‘Real time’ genetic manipulation: a new tool for ecological field studies

Abstract

SUMMARYField experiments with transgenic plants often reveal the functional significance of genetic traits that are important for the performance of the plants in their natural environments. Until now, only constitutive overexpression, ectopic expression and gene silencing methods have been used to analyze gene-related phenotypes in natural habitats. These methods do not allow sufficient control over gene expression for the study of ecological interactions in real time, of genetic traits that play essential roles in development, or of dosedependent effects. We applied the sensitive dexamethasone (DEX)-inducible pOp6/LhGR expression system to the ecological model plant Nicotiana attenuata and established a lanolin-based DEX application method to facilitate ectopic gene expression and RNA interference-mediated gene silencing in the field and under challenging conditions (e.g. high temperature, wind and UV radiation). Fully established field-grown plants were used to silence phytoene desaturase and thereby cause photobleaching only in specific plant sectors, and to activate expression of the cytokinin (CK) biosynthesis gene isopentenyl transferase (ipt). We used ipt expression to analyze the role of CKs in both the glasshouse and the field to understand resistance to the native herbivore Tupiocoris notatus, which attacks plants at small spatial scales. By spatially restricting ipt expression and elevating CK levels in single leaves, damage by T. notatus increased, demonstrating the role of CKs in this plant-herbivore interaction at a small scale. As the arena of most ecological interactions is highly constrained in time and space, these tools will advance the genetic analysis of dynamic traits that matter for plant performance in nature.

Chemical induction of hairpin RNAi molecules to silence vital genes in plant roots

“…However, legumes do contain significant concentrations of phytoestrogens which may be responsible for the expression from the pER8 promoter in Medicago. As observed in the above ground parts of Arabidopsis and tobacco, neither dexamethasone nor estradiol appeared to have effect on the growth of transgenic Medicago roots31011.…”

“…Although the application of dexamethasone system has been reported in leaves3, flowers3 and seeds10, less has been described on application of the inducible silencing systems in roots. Our data provided original quantitative analyses on how this chemically inducible system can silence genes in a temporal and dose-dependent manner in roots and demonstrated how a lethal gene could be chemically silenced and the resultant phenotypes illustrated in roots.…”

“…Transformation vectors with conditionally inducible elements also allow investigators to control the expression of transgenes to a particular stage during development, such as the controlled expression of a cell cycle gene during leaf maturation2. Transgene expression can be similarly restricted to particular plant parts by localized application of chemical inducers, therefore accomplishing spatially controlled expression3.…”

Auxin Is Rapidly Induced by Herbivore Attack and Regulates a Subset of Systemic, Jasmonate-Dependent Defenses

“…Our initial attempts to create transgenic, dexamethasone-inducible plants (Schäfer et al, 2013) harboring a silencing construct for the IAA receptor TIR1 failed, either because of promoter methylation in the F2 crosses (Weinhold et al, 2013) or because the identified TIR1 homolog was inactive. We therefore took advantage of our knowledge on systemic IAA accumulation to devise a series of chemical manipulation experiments.…”

The raison d'être of chemical ecology

“…Once candidate genes have been identified by genetic mapping, they can be validated through knockdown by RNA interference, overexpression, or targeted modification of the genome sequences in the organisms of interest (Kang and Baldwin 2008). This approach has translated successfully to field experiments on chemically mediated plant defense and reproductive ecology in wild tobacco (Nicotiana attenuata) (Baldwin 2010, Scha¨fer et al 2013). An additional benefit of such studies is that selective trait-silencing may reveal unexpected ecological interactions that are preempted by wild-type trait expression.…”