Figure 5

Functionality test of two genes, cloned into the pEG101-SacB/R destination vector, by means of Agrobacterium -mediated transient assays in the leaves of Nicotiana benthamiana. A. tumefaciens GV2260 strains, carrying either pEarleyGate 101 empty vector (A-1) or pEG101-Luc (A-2), were inoculated in N. benthamiana leaves. The A. tumefaciens GV2260 strain carrying pEG101-Luc (B-2) produced a strong luminescent signal under dark conditions after luciferin treatment, while the strain carrying the unmodified vector pEarleyGate 101 (B-1) did not show any signal. The luminescent signal was detected using the Gel Document Image System equipment with a CCD camera. A. tumefaciens strain GV2260 carrying pEG101-Aae2166 (C-2), but not the empty vector pEarleyGate 101 (C-1), was able to trigger programmed cell death in the leaf of N. benthamiana. The fluorescent signal of the Aae2166-GFP fusion protein was detected by fluorescent microscopy (200 × magnifications) (E). As the negative control, GFP signal was not detected in the leaf of N. benthamiana that was inoculated with A. tumefaciens carrying pEarleyGate 101 (D).