , Anfioxos/genética , Herencia Materna , Transcriptoma , Animales , Regulación del Desarrollo de la Expresión Génica , Anfioxos/embriología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Pez CebraRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID 19, continues to evolve since its first emergence in December 2019. Using the complete sequences of 1,932 SARS-CoV-2 genomes, various clustering analyses consistently identified six types of the strains. Independent of the dendrogram construction, 13 signature variations in the form of single nucleotide variations (SNVs) in protein coding regions and one SNV in the 5' untranslated region (UTR) were identified and provided a direct interpretation for the six types (types I to VI). The six types of the strains and their underlying signature SNVs were validated in two subsequent analyses of 6,228 and 38,248 SARS-CoV-2 genomes which became available later. To date, type VI, characterized by the four signature SNVs C241T (5'UTR), C3037T (nsp3 F924F), C14408T (nsp12 P4715L), and A23403G (Spike D614G), with strong allelic associations, has become the dominant type. Since C241T is in the 5' UTR with uncertain significance and the characteristics can be captured by the other three strongly associated SNVs, we focus on the other three. The increasing frequency of the type VI haplotype 3037T-14408T-23403G in the majority of the submitted samples in various countries suggests a possible fitness gain conferred by the type VI signature SNVs. The fact that strains missing one or two of these signature SNVs fail to persist implies possible interactions among these SNVs. Later SNVs such as G28881A, G28882A, and G28883C have emerged with strong allelic associations, forming new subtypes. This study suggests that SNVs may become an important consideration in SARS-CoV-2 classification and surveillance.
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Alelos , Genoma Viral , Genómica , SARS-CoV-2/genética , Geografía , Humanos , Polimorfismo de Nucleótido Simple/genética , Factores de TiempoRESUMEN
Arabidopsis AINTEGUMENTA (ANT), an AP2 transcription factor, is known to control plant growth and floral organogenesis. In this study, our transcriptome analysis and in situ hybridization assays of maize embryonic leaves suggested that maize ANT1 (ZmANT1) regulates vascular development. To better understand ANT1 functions, we determined the binding motif of ZmANT1 and then showed that ZmANT1 binds the promoters of millet SCR1, GNC, and AN3, which are key regulators of Kranz anatomy, chloroplast development, and plant growth, respectively. We generated a mutant with a single-codon deletion and two frameshift mutants of the ANT1 ortholog in the C4 millet Setaria viridis by the CRISPR/Cas9 technique. The two frameshift mutants displayed reduced photosynthesis efficiency and growth rate, smaller leaves, and lower grain yields than wild-type (WT) plants. Moreover, their leaves sporadically exhibited distorted Kranz anatomy and vein spacing. Conducting transcriptomic analysis of developing leaves in the WT and the three mutants we identified differentially expressed genes (DEGs) in the two frameshift mutant lines and found many down-regulated DEGs enriched in photosynthesis, heme, tetrapyrrole binding, and antioxidant activity. In addition, we predicted many target genes of ZmANT1 and chose 13 of them to confirm binding of ZmANT1 to their promoters. Based on the above observations, we proposed a model for ANT1 regulation of cell proliferation and leaf growth, vascular and vein development, chloroplast development, and photosynthesis through its target genes. Our study revealed biological roles of ANT1 in several developmental processes beyond its known roles in plant growth and floral organogenesis.
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Translocador 1 del Nucleótido Adenina/metabolismo , Zea mays/crecimiento & desarrollo , Zea mays/genética , Translocador 1 del Nucleótido Adenina/fisiología , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Cloroplastos/metabolismo , Flores/genética , Flores/crecimiento & desarrollo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/genética , Mijos/genética , Mijos/metabolismo , Organogénesis de las Plantas/genética , Fotosíntesis/genética , Fotosíntesis/fisiología , Desarrollo de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis, kills over 1 million people worldwide annually. Development of drug resistance (DR) in the pathogen is a major challenge for TB control. We conducted whole-genome analysis of seven Taiwan M. tuberculosis isolates: One drug susceptible (DS) and five DR Beijing lineage isolates and one DR Euro-American lineage isolate. Developing a new method for DR mutation identification and applying it to the next-generation sequencing (NGS) data from the 6 Beijing lineage isolates, we identified 13 known and 6 candidate DR mutations and provided experimental support for 4 of them. We assembled the genomes of one DS and two DR Beijing lineage isolates and the Euro-American lineage isolate using NGS data. Moreover, using both PacBio and NGS sequencing data, we obtained a high-quality assembly of an extensive DR Beijing lineage isolate. Comparative analysis of these five newly assembled genomes and two published complete genomes revealed a large number of genetic changes, including gene gains and losses, indels and translocations, suggesting rapid evolution of M. tuberculosis. We found the MazEF toxin-antitoxin system in all the seven isolates studied and several interesting mutations in MazEF proteins. Finally, we used the four assembled Beijing lineage genomes to construct a high-quality Beijing lineage reference genome that is DS and contains all the genes in the four genomes. It contains 212 genes not found in the standard reference H37Rv, which is Euro-American. It is therefore a better reference than H37Rv for the Beijing lineage, the predominant lineage in Asia.
