Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in eukaryotic cells. ZFNs have been used for targeted mutagenesis in model and crop species. In animal and human cells, transient ZFN expression is often achieved by direct gene transfer into the target cells. Stable transformation, however, is the preferred method for gene expression in plant species, and ZFN-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. Here we present an alternative, nontransgenic approach for ZFN delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of intact plants. TRV systemically infected its hosts and virus ZFN-mediated targeted mutagenesis could be clearly observed in newly developed infected tissues as measured by activation of a mutated reporter transgene in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to move to developing buds and regenerating tissues enabled recovery of mutated tobacco and petunia plants. Sequence analysis and transmission of the mutations to the next generation confirmed the stability of the ZFN-induced genetic changes. Because TRV is an RNA virus that can infect a wide range of plant species, it provides a viable alternative to the production of ZFN-mediated mutants while avoiding the use of direct plant-transformation methods.
SummaryIn nature, carotenoid function and mode of action are highly determined by the neighboring protein and lipid molecules. Therefore an understanding of the proteins' involvement in carotenoid sequestration would be of great help in elucidating carotenoid function in vivo. Based on a study of the expression of chromoplastspeci®c carotenoid-associated genes from cucumber corolla (CHRC and CHRD), a working model is presented wherein two major regulatory factors control carotenoid sequestration within the chromoplasts: (i)¯oral tissuespeci®c transcriptional regulators of chromoplastogenesis; and (ii) post-transcriptional regulators related to the amount/type of sequestered carotenoids. This model is supported by the major role transcriptional regulation was found to play in the temporal and spatial expression of the CHRC gene, and by the fact that phytohormones such as gibberellic acid (GA 3 ), abscisic acid and ethylene also acted as transcriptional regulators of CHRC expression. The primary response to GA 3 was localized within the CHRC promoter to a 290 bp fragment. Furthermore, we demonstrated strong down-regulation of CHRC expression at post-transcriptional and translational/post-translational levels resulting from inhibition of carotenoid biosynthesis, thus revealing a close link between carotenoid biosynthetic and sequestration machineries.
SummaryGenome editing, i.e. the ability to mutagenize, insert, delete and replace sequences, in living cells is a powerful and highly desirable method that could potentially revolutionize plant basic research and applied biotechnology. Indeed, various research groups from academia and industry are in a race to devise methods and develop tools that will enable not only site-specific mutagenesis but also controlled foreign DNA integration and replacement of native and transgene sequences by foreign DNA, in living plant cells. In recent years, much of the progress seen in gene targeting in plant cells has been attributed to the development of zinc finger nucleases and other novel restriction enzymes for use as molecular DNA scissors. The induction of double-strand breaks at specific genomic locations by zinc finger nucleases and other novel restriction enzymes results in a wide variety of genetic changes, which range from gene addition to the replacement, deletion and site-specific mutagenesis of endogenous and heterologous genes in living plant cells. In this review, we discuss the principles and tools for restriction enzyme-mediated gene targeting in plant cells, as well as their current and prospective use for gene targeting in model and crop plants.
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