7 February 2012
Transforming DNA into the Arabidopsis germ line presents no real difficulties, but integrating exogenous DNA into specific genome locations in this model organism is no easy feat. Typically, transformed DNA integrates randomly, making gene knockout or replacement a frustrating proposition.
In a technical advance published in The Plant Journal, Even-Faitelson et al. offer a simple strategy for targeted modification of the Arabidopsis genome. The procedure begins with the typical transformation method: exogenous DNA is engineered into Agrobacterium tumefaciens DNA, and delivered into the flower by dipping the budding plant into a solution containing the bacteria.
It is transformation of the egg cell that transmits the exogenous DNA to progeny, so Even-Faitelson et al. directed their attention to an egg cell–based strategy. They hypothesized that egg cell– specific expression of proteins involved in targeted gene mutagenesis or gene targeting would make these strategies easily implementable in Arabidopsis.
To this end, the authors prepared constructs containing a 77–base pair enhancer sequence called the egg apparatus–specific enhancer (EASE), which stimulates expression of adjoining genes in the egg cell or the very early zygote. For gene targeting, the effector protein is RAD54, a chromatin remodeling factor that promotes the homologous recombination of exogenous DNA with its genomic target. For targeted gene mutagenesis, EASE-mediated expression of a zinc finger nuclease is employed to trigger gene mutagenesis by the error-prone non-homologous joining of termini created by the engineered endonuclease.
In tests using reporter vectors to detect successful gene manipulation events, the EASE-regulated expression method yielded rates of gene targeting and targeted gene mutagenesis of 4 x 10-5 and 3 x 10-3, respectively. The same strategy should also be useful for appropriate spatiotemporal expression of other genome-modifying proteins such as transcription activator-like effector nucleases (TALENs), which offer particular flexibility in customizing target sequence recognition.
The advent of the EASE system should help make precise genome modification in Arabidopsis as routine as mutating specific loci in yeast or mice.
1. Even-Faitelson et al. 2011. Localized egg-cell expression of effector proteins for targeted modification of the Arabidopsis genome. Plant J. 68(5):929-37.