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      A virus‐induced gene‐silencing system for functional genetics in a betalainic species, Amaranthus tricolor (Amaranthaceae)

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          Abstract

          Premise of the Study

          Research in Amaranthaceae could be accelerated by developing methods for targeted gene silencing. Most amaranths, including Amaranthus tricolor, produce betalains. However, the physiological and ecological roles of these pigments are uncertain. We sought to establish a virus‐induced gene‐silencing ( VIGS) method for amaranths, using silencing of betalain pigments as a proof‐of‐principle.

          Methods

          We targeted Atri CYP76 AD1 , a putative cytochrome P450 component of the betalain biosynthetic pathway, using VIGS, and compared two different methods of introducing the VIGS construct into plants. We measured transcript abundance and concentrations of betalains and their lDOPA precursor in VIGS‐treated plants, and compared these to controls.

          Results

          We observed that when Atri CYP76 AD1 was targeted by VIGS in normally red plants, Atri CYP76 AD1 and the related genes Atri CYP76 AD6 and Atri CYP76 AD5 had diminished transcript abundance. Furthermore, newly emergent petioles and leaves of VIGS‐treated plants appeared green, betacyanin accumulation was strongly reduced, and lDOPA accumulation was increased. No betaxanthin could be detected in this variety of A. tricolor, either before or after VIGS treatment.

          Discussion

          These results help to establish the genetic basis of betalain synthesis in amaranths. Furthermore, this is the first report of VIGS in amaranths and demonstrates the potential of this technique for basic and applied research in these species.

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          Most cited references38

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          Virus-induced gene silencing in tomato.

          We have previously demonstrated that a tobacco rattle virus (TRV)-based vector can be used in virus-induced gene silencing (VIGS) to study gene function in Nicotiana benthamiana. Here we show that recombinant TRV infects tomato plants and induces efficient gene silencing. Using this system, we suppressed the PDS, CTR1 and CTR2 genes in tomato. Suppression of CTR1 led to a constitutive ethylene response phenotype and up-regulation of an ethylene response gene, CHITINASE B. This phenotype is similar to Arabidopsis ctr1 mutant plants. We have constructed a modified TRV vector based on the GATEWAY recombination system, allowing restriction- and ligation-free cloning. Our results show that tomato expressed sequence tags (ESTs) can easily be cloned into this modified vector using a single set of primers. Using this vector, we have silenced RbcS and an endogenous gene homologous to the tomato EST cLED3L14. In the future, this modified vector system will facilitate large-scale functional analysis of tomato ESTs.
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            Data access for the 1,000 Plants (1KP) project

            The 1,000 plants (1KP) project is an international multi-disciplinary consortium that has generated transcriptome data from over 1,000 plant species, with exemplars for all of the major lineages across the Viridiplantae (green plants) clade. Here, we describe how to access the data used in a phylogenomics analysis of the first 85 species, and how to visualize our gene and species trees. Users can develop computational pipelines to analyse these data, in conjunction with data of their own that they can upload. Computationally estimated protein-protein interactions and biochemical pathways can be visualized at another site. Finally, we comment on our future plans and how they fit within this scalable system for the dissemination, visualization, and analysis of large multi-species data sets.
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              Technical Advance. Tobacco rattle virus as a vector for analysis of gene function by silencing.

              Virus vectors carrying host-derived sequence inserts induce silencing of the corresponding genes in infected plants. This virus-induced gene silencing (VIGS) is a manifestation of an RNA-mediated defence mechanism that is related to post-transcriptional gene silencing (PTGS) in transgenic plants. Here we describe an infectious cDNA clone of tobacco rattle virus (TRV) that has been modified to facilitate insertion of non-viral sequence and subsequent infection to plants. We show that this vector mediates VIGS of endogenous genes in the absence of virus-induced symptoms. Unlike other RNA virus vectors that have been used previously for VIGS, the TRV construct is able to target host RNAs in the growing points of plants. These features indicate that the TRV vector will have wide application for gene discovery in plants.
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                Author and article information

                Contributors
                dinesh.adhikary@ubc.ca
                Journal
                Appl Plant Sci
                Appl Plant Sci
                10.1002/(ISSN)2168-0450
                APS3
                Applications in Plant Sciences
                John Wiley and Sons Inc. (Hoboken )
                2168-0450
                07 February 2019
                February 2019
                : 7
                : 2 ( doiID: 10.1002/aps3.2019.7.issue-2 )
                : e01221
                Affiliations
                [ 1 ] Department of Biology University of British Columbia Kelowna British Columbia Canada
                [ 2 ] Agricultural, Food, and Nutritional Science Department University of Alberta Edmonton Canada
                [ 3 ] Department of Chemistry University of British Columbia Kelowna British Columbia Canada
                Author notes
                [*] [* ]Author for correspondence: dinesh.adhikary@ 123456ubc.ca
                Author information
                https://orcid.org/0000-0001-6669-2562
                Article
                APS31221
                10.1002/aps3.1221
                6384298
                8848193f-f32e-4533-89be-0e69b9207482
                © 2019 Adhikary et al. Applications in Plant Sciences is published by Wiley Periodicals, Inc. on behalf of the Botanical Society of America

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 30 October 2018
                : 18 December 2018
                Page count
                Figures: 5, Tables: 0, Pages: 9, Words: 7444
                Funding
                Funded by: Natural Sciences and Engineering Research Council (NSERC) of Canada
                Categories
                Application Article
                Application Articles
                Custom metadata
                2.0
                aps31221
                February 2019
                Converter:WILEY_ML3GV2_TO_NLMPMC version:5.5.9 mode:remove_FC converted:21.02.2019

                amaranthaceae,amaranthus tricolor,atricyp76ad1,betalain,l‐dopa,virus‐induced gene‐silencing (vigs)

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