Agrobacterium tumefaciens does not transform guard cells in Nicotiana benthamiana

The bacterium Agrobacterium tumefaciens is widely used to genetically transform leaf cells of Nicotiana benthamiana using the agroinfiltration method (http://www.youtube.com/watch?v=GHc7PU_jG2M). Interestingly, A. tumefaciens does not transform guard cells, whereas it transforms efficiently pavement cells. The high chlorophyll content makes chloroplasts autofluorescent. In the case of live-cell imaging experiments with leaf cells transiently expressing fluorescent markers targeted to chloroplasts, it is hard to differenciate the fluorescent marker signal from chlorophyll autofluorescence. Here, I show that guard cells can be used to differentiate those. A GFP targeted to the chloroplast stroma was transiently expressed in N. benthamiana leaf cells, and epidermis cells were observed by live-cell imaging with a laser-scanning confocal microscope two days after the agro-infiltration. A 488 nm wavelength was used to illuminate the tissues, and GFP and chlorophyll signals were collected between 505-525 nm and 680-700 nm, respectively. The asterisks mark the guard cells, the cross marks a non-transformed pavement cell. The white rectangle on the merged image delimitates the close-ups presented in the lower panel. Note the absence of GFP signal in chloroplasts from the guard cells and from the non-transformed pavement cell.