Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

This method can help answer key questions in the plant-microbe interaction field such as how pathogenic bacteria can disturb the optimal condition of their host plant cells by using proteins called effectors secreted into plant cells. The main advantage of this technique is the bacterial effector protein is expressed in the bacterial cell using their native expression system and then delivered to the plant cell through the bacterial type III secretion system. To begin this procedure, prepare Nicotiana benthamiana and Arabidopsis thaliana plants as outlined in the text protocol.

Next, use standard electroporation to transform the plasmid carrying and effector gene fused to the sfGFP11 tag to Pseudomonas syringae pathovar tomato CUCPB5500 strain. The optimal time point and expression level, or reconstitute sfGFP, really depends on your effector protein. We recommend to optimize inoculation or observation conditions.

Gently spread the transformed bacterial cells over the surface of King's B Agar plates. Then incubate the plates at 28 degrees Celsius for two days. After this, inoculate one bacterial colony into King's B liquid medium with an antibiotic appropriate for the vector.

Incubate overnight at 28 degrees Celsius with shaking at 200 rpm. The next day, add autoclaved glycerol to the inoculated medium to a final concentration of 50%Store at negative 80 degrees Celsius. To begin, transform the plasmid into Agrobacterium tumefaciens strain GV3101 cells as outlined in the text protocol.

Grow the cells on supplemented LB Agar medium at 28 degrees Celsius for two days. Using a single colony from the LB Agar medium, inoculate 5 milliliters of supplemented liquid LB medium. Then, grow the cells overnight at 28 degrees Celsius with shaking at 200 rpm.

After this, centrifuge at 3000 G for 10 minutes to harvest the cells. Pour off the supernatant and re-suspend the pellet in 1 milliliter of freshly made infiltration buffer. Using a spectrophotometer, determine the quantity of Agrobacterium cells by measuring the optical density value at an absorbance of 600 nanometers.

Then, use infiltration buffer to adjust the OD 600 of the bacterial cells to 0.5. Leave the culture on a gentle rocker at room temperature for one-to-five hours. To infiltrate the leaves, use 10-microliter pipette tip to poke a hole in the center of each leaf.

Use a needleless 1-milliliter syringe to carefully inject 500 microliters of the Agrobacterium suspension into the adaxial side of the leaf. Wipe the remaining bacterial suspension off of the leaves and mark the boundary of the infiltrated region. Keep the infiltrated plants in a growth chamber at 25 degrees Celsius and 60%humidity for two days.

Streak the transformed Pseudomonas strain from the glycerol stock onto King's B Agar medium with the appropriate antibiotic. Incubate at 28 degrees Celsius for two days. Inoculate a loop of Pseudomonas cells in mannitol glutamate liquid medium.

Incubate the culture overnight at 28 degrees Celsius with shaking at 200 rpm. After this, centrifuge at 3000 G for 10 minutes to harvest the cells. Pour off the supernatant and re-suspend the pellet in 10-millimolar magnesium chloride solution.

Then, adjust the optical density to 0.02 for Nicotiana benthamiana leaves and to 0.002 for Arabidopsis leaves. For the Nicotiana benthamiana leaves, infiltrate the Pseudomonas suspension into the same area as the Agrobacterium was infiltrated two days earlier. For the transgenic Arabidopsis, infiltrate the Pseudomonas suspension into two four-week-old short day-grown leaves.

Finally, cut out a leaf disk for 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 disks 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.

In this study, a fluorescent protein-based approach is used to monitor effectors secreted by bacteria into host cells. A confocal laser scan of a Nicotiana benthamiana leaf three hours after infection is shown here. Cells expressing optimized sfGFP with AvrB only do not show any fluorescent signal.

However, GFP signals are seen from the infected cells containing AvrB, sfGFP11. This verifies that AvrB sfGFP11 complex is reconstituted with optimized sfGFP in the cytosol and then translocates to the plasma membrane. While attempting this procedure, it's important to remember to keep the plant materials healthy and to prepare fresh bacteria culture for active cells.

Following this procedure, other methods like resin blood analysis can be preponed to understand false translation modification of bacteria effector proteins in plant cells during plant immune responses. After watching this video, you should have a good understanding of how to visualize the type III effector delivery in infected plant cells. Plant materials and bacteria cultures should be disposed of following your lab's criteria for biohazardous waste and don't forget to wear your PPE.