Development of a tightly regulated copper-inducible transient gene expression system in Nicotiana benthamiana incorporating a suicide exon and Cre recombinase

• Chemical-inducible gene expression systems are commonly used to regulate gene expression for functional genomics in various plant species. However, a convenient system that can tightly regulate transgene expression in Nicotiana benthamiana is still lacking.
• In this study, we developed a tightly regulated copper-inducible system that can control transgene expression and conduct cell death assays in N. benthamiana. We tested several chemical-inducible systems using Agrobacterium-mediated transient expression and found that the copper-inducible system exhibited the least concerns regarding leakiness in N. benthamiana. Although the copper-inducible system can control the expression of some tested reporters, it is not sufficiently tight to regulate certain tested hypersensitive cell death responses. Using the MoClo-based synthetic biology approach, we incorporated the suicide exon HyP5SM/OsL5 and Cre/LoxP as additional regulatory elements to enhance the tightness of the regulation.
• This new design allowed us to tightly control the hypersensitive cell death induced by several tested leucine-rich repeat-containing proteins and their matching avirulence factors, and it can be easily applied to regulate the expression of other transgenes in transient expression assays.
• Our findings offer new approaches for both fundamental and translational studies in plant functional genomics.