Split GFP assay to show chloroplast targeting ofAgrobacteriumVirD2 protein

Abstract

Regenerating fertileArabidopsis thalianaplants from tissue culture cells with transformed plastid genomes is difficult, because of somaclonal variation in tissue culture cells. For nuclear gene transformation, tissue culture limitations were overcome in Arabidopsis by direct transformation of the female gametes using the floral dip protocol and identification of transgenic events in the seed progeny. DuringAgrobacteriumtransformation the VirD2 protein guides the T-complex, consisting of single stranded transferred-DNA (T-DNA) coated with VirE2 proteins, to the plant nucleus. To enable floral dip transformation of the plastid DNA, we retargeted VirD2 to chloroplasts. We show plastid targeting of VirD2 in a split GFP assay, where VirD2-GFP11complements GFP1-10in chloroplasts. Floral dip transformation of plastids will avoid tissue culture altogether, making plastid transformation readily available for the research community.