This protocol is significant because it allows scientists to perform straightforward genetic analysis of bacterial wilt disease in tomato. The main advantage of this technique is that the manipulation of gene expression and subsequent assays can be performed in a short time and with the small requirements of equipment and plant growth space. This is method is very versatile, and it may be applied to other crop plants to perform both pathogen inoculation and other physiological assays.
To maintain the sterility during manipulation of seedlings in vitro, it is important to keep the plates closed when they are outside of the flow hood. This is an easy technique, but it requires multiple step for manipulation. The visualization of this method will help to see small tricks that are difficult to explain in the manuscript, helping other researchers to implement the method in their labs.
Helping to demonstrate this procedure will be Achen Zhao, a graduate student. After sterilizing and washing the tomato seeds, transfer them to half-strength Murashige and Skoog medium without sucrose. Keep the seeds in the dark at a temperature between 25 and 28 degrees Celsius for three days.
Place autoclaved 8.5-square-centimeter filters inside nine-square-centimeter Petri dishes containing half-strength Murashige and Skoog medium, and place six germinated tomato seeds on each plate. Seal the plate with Micropore tape, and incubate the germinated seeds at 25 to 28 degrees Celsius for three to four days. Grow the Agrobacterium rhizogenes MSU440 in solid LB medium with appropriate antibiotics for two days at 28 degrees Celsius before plant transformation.
Using a sterile scalpel, cut the radicle and the bottom of the hypocotyl of the tomato seedlings. Use plastic tips or a scalpel blade to harvest A.rhizogenes biomass from the surface of the LB medium, and carefully dip the cut tomato seedlings in the bacterial biomass. After this, cover the tomato seedlings with a two-centimeter-by-four-centimeter semicircular filter paper in order to keep a high humidity and facilitate survival and new root development.
It is important to keep the seedlings in high humidity in an environment that allows transpiration. To do this, cover the seedlings with filter paper, and close the plate with Micropore tape. Store the transformed tomato seedlings for six to seven days in a growth chamber at a temperature between 25 and 28 degrees Celsius.
Then use a sterile scalpel to cut the new emerging hairy roots, and allow the seedlings to produce new hairy roots. Once the second generation of new hairy roots appears, remove the filter paper on top of the seedlings, and seal the plate with Micropore tape again. To visualize DsRed fluorescence or any other equipment for plant in vivo imaging, mark the positive transformed roots, which can be identified by the red fluorescence.
Use a scalpel to remove the negative non-transformed roots, which can be identified by their lack of red fluorescence. Transfer the seedlings that show red fluorescence to a new plate containing half-strength Murashige and Skoog medium in order to facilitate the development of the transformed root as the main root. Keep the seedlings that do not show red fluorescence in the same plate to check the emergence of fluorescent roots in later time points.
Cover the seedlings with a two-centimeter-by-four-centimeter semicircle filter paper, seal the plate, and incubate the seedlings to let them develop new hairy roots. Prepare the inoculation pots where the surface of the roots will be exposed to the bacterial inoculum by first soaking the inoculation pots with water, pouring off any excess water, and then placing them in a plastic planting tray. Using tweezers, transfer the selected seedlings with transformed roots to the inoculation pots.
Cover the tray with plastic wrap or a transparent lid, and keep them at 25 to 28 degrees Celsius with 65%humidity. Remove the cover after five or six days. Grow Ralstonia in five LB liquid medium in an orbital shaker at 28 degrees Celsius, with shaking at 200 rpm, until the stationary phase.
Measure the optical density at 600 nanometers to determine the bacterial numbers. Dilute the bacterial culture with water to an OD600 of 1. Place 16 to 20 inoculation pots that contain transformed tomato plants into an inoculation tray.
Then pour 300 milliliters of diluted bacterial inoculum into the tray, and allow the plants to soak in the inoculum for 20 minutes. After this, prepare a new tray with a layer of potting soil. Move the inoculated pots into the new tray, and place the trays in a growth chamber with 75%humidity, a temperature between 26 and 28 degrees Celsius, and a photoperiod of 12 hours of light and 12 hours of darkness.
Here, tomato roots are transformed and inoculated with Ralstonia to perform straightforward genetic analysis for the study of bacterial wilt disease. The development of disease symptoms is tracked in tomato plants with roots transformed with an empty vector and those transformed with an RAI construct targeting tomato CESA6. The disease index data are collected from the same experimental unit over time according to an arbitrary scale from zero to four and do not follow a Gaussian distribution, ruling out the use of standard tests for parametric data.
As a standard approach, a U Mann-Whitney, two-tailed, non-parametric test to compare both the control and infection curves is used. According with this analysis, the difference between the medians of both curves appears to be non-significant. It is also possible to quantify the area under the disease progress curve, which allows combining multiple observations of disease progress into a single value.
The area under the disease progress curve shows a higher value for control plants compared to tomato CESA6 RNAi plants at the end of the infection process, indicating that tomato CESA6-silenced plants are more resistant to Ralstonia infection than control plants. Confidence intervals offer a way of estimating, with high probability, a range of values in which the population value of a given variable is found. As seen here, the area of the 95%confidence interval for the control and infection curves estimate a higher chance of resistance when CESA6 is silenced.
Disease index values can be transformed into binary data, considering a disease index lower than two corresponding to zero and a disease index equal or higher than two corresponding to one. This allows the representation of a survival curve after Ralstonia inoculation. The differences in the survival rate between the control and the infected plants are not statistically significant according to the Gehan-Breslow-Wilcoxon statistical test.
The expression of CESA6 is then analyzed in two randomly selected transformed roots before the inoculation step, showing that the enhanced resistance to Ralstonia correlates with the reduced expression of CESA6. After two rounds of selection, a transformation rate of 35 to 40%can be obtained, and this value can be increased by performing additional selection rounds. While performing this procedure, it is important to keep the seedlings covered with a piece of filter paper to maintain high humidity and to seal the plates with Micropore to ensure gas exchange.
After the root transformation, many of the treatments can be applied to study the plant response by observation of root physiology or using molecular biology, cell biology, or biochemistry. Mainly, this technique will allow researchers with limited resources to perform genetic analysis of bacterial infection in tomato roots.
