Tissue‐specific inactivation by cytosine deaminase/uracil phosphoribosyl transferase as a tool to study plant biology

Abstract

Recent advances in the study of plant developmental and physiological responses have benefited from tissue-specific approaches, revealing the role of some cell types in these processes. Such approaches have relied on the inactivation of target cells using either toxic compounds or deleterious genes; however, both tissue-specific and truly inducible tools are lacking in order to precisely target a developmental window or specific growth response. We engineered the yeast fluorocytosine deaminase (FCY1) gene by creating a fusion with the bacterial uracil phosphoribosyl transferase (UPP) gene. The recombinant protein converts the precursor 5-fluorocytosine (5-FC) into 5-fluorouracyl, a drug used in the treatment of a range of cancers, which triggers DNA and RNA damage. We expressed the FCY-UPP gene construct in specific cell types using enhancer trap lines and promoters, demonstrating that this marker acts in a cell-autonomous manner. We also showed that it can inactivate slow developmental processes like lateral root formation by targeting pericycle cells. It also revealed a role for the lateral root cap and the epidermis in controlling root growth, a faster response. The 5-FC precursor acts systemically, as demonstrated by its ability to inhibit stomatal movements when supplied to the roots in combination with a guard cell-specific promoter. Finally, we demonstrate that the tissular inactivation is reversible, and can therefore be used to synchronize plant responses or to determine cell type-specific functions during different developmental stages. This tool will greatly enhance our capacity to understand the respective role of each cell type in plant physiology and development. Tissue-specific genetic ablation 733Histochemical analysis and microscopy GUS staining was performed as previously described (P eret et al., 2007). Plants were cleared for 24 h in 1 M chloral hydrate and 33% glycerol. Seedlings were mounted in 50% glycerol and observed

Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants

“…Therefore, 5‐FC only becomes toxic to plants in the presence of the FCY‐UPP gene; a similar fusion was recently used for tissue‐specific genetic ablation (Leonhardt et al , 2020). We wanted to avoid working under sterile conditions and therefore first tested toxicity of 5‐FC on quartz sand plates supplemented with MS solution (Davis et al , 2009) using a previously reported FCY‐UPP ‐transgenic Arabidopsis line (under 35S promoter control; Leonhardt et al , 2020). 5‐FC did not have any adverse effects on the growth of wild‐type plants, but became toxic to the transgenic line at concentrations of 1 m m or higher (Figure S4).…”

“…The FCY‐UPP marker is probably the most versatile counter‐selection selection system we used (Figure 2a,c). The selection strategy is not new (Leonhardt et al , 2020; Perera et al , 1993; Stougaard, 1993, and references therein), but it had previously not been considered in genome editing applications. In our hands, the FCY‐UPP marker worked flawlessly for counter‐selection.…”

Highly efficient multiplex editing: One-shot generation of 8xNicotiana benthamianaand 12x Arabidopsis mutants

“…Therefore, 5-FC only becomes toxic to plants in presence of the FCY-UPP gene; a similar fusion was recently used for tissue-specific genetic ablation (Leonhardt et al , 2020). We wanted to avoid working under sterile conditions and therefore first tested toxicity of 5-FC on quartz sand plates supplemented with MS solution (Davis et al , 2009) using a previously reported FCY-UPP -transgenic Arabidopsis line (under 35S promoter control; Leonhardt et al , 2020). 5-FC did not have any adverse effects on the growth of wild-type plants, but became toxic to the transgenic line at concentrations of 1mM or higher (Figure S4).…”

“…The FCY-UPP marker is probably the most versatile counter-selection selection system we used (Figure 2a,c). The selection strategy is not new (Perera et al , 1993, Stougaard, 1993, Leonhardt et al , 2020 and references therein), but it had previously not been considered in genome editing applications. In our hands, the FCY-UPP marker worked flawlessly for counter-selection.…”

“…Arabidopsis wild-type or FCY-UPP transgenic plants (Leonhardt et al, 2020) were grown on quartz sand plates supplemented with 1/4 MS solution containing the indicated concentrations of 5FC. Plates were documented after 18 days.…”