This high throughput protocol allows rapid screening of tomato seedlings from wild accessions to the bacterial pathogen Pseudomonas syringae. The seedling flood assay minimizes plant growth time and growth chamber needs, allows rapid turnover of plants, and allows large sample sizes to be tested. This assay is a powerful tool that can be used to screen for resistance in wild accessions and other lines with complex genetic backgrounds.
The protocol provides specific directions for flood innoculation of two Pseudomonas syringae strains. However, it is versatile and can be modified to detect host resistance to other bacterial pathogens. Demonstrating the procedure will be Yana Hasan, a research specialist in my laboratory.
Begin by growing the tomato seedlings. Place tomato seeds in a 2.2 milliliter microcentrifuge tube and add two milliliters of 50%bleach solution. Rock the tube for 25 minutes then remove the bleach solution with a pipette.
Add two milliliters of ultra pure water and wash the seeds by inverting the tube five times. Aspirate the liquid from the tube, and repeat the wash four more times. After the final wash, add two milliliters of ultra-pure water and pour the seeds into a sterile Petri dish.
Sterilize forceps by flaming them in ethanol then use them to transfer five to seven seeds onto a 100 by 25 milliliter plate with 0.5 XMS plus 0.8 percent agar media. Seal the edges of the plate with surgical tape. Ensure that the plates are stacked flat and face up.
Stratify the sterilized seeds at four degrees Celsius in the dark for at least three days to synchronize germination. After three days, vertically orient the plates so that the roots will grow down along the surface of the plate and the line of seeds is oriented horizontally. Transfer the seeds to a growth chamber set to 22 degrees Celsius with a 16-hour light, 8-hour dark cycle.
Grow the seedlings for ten days in the chamber, at which point they will typically display fully emerged and expanded cotyledons and emerging first true leaves. Streak fresh bacteria onto appropriate KB agar with a flat, sterile toothpick. For PstT1, incubate the plate at 28 degrees Celsius for 48 hours prior to using the bacteria in the flood experiment.
To prepare the inoculum, aseptically resuspend the bacteria in sterile, ten millimolar magnesium chloride to an optical density of 0.1. Then make two serial dilutions to obtain a working concentration with an OD600 of 0075. Also prepare a one to ten dilution of non-ionic organosilicone surfactant copolymer in ten millimolar magnesium chloride.
Vortex it and add it to the bacteria, swirling the tube to mix. Transfer the plates with the ten-day-old seedlings from the growth chamber to the biosafety cabinet and remove the surgical tape. Then transfer six milliliters of inoculum to each plate.
Gently push the seedlings into the inoculum with a pipette tip and start a timer for three minutes. Hold one plate in each hand and tilt the front of the plate down to submerge the cotyledons and leaves of the seedlings. Swish the inoculum side to side five to seven times, then tip the plates back to cover the roots with the inoculum.
Tilt the plates down again and repeat the cycle for a total of three minutes. Pour the inoculum off the plates, set them down on a flat surface, and pour off any residual inoculum for a second time. Re-wrap the plates and place them back in the growth chamber.
Moneymaker-PtoR and Moneymaker-PtoS cultivars were flooded with PSTDC3000 and phenotyped seven to ten days after flooding. Moneymaker-PtoR seedlings carry the PtoPRF gene cluster and were resistant to the PSTDC3000. While near-isogenic Moneymaker-PtoS seedlings, which cannot recognize the PSTDC3000 effectors AVRPto or AVRPto-B died quickly, within seven days after flooding.
Ten day old seedlings were flooded with PstT1 and phenotyped at least ten days after flooding. Susceptible accessions were dead, had brown apical meristems and lacked new growth. In contrast, resistant seedlings displayed a high level of new green growth and survived infection with PstT1.
Bacterial growth assays were carried out to quantitatively confirm PstT1 resistance and Solanum NeoRici LA1329. There was a 1.7 log difference in bacterial growth between resistant and susceptible LA1329 and a 1.6 log difference between resistant LA1329 and Moneymaker-PtoS, which correlated with the phenotypic results. When performing this assay, the most important things to remember are to pay close attention to aseptic technique throughout the protocol and ensure that all seedlings on the plate are thoroughly flooded.
Be sure to autoclave infected plant tissue and bacteria and dispose of materials in accordance with appropriate regulations.