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Genoma Bacteriano/genética , Mycobacterium tuberculosis/genética , Proteínas Bacterianas/genética , Beijing , Farmacorresistencia Bacteriana Múltiple/genética , Mutación INDEL/genética , Mutación/genética , Mycobacterium tuberculosis/clasificación , FilogeniaRESUMEN
Pyricularia is a fungal genus comprising several pathogenic species causing the blast disease in monocots. Pyricularia oryzae, the best-known species, infects rice, wheat, finger millet, and other crops. As past comparative and population genomics studies mainly focused on isolates of P. oryzae, the genomes of the other Pyricularia species have not been well explored. In this study, we obtained a chromosomal-level genome assembly of the finger millet isolate P. oryzae MZ5-1-6 and also highly contiguous assemblies of Pyricularia sp. LS, P. grisea, and P. pennisetigena. The differences in the genomic content of repetitive DNA sequences could largely explain the variation in genome size among these new genomes. Moreover, we found extensive gene gains and losses and structural changes among Pyricularia genomes, including a large interchromosomal translocation. We searched for homologs of known blast effectors across fungal taxa and found that most avirulence effectors are specific to Pyricularia, whereas many other effectors share homologs with distant fungal taxa. In particular, we discovered a novel effector family with metalloprotease activity, distinct from the well-known AVR-Pita family. We predicted 751 gene families containing putative effectors in 7 Pyricularia genomes and found that 60 of them showed differential expression in the P. oryzae MZ5-1-6 transcriptomes obtained under experimental conditions mimicking the pathogen infection process. In summary, this study increased our understanding of the structural, functional, and evolutionary genomics of the blast pathogen and identified new potential effector genes, providing useful data for developing crops with durable resistance.
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Evolución Biológica , Genoma Fúngico , Familia de Multigenes , Pyricularia grisea/genética , Cromosomas Fúngicos , Metaloproteasas/genética , Mijos/microbiología , Enfermedades de las Plantas , Homología de Secuencia de Ácido Nucleico , TranscriptomaRESUMEN
Degradome sequencing provides large amounts of data regarding RNA degradation. The degradome library construction described here is modified from the 5'-rapid amplification of cDNA ends (5'-RACE), and each degradome cDNA is sequenced by next-generation sequencing (NGS). Degradome profiles provide information confirming miRNA-mediated cleavage of target genes and allow the identification of novel targets. Furthermore, degradome sequencing provides additional information for the study of RNA processing, such as information regarding RNA-binding proteins. In this chapter, we describe a detailed optimized protocol for constructing a degradome library with high yield and quality, along with NGS and data mining procedures. We hope that the degradome approach will be applied to further studies of non-model organisms.
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Plantas/genética , Estabilidad del ARN/genética , ARN de Planta/genética , Análisis de Secuencia de ARN/métodos , Secuencia de Bases , ADN Complementario/genética , Regulación de la Expresión Génica de las Plantas/genética , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , MicroARNs/genética , Proteínas de Plantas/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Time-series transcriptomes of a biological process obtained under different conditions are useful for identifying the regulators of the process and their regulatory networks. However, such data are 3D (gene expression, time, and condition), and there is currently no method that can deal with their full complexity. Here, we developed a method that avoids time-point alignment and normalization between conditions. We applied it to analyze time-series transcriptomes of developing maize leaves under light-dark cycles and under total darkness and obtained eight time-ordered gene coexpression networks (TO-GCNs), which can be used to predict upstream regulators of any genes in the GCNs. One of the eight TO-GCNs is light-independent and likely includes all genes involved in the development of Kranz anatomy, which is a structure crucial for the high efficiency of photosynthesis in C4 plants. Using this TO-GCN, we predicted and experimentally validated a regulatory cascade upstream of SHORTROOT1, a key Kranz anatomy regulator. Moreover, we applied the method to compare transcriptomes from maize and rice leaf segments and identified regulators of maize C4 enzyme genes and RUBISCO SMALL SUBUNIT2 Our study provides not only a powerful method but also novel insights into the regulatory networks underlying Kranz anatomy development and C4 photosynthesis.
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Redes Reguladoras de Genes/genética , Fotosíntesis/genética , Hojas de la Planta/genética , Transcriptoma/genética , Regulación de la Expresión Génica de las Plantas/genética , Oryza/genética , Fotoperiodo , Proteínas de Plantas , Ribulosa-Bifosfato Carboxilasa/genética , Zea mays/genéticaRESUMEN
Cellular responses to oxygen deprivation are essential for survival during energy crises in plants and animals. Hypoxia caused by submergence results in reprogramming of translation dynamic in plants, but the molecular mechanisms are not well understood. Here we show that Arabidopsis Snf1-related protein kinase 1 (SnRK1) phosphorylates the translation initiation factor eIFiso4G to regulate translation dynamic under submergence. In Arabidopsis, there are two eIFiso4G genes, eIFiso4G1 and eIFiso4G2, which belong to the eIF4G family. Both eIFiso4Gs were phosphorylated by SnRK1 under submergence. Interestingly, the eIFiso4G1 knockout mutant, but not the eIFiso4G2 mutant, became more sensitive to submergence, implying that eIFiso4G1 is involved in regulating submergence tolerance in Arabidopsis. Comparison of RNA sequences in the polysome fraction and the RNAs immunoprecipitated by eIFiso4G1 from Col-0 and the SnRK1 and eIFiso4G1 mutants revealed that lack of eIFiso4G1 phosphorylation disrupts the translation of specific mRNAs under submergence. Taken together, our findings suggest that the SnRK1-eIFiso4G1 relay controls the translation of an array of genes under hypoxia, including core hypoxia response genes and genes related to stress response and biosynthetic process.