scholarly journals The Zymoseptoria tritici ORFeome: A Functional Genomics Community Resource

2019 ◽  
Vol 32 (12) ◽  
pp. 1564-1570
Author(s):  
Yogesh Chaudhari ◽  
Timothy C. Cairns ◽  
Yaadwinder Sidhu ◽  
Victoria Attah ◽  
Graham Thomas ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Open Reading Frames ◽  
Zymoseptoria Tritici ◽  
Entry Vector ◽  
Plant Infection ◽  
Plasmid Library ◽  
A Genome ◽  
Genomic Characterization ◽  
Serious Disease ◽  
Gateway Entry Vector

Libraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for Zymoseptoria tritici, which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4,075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, were cloned into the Gateway entry vector pDONR207, and were sequenced, providing a total of 3,022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors (n = 410) and secondary metabolite biosynthetic proteins (n = 171) in addition to genes residing at dispensable chromosomes (n = 122) or those that are preferentially expressed during plant infection (n = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The Z. tritici ORFeome constitutes a powerful resource for functional genomics and offers unparalleled opportunities to understand the biology of Z. tritici. [Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

scholarly journals The Zymoseptoria tritici ORFeome: a functional genomics community resource

2019 ◽  
Author(s):  
Yogesh Chaudhari ◽  
Timothy C. Cairns ◽  
Yaadwinder Sidhu ◽  
Victoria Attah ◽  
Graham Thomas ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Open Reading Frames ◽  
Zymoseptoria Tritici ◽  
Entry Vector ◽  
Plant Infection ◽  
Plasmid Library ◽  
A Genome ◽  
Protein Encoding ◽  
Genomic Characterization ◽  
Serious Disease

AbstractLibraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for Zymoseptoria tritici, which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, cloned into the Gateway® Entry Vector pDONR207, and sequenced, providing a total of 3022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors (n = 410) and secondary metabolite biosynthetic proteins (n = 171), in addition to genes residing at dispensable chromosomes (n= 122), or those that are preferentially expressed during plant infection (n = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway® Destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The Z. tritici ORFeome constitutes a powerful resource for functional genomics, and offers unparalleled opportunities to understand the biology of Z. tritici.

scholarly journals Construction and Expression of Sugar Kinase Transcriptional Gene Fusions by Using the Sinorhizobium meliloti ORFeome

2008 ◽  
Vol 74 (21) ◽  
pp. 6756-6765 ◽  
Author(s):  
Jodi L. Humann ◽  
Brenda K. Schroeder ◽  
Michael W. Mortimer ◽  
Brent L. House ◽  
Svetlana N. Yurgel ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Fluorescent Protein ◽  
Carbon Sources ◽  
Open Reading Frames ◽  
Vector System ◽  
Destination Vector ◽  
Entry Vector ◽  
Gateway Entry Vector ◽  
Sugar Kinase

ABSTRACT The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to β-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput β-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed.

scholarly journals Genomic Characterization Provides an Insight into the Pathogenicity of the Poplar Canker Bacterium Lonsdalea populi

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 246
Author(s):  
Xiaomeng Chen ◽  
Rui Li ◽  
Yonglin Wang ◽  
Aining Li
Keyword(s):  
Genome Sequence ◽  
Extracellular Enzymes ◽  
De Novo ◽  
Whole Genome Sequence ◽  
Hybrid Poplars ◽  
A Genome ◽  
Conserved Genes ◽  
Genomic Characterization ◽  
Molecular Bases ◽  
Insight Into

An emerging poplar canker caused by the gram-negative bacterium, Lonsdalea populi, has led to high mortality of hybrid poplars Populus × euramericana in China and Europe. The molecular bases of pathogenicity and bark adaptation of L. populi have become a focus of recent research. This study revealed the whole genome sequence and identified putative virulence factors of L. populi. A high-quality L. populi genome sequence was assembled de novo, with a genome size of 3,859,707 bp, containing approximately 3434 genes and 107 RNAs (75 tRNA, 22 rRNA, and 10 ncRNA). The L. populi genome contained 380 virulence-associated genes, mainly encoding for adhesion, extracellular enzymes, secretory systems, and two-component transduction systems. The genome had 110 carbohydrate-active enzyme (CAZy)-coding genes and putative secreted proteins. The antibiotic-resistance database annotation listed that L. populi was resistant to penicillin, fluoroquinolone, and kasugamycin. Analysis of comparative genomics found that L. populi exhibited the highest homology with the L. britannica genome and L. populi encompassed 1905 specific genes, 1769 dispensable genes, and 1381 conserved genes, suggesting high evolutionary diversity and genomic plasticity. Moreover, the pan genome analysis revealed that the N-5-1 genome is an open genome. These findings provide important resources for understanding the molecular basis of the pathogenicity and biology of L. populi and the poplar-bacterium interaction.

