Genome-Wide Insertional Mutagenesis of Arabidopsis thaliana

Science ◽  
2003 ◽  
Vol 301 (5633) ◽  
pp. 653-657 ◽  
Author(s):  
J. M. Alonso
Keyword(s):  
Arabidopsis Thaliana ◽  
Insertional Mutagenesis ◽  
Genome Wide

scholarly journals Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
Nicholas S. Eyre ◽  
Stephen M. Johnson ◽  
Auda A. Eltahla ◽  
Maria Aloi ◽  
Amanda L. Aloia ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Insertional Mutagenesis ◽  
High Resolution Imaging ◽  
Ns1 Protein ◽  
Reporter Virus ◽  
Genome Wide ◽  
Infected Cells ◽  
Resolution Imaging ◽  
Virus Replication Cycle

ABSTRACT Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3′ untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and β-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies. IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as “vesicle packets” (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools.

scholarly journals Genome-Wide Characterization and Expression Analysis of KH Family Genes Response to ABA and SA in Arabidopsis thaliana

2022 ◽  
Vol 23 (1) ◽  
pp. 511
Author(s):  
Yanjie Zhang ◽  
Yu Ma ◽  
Ruiqi Liu ◽  
Guanglin Li
Keyword(s):  
Arabidopsis Thaliana ◽  
Seed Germination ◽  
Nucleic Acid Binding ◽  
Phylogenetic Tree Analysis ◽  
Genome Wide ◽  
Binding Factor ◽  
Pathogenesis Related ◽  
Kh Domain ◽  
Family Based ◽  
Related Proteins

K-homologous (KH) family is a type of nucleic acid-binding protein containing the KH domain and has been found to affect splicing and transcriptional regulation. However, KH family genes haven’t been investigated in plant species systematically. In this study, we identified 30 genes that belonged to the KH family based on HMM of the KH domain in Arabidopsis thaliana. Phylogenetic tree analysis showed that the KH family is grouped into three subgroups. Synteny analysis showed that AtKH9 and AtKH29 have the conserved synteny relationship between A. thaliana and the other five species. The AtKH9 and AtKH29 were located in the cytoplasm and nucleus. The seed germination rates of the mutants atkh9 and atkh29 were higher than wild-type after abscisic acid (ABA) and salicylic acid (SA) treatments. In addition, the expression of ABA-related genes, such as ABRE-binding factor 2 (ABF2), ABRE-binding factor 4 (ABF4), and delta 1-pyrroline-5-carboxylate synthase (P5CS), and an SA-related gene pathogenesis-related proteins b (PR1b) were downregulated after ABA and SA treatments, respectively. These results suggested that atkh9 and atkh29 mutants inhibit the effect of ABA and SA on seed germination. In conclusion, our results provide valuable information for further exploration of the function of KH family genes and propose directions and ideas for the identification and characterization of KH family genes in other plants.

scholarly journals Genome wide analysis of stress responsive WRKY transcription factors in Arabidopsis thaliana

2016 ◽  
Vol 4 (4) ◽  
pp. 279 ◽  
Author(s):  
Shaiq Sultan ◽  
Muhammad Amjid Ali ◽  
Rana Muhammad Atif ◽  
Farrukh Azeem ◽  
Habibullah Nadeem ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Transcription Factors ◽  
Chromosomal Localization ◽  
Evolutionary Relationship ◽  
Wrky Transcription Factors ◽  
Specific Sequence ◽  
Multiple Sequence ◽  
Genome Wide Analysis ◽  
Functional Homology ◽  
Genome Wide

WRKY transcription factors are a class of DNA-binding proteins that bind with a specific sequence C/TTGACT/C known as W-Box found in promoters of genes which are regulated by these WRKYs. From previous studies, 43 different stress responsive WRKY transcription factors in Arabidopsis thaliana, identified and then categorized in three groups viz., abiotic, biotic and both of these stresses. A comprehensive genome wide analysis including chromosomal localization, gene structure analysis, multiple sequence alignment, phylogenetic analysis and promoter analysis of these WRKY genes was carried out in this study to determine the functional homology in Arabidopsis. This analysis led to the classification of these WRKY family members into 3 major groups and subgroups and showed evolutionary relationship among these groups on the base of their functional WRKY domain, chromosomal localization and intron/exon structure. The proposed groups of these stress responsive WRKY genes and annotation based on their position on chromosomes can also be explored to determine their functional homology in other plant species in relation to different stresses. The result of the present study provides indispensable genomic information for the stress responsive WRKY transcription factors in Arabidopsis and will pave the way to explain the precise role of various AtWRKYs in plant growth and development under stressed conditions.

