MultiSite Gateway-Compatible Cell Type-Specific Gene-Inducible System for Plants

Abstract

A powerful method to study gene function is expression or overexpression in an inducible, cell type-specific system followed by observation of consequent phenotypic changes and visualization of linked reporters in the target tissue. Multiple inducible gene overexpression systems have been developed for plants, but very few of these combine plant selection markers, control of expression domains, access to multiple promoters and protein fusion reporters, chemical induction, and high-throughput cloning capabilities. Here, we introduce a MultiSite Gateway-compatible inducible system for Arabidopsis (Arabidopsis thaliana) plants that provides the capability to generate such constructs in a single cloning step. The system is based on the tightly controlled, estrogen-inducible XVE system. We demonstrate that the transformants generated with this system exhibit the expected cell type-specific expression, similar to what is observed with constitutively expressed native promoters. With this new system, cloning of inducible constructs is no longer limited to a few special cases but can be used as a standard approach when gene function is studied. In addition, we present a set of entry clones consisting of histochemical and fluorescent reporter variants designed for gene and promoter expression studies.

Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI

“…The β‐estradiol inducible transgene WOLprom:XVE >> VIR RNAi and UBQ10prom:XVE >> MTB RNAi constructs were made as described previously (Mähönen et al ., 2014; Siligato et al ., 2016), inserting the regions detailed in Table S1 (Supporting Information) (table of primers used) in the sense and antisense orientation into entry clones with restriction enzyme‐mediated cloning. The promoter UBQ10 was chosen because it directs stable, widespread expression and is resistant to silencing (Geldner et al ., 2009).…”

De novo assembly of plant body plan: a step ahead of Deadpool

“…It is interesting to note that similar or the same set of factors in different contexts can regulate distinct modes of regeneration. Ectopic overexpression of WUS or PLT5 can induce de novo shoot regeneration (Gallois et al, 2004;Kareem et al, 2015) and somatic embryogenesis (Zuo, Niu, Frugis, & Chua, 2002;Tsuwamoto, Yokoi, & Takahata, 2010;Siligato et al, 2016). Similarly, ALF4 plays significant role in callus formation (Sugimoto et al, 2010) as well as wound healing during grafting (Melnyk et al 2015).…”

Visualizing protein-protein interactions in plants by rapamycin-dependent delocalization

“…Similar to KS in animals (Robinson et al, 2010), KSP might become a conditional in planta knockout tool, which could be used to study otherwise lethal mutants (Lloyd and Meinke, 2012;Lloyd et al, 2015). Even more, combining KSP with tissue-specific promoters (Siligato et al, 2016), would allow to live-image study the immediate effects of inducible protein knockouts in in particular plant cell types, tissues, and organs, while avoiding the pleiotropic effects of gene loss by conventional approaches. This approach might overcome limitations in terms of capacity and/or timing observed by conditionally delocalizing functional proteins using nanobody expression (Winkler et al, 2020).…”