This study demonstrates a method for visualizing the delivery of effector proteins from Pseudomonas bacteria into plant cells using genetically modified leaves. The technique employs fluorescence to confirm successful bacterial infection and protein translocation.
Begin with an experimental plant with genetically modified leaf regions expressing large non-fluorescent fragments of superfolder green fluorescent protein or sfGFP.
Inject these modified areas with Pseudomonas bacteria containing effector proteins tagged with the small non-fluorescent fragment of sfGFP.
Incubate to allow interaction between the bacteria and the leaf cells.
Using a specialized secretion system, Pseudomonas delivers the tagged effector proteins into the leaf’s cytosol.
The proximity between the large and small GFP fragments enables their assembly into functional fluorescent sfGFP, which then translocates to the plasma membrane.
After incubation, excise leaf discs from the injected areas.
Visualize these discs using a laser scanning confocal microscope.
Initially, image the leaf disc at low laser intensity to minimize autofluorescence. Then, increase the laser power to enhance visibility.
The green fluorescence confirms successful bacterial infection and effector protein delivery into the plant cells.
For the Nicotiana benthamiana leaves, infiltrate the Pseudomonas suspension into the same area as the Agrobacterium was infiltrated two days earlier.
Finally, cut out a leaf disc from the Pseudomonas-inoculated leaves. At specific time points after infiltration of Pseudomonas, use a confocal laser scanning system to image two, two square centimeter leaf discs from the single plant. Observe the cells away from the infiltration hole to avoid dead cells killed by wounding.
Translocated effectors from the bacteria are present only in very small amounts. Therefore, you may increase the laser power and gain a fluorescence emission to detect a fluorescent signal. However, don't overpower to avoid capturing false signal from autofluorescence from the plant cells.
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