scholarly journals Learning a genome-wide score of human–mouse conservation at the functional genomics level

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Soo Bin Kwon ◽  
Jason Ernst
Keyword(s):  
Mouse Model ◽  
Functional Genomics ◽  
Functional Genomic ◽  
Transcriptomic Data ◽  
Model Studies ◽  
Genome Wide ◽  
A Genome ◽  
Important Challenge ◽  
Genomic Regions ◽  
Human And Mouse

AbstractIdentifying genomic regions with functional genomic properties that are conserved between human and mouse is an important challenge in the context of mouse model studies. To address this, we develop a method to learn a score of evidence of conservation at the functional genomics level by integrating information from a compendium of epigenomic, transcription factor binding, and transcriptomic data from human and mouse. The method, Learning Evidence of Conservation from Integrated Functional genomic annotations (LECIF), trains neural networks to generate this score for the human and mouse genomes. The resulting LECIF score highlights human and mouse regions with shared functional genomic properties and captures correspondence of biologically similar human and mouse annotations. Analysis with independent datasets shows the score also highlights loci associated with similar phenotypes in both species. LECIF will be a resource for mouse model studies by identifying loci whose functional genomic properties are likely conserved.

scholarly journals Identification of Bacterial Blight Resistance Loci in Rice (Oryza sativa L.) against Diverse Xoo Thai Strains by Genome-Wide Association Study

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 518
Author(s):  
Siriporn Korinsak ◽  
Clive T. Darwell ◽  
Samart Wanchana ◽  
Lawan Praphaisal ◽  
Siripar Korinsak ◽  
...  
Keyword(s):  
Association Study ◽  
Genome Wide Association Study ◽  
Oryza Sativa L ◽  
Genome Wide Association ◽  
Rice Agriculture ◽  
Large Numbers ◽  
Genome Wide ◽  
Resistant Varieties ◽  
A Genome ◽  
Serious Disease

Bacterial leaf blight (BLB) is a serious disease affecting global rice agriculture caused by Xanthomonas oryzae pv. oryzae (Xoo). Most resistant rice lines are dependent on single genes that are vulnerable to resistance breakdown caused by pathogen mutation. Here we describe a genome-wide association study of 222 predominantly Thai rice accessions assayed by phenotypic screening against 20 Xoo isolates. Loci corresponding to BLB resistance were detected using >142,000 SNPs. We identified 147 genes according to employed significance thresholds across chromosomes 1–6, 8, 9 and 11. Moreover, 127 of identified genes are located on chromosomal regions outside estimated Linkage Disequilibrium influences of known resistance genes, potentially indicating novel BLB resistance markers. However, significantly associated SNPs only occurred across a maximum of six Xoo isolates indicating that the development of broad-spectrum Xoo strain varieties may prove challenging. Analyses indicated a range of gene functions likely underpinning BLB resistance. In accordance with previous studies of accession panels focusing on indica varieties, our germplasm displays large numbers of SNPs associated with resistance. Despite encouraging data suggesting that many loci contribute to resistance, our findings corroborate previous inferences that multi-strain resistant varieties may not be easily realised in breeding programs without resorting to multi-locus strategies.

scholarly journals A genome-wide screen in the mouse liver reveals sex-specific and cell non-autonomous regulation of cell fitness

2021 ◽  
Author(s):  
Heather R. Keys ◽  
Kristin A. Knouse
Keyword(s):  
Functional Genomics ◽  
High Throughput ◽  
Mouse Liver ◽  
Ex Vivo ◽  
Screening Method ◽  
Living Organism ◽  
Cellular Processes ◽  
Autonomous Regulation ◽  
Genome Wide ◽  
A Genome

