Isolation of Plant Genes by T-DNA and Transposon Mutagenesis — Gene Tagging

Abstract Members of the Tourist family of miniature inverted-repeat transposable elements (MITEs) are very abundant among a wide variety of plants, are frequently found associated with normal plant genes, and thus are thought to be important players in the organization and evolution of plant genomes. In Arabidopsis, the recent discovery of a Tourist member harboring a putative transposase has shed new light on the mobility and evolution of MITEs. Here, we analyze a family of Tourist transposons endogenous to the genome of the nematode Caenorhabditis elegans (Bristol N2). One member of this large family is 7568 bp in length, harbors an ORF similar to the putative Tourist transposase from Arabidopsis, and is related to the IS5 family of bacterial insertion sequences (IS). Using database searches, we found expressed sequence tags (ESTs) similar to the putative Tourist transposases in plants, insects, and vertebrates. Taken together, our data suggest that Tourist-like and IS5-like transposons form a superfamily of potentially active elements ubiquitous to prokaryotic and eukaryotic genomes.

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Simultaneous silencing of two different Arabidopsis genes with a novel virus-induced gene silencing vector

Plant Methods ◽

10.1186/s13007-020-00701-6 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Kunxin Wu ◽

Yadan Wu ◽

Chunwei Zhang ◽

Yan Fu ◽

Zhixin Liu ◽

...

Keyword(s):

Gene Silencing ◽

Double Mutant ◽

Target Genes ◽

Rna Binding ◽

Protein Product ◽

Antiviral Defense ◽

Virus Induced Gene Silencing ◽

Argonaute 2 ◽

Plant Genes ◽

Silencing Pathways

Abstract Background Virus-induced gene silencing (VIGS) is a useful tool for functional characterizations of plant genes. However, the penetrance of VIGS varies depending on the genes to be silenced, and has to be evaluated by examining the transcript levels of target genes. Results In this report, we report the development of a novel VIGS vector that permits a preliminary assessment of the silencing penetrance. This new vector is based on an attenuated variant of Turnip crinkle virus (TCV) known as CPB that can be readily used in Arabidopsis thaliana to interrogate genes of this model plant. A CPB derivative, designated CPB1B, was produced by inserting a 46 nucleotide section of the Arabidopsis PHYTOENE DESATURASE (PDS) gene into CPB, in antisense orientation. CPB1B induced robust PDS silencing, causing easily visible photobleaching in systemically infected Arabidopsis leaves. More importantly, CPB1B can accommodate additional inserts, derived from other Arabidopsis genes, causing the silencing of two or more genes simultaneously. With photobleaching as a visual marker, we adopted the CPB1B vector to validate the involvement of DICER-LIKE 4 (DCL4) in antiviral defense against TCV. We further revealed the involvement of ARGONAUTE 2 (AGO2) in PDS silencing and antiviral defense against TCV in dcl2drb4 double mutant plants. These results demonstrated that DOUBLE-STRANDED RNA-BINDING PROTEIN 4 (DRB4), whose protein product (DRB4) commonly partners with DCL4 in the antiviral silencing pathway, was dispensable for PDS silencing induced by CPB1B derivative in dcl2drb4 double mutant plants. Conclusions The CPB1B-based vector developed in this work is a valuable tool with visualizable indicator of the silencing penetrance for interrogating Arabidopsis genes, especially those involved in the RNA silencing pathways.

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Progress in sequencing plant genes

Plant Molecular Biology Reporter ◽

10.1007/bf02670900 ◽

1995 ◽

Vol 13 (3) ◽

pp. 219-226

Author(s):

Ellen M. Reardon ◽

Carl A. Price

Keyword(s):

Plant Genes

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Tn5 transposon mutagenesis in Acidovorax citrulli for identification of genes required for pathogenicity on cucumber

Plant Pathology ◽

10.1111/j.1365-3059.2011.02519.x ◽

2011 ◽

Vol 61 (2) ◽

pp. 364-374 ◽

Cited By ~ 2

Author(s):

J. Liu ◽

S. Z. Luo ◽

Q. Zhang ◽

Q. H. Wang ◽

J. F. Chen ◽

...

Keyword(s):

Transposon Mutagenesis ◽

Tn5 Transposon ◽

Acidovorax Citrulli

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Transposition of Tn5367 in Mycobacterium marinum, Using a Conditionally Recombinant Mycobacteriophage

Journal of Bacteriology ◽

10.1128/jb.185.5.1745-1748.2003 ◽

2003 ◽

Vol 185 (5) ◽

pp. 1745-1748 ◽

Cited By ~ 9

Author(s):

Jan Rybniker ◽

Martina Wolke ◽

Christiane Haefs ◽

Georg Plum

Keyword(s):

Mycobacterium Tuberculosis ◽

Skin Infection ◽

Model Organism ◽

Transposon Mutagenesis ◽

Mycobacterium Marinum ◽

Close Relative ◽

Mutant Library ◽

Human Pathogen ◽

Valid Model ◽

Highly Efficient

ABSTRACT Mycobacterium marinum is a close relative of the obligate human pathogen Mycobacterium tuberculosis. As with M. tuberculosis, M. marinum causes intracellular infection of poikilothermic vertebrates and skin infection in humans. It is considered a valid model organism for the study of intracellular pathogenesis of mycobacteria. Low transformation efficiencies for this species have precluded approaches using mutant libraries in pathogenesis studies. We have adapted the conditionally replicating mycobacteriophage phAE94, originally developed as a transposon mutagenesis tool for M. tuberculosis, to meet the specific requirements of M. marinum. Conditions permissive for phage replication in M. tuberculosis facilitated highly efficient transposon delivery in M. marinum. Using this technique we succeeded in generating a representative mutant library of this species, and we conclude that TM4-derived mycobacteriophages are temperature-independent suicide vectors for M. marinum.

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