treatment use 20 plants and the experiments should be
repeated three times.- Transfer the plants to the greenhouse and inspect the plants
weekly (up to 3 weeks) for appearance of symptoms. - Determine the degree of disease severity and disease incidence.
In the case ofXhpa four-grade scale for disease severity was
defined (seeNote 7). - For disease incidence determine the percentage of symptomatic
plants in each treatment at the end of the experiments (in the
case ofXhp21 days post inoculation).
3.4 Plant
Colonization
- After 15–21 days post inoculation, cut pieces of 1 cm with a
sterile scissor along the plant stem: at the inoculation site,
1.5 cm above the inoculation site, and at the apex (seeNote 8). - Weight each stem sections and transfer them to sterile plastic bags.
- Macerate manually the plant samples with a hammer and a
crusher until the plant tissue is completely disintegrated. - Add 1 ml of sterile water to the sample bag and shake it gently
at an orbital shaker for 5 min. - Remove with a pipette, 0.9 ml of suspension and place in a
sterile 1.5 ml tube. - Prepare tenfold serial dilutions of these suspensions with sterile
water. - Spread 100μl of each dilution on three LB agar plates amended
with the appropriate antibiotics. - Incubate the plates at 28C for 48 h and calculate the number
of CFU (colony forming units) per gram of plant fresh weight.
3.5 Movement
of GFP-Labeled
Pathogen In Planta
To study the effect of QS on virulence it is important to determine
the movement of the pathogen in its host as compared to its QS
mutants in different tissues and time after inoculation.- Inoculate plants with the wild type and its QS-mutant strains
labeled with GFP as described in Subheading3.3. - At the defined time point after inoculation, excise with a sterile
scissor symptomatic tissues (root, stem, leaves, fruit, and flower). - Prepare plant samples by cutting thin transversal and longitu-
dinal sections with a razor. - Place the samples on a glass microscope, add 1–2 drops of
water, and cover with a cover glass. - Proceed to confocal microscopy examination by placing the
sample with the cover glass facing down. - Examine several tissue samples (at least 4) taken from each
plant.
248 Shulamit Manulis-Sasson and Laura Chalupowicz