ABSTRACTOur ability to understand and modulate mammalian physiology and disease requires knowing how all genes contribute to any given phenotype in the organism. Genome-wide screening using CRISPR-Cas9 has emerged as a powerful method for the genetic dissection of cellular processes1,2, but the need to stably deliver single guide RNAs to millions of cells has restricted its implementation to ex vivo systems. These ex vivo systems cannot reproduce all of the cellular phenotypes observed in vivo nor can they recapitulate all of the factors that influence these phenotypes. There thus remains a pressing need for high-throughput functional genomics in a living organism. Here, we establish accessible genome-wide screening in the mouse liver and use this approach to uncover the complete regulation of cellular fitness in a living organism. We discover novel sex-specific and cell non-autonomous regulation of cell growth and viability. In particular, we find that the class I major histocompatibility complex is essential for preventing immune-mediated clearance of hepatocytes. Our approach provides the first comprehensive picture of cell fitness in a living organism and highlights the importance of investigating cellular phenomena in their native context. Our screening method is robust, scalable, and easily adapted to examine diverse cellular processes using any CRISPR application. We have hereby established a foundation for high-throughput functional genomics in a living mammal, enabling unprecedented insight into mammalian physiology and disease.

scholarly journals The Novel Agrotis ipsilon Nora Virus Confers Deleterious Effects to the Fitness of Spodoptera frugiperda (Lepidoptera: Noctuidae)

2021 ◽  
Vol 12 ◽  
Author(s):  
Tong Li ◽  
Ruobing Guan ◽  
Yuqing Wu ◽  
Su Chen ◽  
Guohui Yuan ◽  
...  
Keyword(s):  
Spodoptera Frugiperda ◽  
Rna Virus ◽  
Open Reading Frames ◽  
Agrotis Ipsilon ◽  
The Novel ◽  
Black Cutworm ◽  
A Genome ◽  
Nora Virus ◽  
Novel Host ◽  
Lepidoptera Noctuidae

In the present study, we identified a novel, positive-sense single-stranded RNA virus in the Chinese black cutworm, Agrotis ipsilon. It has a genome length of 11,312 nucleotides, excluding the poly(A) tails, and contains five open reading frames. The ORF2 encodes the conserved domains of RNA helicase and RNA-dependent RNA polymerase, while ORF4 and 5 encode three viral proteins. Herein, the A. ipsilon virus was clustered with a Helicoverpa armigera Nora virus and was thus provisionally named “Agrotis ipsilon Nora virus” (AINV). AINV was successfully transmitted into a novel host, Spodoptera frugiperda, through injection, causing a stable infection. This found the possibility of horizontal AINV transmission among moths belonging to the same taxonomic family. Nonetheless, AINV infection was deleterious to S. frugiperda and mainly mediated by antiviral and amino acid metabolism-related pathways. Furthermore, the infection significantly increased the S. frugiperda larval period but significantly reduced its moth eclosion rate. It suggests that AINV is probably to be a parasitic virus of S. frugiperda.

scholarly journals Episomal tools for RNAi in the diatom Phaeodactylum tricornutum

2017 ◽  
Author(s):  
Vincent A Bielinski ◽  
Tayah M Bolt ◽  
Christopher L Dupont ◽  
Philip D Weyman
Keyword(s):  
Phaeodactylum Tricornutum ◽  
Expression Vectors ◽  
Dna Assembly ◽  
Destination Vector ◽  
Genetic Tools ◽  
Entry Vector ◽  
Polymerase Iii ◽  
Recombination System ◽  
Low Efficiency ◽  
Gateway Entry Vector