scholarly journals QTL and candidate genes associated with leaf anion concentrations in response to phosphate supply in Arabidopsis thaliana

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Mohamed El-Soda ◽  
Charles Neris Moreira ◽  
Nakai Goredema-Matongera ◽  
Diaan Jamar ◽  
Maarten Koornneef ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Candidate Genes ◽  
Long Distance ◽  
Gene Encoding ◽  
Significant Treatment ◽  
P Deficiency ◽  
Sulphate Concentration ◽  
Genome Wide ◽  
Pi Starvation ◽  
Phosphatase 2A

Abstract Background Phosphorus is often present naturally in the soil as inorganic phosphate, Pi, which bio-availability is limited in many ecosystems due to low soil solubility and mobility. Plants respond to low Pi with a Pi Starvation Response, involving Pi sensing and long-distance signalling. There is extensive cross-talk between Pi homeostasis mechanisms and the homeostasis mechanism for other anions in response to Pi availability. Results Recombinant Inbred Line (RIL) and Genome Wide Association (GWA) mapping populations, derived from or composed of natural accessions of Arabidopsis thaliana, were grown under sufficient and deficient Pi supply. Significant treatment effects were found for all traits and significant genotype x treatment interactions for the leaf Pi and sulphate concentrations. Using the RIL/QTL population, we identified 24 QTLs for leaf concentrations of Pi and other anions, including a major QTL for leaf sulphate concentration (SUL2) mapped to the bottom of chromosome (Chr) 1. GWA mapping found 188 SNPs to be associated with the measured traits, corresponding to 152 genes. One of these SNPs, associated with leaf Pi concentration, mapped to PP2A-1, a gene encoding an isoform of the catalytic subunit of a protein phosphatase 2A. Of two additional SNPs, associated with phosphate use efficiency (PUE), one mapped to AT5G49780, encoding a leucine-rich repeat protein kinase involved in signal transduction, and the other to SIZ1, a gene encoding a SUMO E3 ligase, and a known regulator of P starvation-dependent responses. One SNP associated with leaf sulphate concentration was found in SULTR2;1, encoding a sulphate transporter, known to enhance sulphate translocation from root to shoot under P deficiency. Finally, one SNP was mapped to FMO GS-OX4, a gene encoding glucosinolate S-oxygenase involved in glucosinolate biosynthesis, which located within the confidence interval of the SUL2 locus. Conclusion We identified several candidate genes with known functions related to anion homeostasis in response to Pi availability. Further molecular studies are needed to confirm and validate these candidate genes and understand their roles in examined traits. Such knowledge will contribute to future breeding for improved crop PUE .

The COP9 signalosome influences the epigenetic landscape of Arabidopsis thaliana

2018 ◽  
Vol 35 (16) ◽  
pp. 2718-2723 ◽  
Author(s):  
Tamir Tuller ◽  
Alon Diament ◽  
Avital Yahalom ◽  
Assaf Zemach ◽  
Shimshi Atar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Arabidopsis Thaliana ◽  
Gene Expression Regulation ◽  
Developmental Pathways ◽  
Cop9 Signalosome ◽  
Supplementary Information ◽  
Loss Of Function ◽  
Genome Wide ◽  
High Level

Abstract Motivation The COP9 signalosome is a highly conserved multi-protein complex consisting of eight subunits, which influences key developmental pathways through its regulation of protein stability and transcription. In Arabidopsis thaliana, mutations in the COP9 signalosome exhibit a number of diverse pleiotropic phenotypes. Total or partial loss of COP9 signalosome function in Arabidopsis leads to misregulation of a number of genes involved in DNA methylation, suggesting that part of the pleiotropic phenotype is due to global effects on DNA methylation. Results We determined and analyzed the methylomes and transcriptomes of both partial- and total-loss-of-function Arabidopsis mutants of the COP9 signalosome. Our results support the hypothesis that the COP9 signalosome has a global genome-wide effect on methylation and that this effect is at least partially encoded in the DNA. Our analyses suggest that COP9 signalosome-dependent methylation is related to gene expression regulation in various ways. Differentially methylated regions tend to be closer in the 3D conformation of the genome to differentially expressed genes. These results suggest that the COP9 signalosome has a more comprehensive effect on gene expression than thought before, and this is partially related to regulation of methylation. The high level of COP9 signalosome conservation among eukaryotes may also suggest that COP9 signalosome regulates methylation not only in plants but also in other eukaryotes, including humans. Supplementary information Supplementary data are available at Bioinformatics online.

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