Fig. 1: Agrobacterium-mediated transient expression of GUS and CRISPR/Cas9 genes in tobacco.

a, b T-DNA constructs for (a) GUS and (b) CRISPR/Cas9 (Cas9 and a PDS-targeted sgRNA) expression. The GUS construct consists of a GUSPlus gene interrupted by a cas1 intron (in), to prevent bacterial expression, under the control of a CaMV 35S promoter, and a kanamycin resistance gene (Kan^R). b The CRISPR construct (hCas9-NtPDS) consists of a Cas9 gene interrupted with an IV2 intron (in), an sgRNA targeting the tobacco PDS gene under the control of a U6 promoter, and a kanamycin (Kan^R) resistance gene. Primer sets 1, 2, and 3 were used for PCR analysis to determine the presence of T-DNA fragments stably integrated into the tobacco genome. c The fourth exon of the tobacco PDS gene was selected as the target site for the sgRNA. d, e Histochemical staining of GUS activity in tobacco leaf discs (d) without or (e) with timentin to suppress Agrobacterium growth. d GUS activity in tobacco leaf discs 2–6 days following infection by Agrobacterium. e Reduction in GUS activity in tobacco explants transferred to timentin-containing media, to suppress Agrobacterium growth, for an additional 5 days following the initial 2–6 day co-incubation. The difference in GUS expression activities between explants in d and those in e indicated transient GUS expression demonstrating that transient GUS expression peaked at 3–4 days post infection