Background. The diatom Phaeodactylum tricornutum is a model photosynthetic organism. Functional genomic work in this organism has established a variety of genetic tools including RNA interference (RNAi). RNAi is a post-transcriptional regulatory process that can be utilized to knockdown expression of genes of interest in eukaryotes. RNAi has been previously demonstrated in P. tricornutum, but in practice the efficiency of inducing RNAi is low. Methods. We developed an efficient method for construction of inverted repeat hairpins based on Golden Gate DNA assembly into a Gateway entry vector. The hairpin constructs were then transferred to a variety of destination vectors through the Gateway recombination system. After recombining the hairpin into the destination vector, the resulting expression vector was mobilized into P. tricornutum using direct conjugation from E. coli. Because the hairpin expression vectors had sequences allowing for episomal maintenance in P. tricornutum, we tested whether a consistent, episomal location for hairpin expression improved RNAi induction efficiency. Results. We successfully demonstrated that RNAi could be induced using hairpin constructs expressed from an episome. After testing two different reporter targets and a variety of hairpin sequences with 3 polymerase II and 2 polymerase III promoters, we achieved a maximal RNAi induction efficiency of 25% of lines displaying knockdown of reporter activity by 50% or more. We created many useful genetic tools through this work including Gateway destination vectors for P. tricornutum expression from a variety of polymerase II and III promoters including the P. tricornutum FCPB, H4, and 49202 polymerase II promoters as well as the U6 and snRNA polymerase III promoters. We also created Gateway destination vectors that allow a cassette cloned in an entry vector to be easily recombined into a transcriptional fusion with either ShBle or, for polymerase III promoters, the green fluorescent Spinach aptamer. Such transcriptional fusions allow for linkage of expression with a marker such as bleomycin resistance or fluorescence from the Spinach aptamer to easily select or screen for lines that maintain transgene expression. Discussion. While RNAi can be used as an effective tool for P. tricornutum genetics, especially where targeted knockouts may be lethal to the cell, induction of this process remains low efficiency. Techniques resulting in higher efficiency establishment of RNAi would be of great use to the diatom genetics community and would enable this technique to be used as a forward genetic tool for discovery of novel gene function.

Phenotypic Screen and Transcriptomics Approach Complement Each Other in Functional Genomics of Defensive Stink Gland Physiology

2021 ◽  
Author(s):  
Sabrina Lehmann ◽  
Bibi Atika ◽  
Daniela Grossmann ◽  
Christian Schmitt-Engel ◽  
Nadi Strohlein ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Large Scale ◽  
Reverse Genetics ◽  
Expression Profiles ◽  
Forward Genetics ◽  
Large Set ◽  
Knock Down ◽  
Genome Wide ◽  
Phenotypic Screen ◽  
A Genome

Abstract Background Functional genomics uses unbiased systematic genome-wide gene disruption or analyzes natural variations such as gene expression profiles of different tissues from multicellular organisms to link gene functions to particular phenotypes. Functional genomics approaches are of particular importance to identify large sets of genes that are specifically important for a particular biological process beyond known candidate genes, or when the process has not been studied with genetic methods before. Results Here, we present a large set of genes whose disruption interferes with the function of the odoriferous defensive stink glands of the red flour beetle Tribolium castaneum. This gene set is the result of a large-scale systematic phenotypic screen using a reverse genetics strategy based on RNA interference applied in a genome-wide forward genetics manner. In this first-pass screen, 130 genes were identified, of which 69 genes could be confirmed to cause knock-down gland phenotypes, which vary from necrotic tissue and irregular reservoir size to irregular color or separation of the secreted gland compounds. The knock-down of 13 genes caused specifically a strong reduction of para-benzoquinones, suggesting a specific function in the synthesis of these toxic compounds. Only 14 of the 69 confirmed gland genes are differentially overexpressed in stink gland tissue and thus could have been detected in a transcriptome-based analysis. Moreover, of the 29 previously transcriptomics-identified genes causing a gland phenotype, only one gene was recognized by this phenotypic screen despite the fact that 13 of them were covered by the screen. Conclusion Our results indicate the importance of combining diverse and independent methodologies to identify genes necessary for the function of a certain biological tissue, as the different approaches do not deliver redundant results but rather complement each other. The presented phenotypic screen together with a transcriptomics approach are now providing a set of close to hundred genes important for odoriferous defensive stink gland physiology in beetles.

scholarly journals Genome Sequence of a T4-like Phage, Escherichia Phage vB_EcoM-Sa45lw, Infecting Shiga Toxin-Producing Escherichia coli Strains

2019 ◽  
Vol 8 (32) ◽  
Author(s):  
Yen-Te Liao ◽  
Yujie Zhang ◽  
Alexandra Salvador ◽  
Vivian C. H. Wu
Keyword(s):  
Escherichia Coli ◽  
Surface Water ◽  
Genome Size ◽  
Shiga Toxin ◽  
Open Reading Frames ◽  
Content Type ◽  
E Coli ◽  
Double Stranded Dna ◽  
A Genome ◽  
Reading Frames

Escherichia phage vB_EcoM-Sa45lw, a new member of the T4-like phages, was isolated from surface water in a produce-growing area. The phage, containing double-stranded DNA with a genome size of 167,353 bp and 282 predicted open reading frames (ORFs), is able to infect generic Escherichia coli and Shiga toxin-producing E. coli O45 and O157 strains.

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