2. MATERIALS AND METHODS
2.2. Methods
2.2.6. Pathogen-associated disease phenotype of ABA biosynthetic and signaling
Two different virulent pathogens were used in this investigation to evaluate the pathogen-associated disease phenotype of the tested ABA biosynthetic and signaling single and double mutants: aba1-101, aba2-1, snrk2d, snrk2e, snrk2i, snrk2d snrk2e, snrk2d snrk2i, and snrk2e snrk2i. Spores of the oomycete Noco2 were collected at five to six days after infection of A. thaliana Col-0 seedlings. Next, the sporulated shoots of the infected Col-0 plants were harvested in 50 ml falcon tubes soon after the transfer of the infected pots from the high humidity chamber to the relatively low humidity laboratory. Subsequently, 20 ml of sterile distilled H2O (room temperature) was added, followed by gentle shaking to release the spores into the water. The filtered spore solution was counted using a hemocytometer chamber (Labor Optik, United Kingdom) and diluted to 5 x 104 spores per ml. Next, two-week-old mutant and the susceptible wildtype Col-0 plants (20 plants / 8.0 cm2 pot), were sprayed with the spore solution.
The hyper-susceptible mutant eds1 (enhanced disease susceptibility 1) (Parker et al.
1996), and the highly disease resistant mutant snc1 (Li et al. 2001) were used as positive and negative controls, respectively. Noco2 infected plants were kept in covered trays and maintained in a growth chamber under short-day conditions for 5-6 days. Subsequently, plants were harvested in a weighed 50 mL falcon tube. Then 5 µL H2O /mg plant tissue was added and subsequently the samples were vortexed for 15 sec. The spore density was determined using a hemocytometer chamber. Spore counting was repeated four times for each replicate.
For inoculation with G. orontii spores, five-week-old plants were brush-inoculated with the fungus. The G. orontii spores were collected at 10 days after infection for spore counting. Five plants obtained from one pot were collected in a weighed 50 mL tube.
Then 5.0 µL H2O were added per mg plant material and subsequently the samples were vortexed for 15 sec. Using the hemocytometer chamber, the spores were counted
53
four times per replicate. The previously mentioned mutants were tested in addition to the hypersusceptible mutant eds1 (enhanced disease susceptibility 1) (Parker et al.
1996), hyperresistant mutant edr1 (enhanced disease resistance 1) (Frye & Innes 1998) and the susceptible wildtype Col-0.
2.2.7. Plant-microbe interaction experiments to map pathogen-mediated hormone signaling
2.2.7.1. H. arabidopsidis-Noco2 and -Emwa1 infection experiments
To address the hormone signatures during interaction with the oomycete H.
arabidopsidis, the T4 generations of COLORFUL-ABA, -SA, -JA, -JA/ET reporter lines
#1 and #2 and the F4 generations of COLORFUL-ABA line #1 in aba1-101, snrk2d, snrk2i, snrk2d snrk2i, ahg3-1 and pp2ca1 backgrounds, as well as COLORFUL-SA line #1 in ahg3-1 and pp2ca1 background were used for oomycetes inoculation. Seedlings were grown under short-day conditions covered with a clear plastic hood and transplanted into individual pots (4.5 cm in diameter), three seedlings per pot, for an additional two weeks. Subsequently, three-week-old plants, as mentioned earlier, were sprayed with the Emwa1 and Noco2 spore solutions adjusted to 5 x 104 spores per ml as mentioned above in comparison with unchallenged plants, which were sprayed with water. Challenged and mock plants then were kept in covered trays and maintained in a growth chamber under short-day conditions. The 5th leaf on the rosette was consistently selected for CLSM (Leica, Germany) at 1 and 2 dpi.
2.2.7.2. G. orontii infection experiments
To map the hormone signaling during interaction with G. orontii, five-week-old plants were used for inoculation. Seeds of T4 generations of the reporter lines COLORFUL-SA line #1, -ABA line #1 and the F4 generations of COLORFUL-ABA line #1 in aba1-101, snrk2d, snrk2i, snrk2d snrk2i, ahg3-1 and pp2ca1 backgrounds were sown onto 8.0 cm2 pots and incubated in the dark at 4.0 °C for three days. The seedlings were grown for a week covered with a clear plastic hood and transplanted into individual 8.0 cm2 pots, five seedlings per pot, for an additional four weeks under the short-day
54
chamber conditions. Spores of heavily infected hyper-susceptible A. thaliana eds1 plants were brushed directly upon the five-week-old plants of the aforementioned genotypes. Uninfected plants were used as control. Infected and mock plants were randomized and incubated in uncovered trays in two separate growth chambers adjusted to the short-day conditions (mentioned above). The 5th rosette leaf of plants infected with G. orontii, as well as corresponding uninfected mock plants were consistently selected for CLSM (Leica, Germany) at 1 and 2 dpi.
2.2.7.3. Visualization of pathogens during microscopy
To allocate different spores of H. arabidopsidis and G. orontii used in this study, to help define the real penetration sites, and help monitor the progression of these different pathogens, a fast stain named Fluorescent Brightener 28 (FB-28) (Sigma-Aldrich, Germany) was used. A stock solution of 10 mg/ml FB-28 was diluted to 1:1000 for a final solution concentration of 10 µg/ml. The adaxial surface of the 5th leaf was immerged in a drop of FB-28 for 30 sec to stain the spores. The midrib of the leaves was excised, and the leaves were placed on microscopic slides (Thermo Fisher Scientific, Germany) and mounted in the FB 28 solution.
2.2.7.4. Evaluation of growth dynamics of Emwa1 and Noco2
To address the invasion dynamics of Noco2 and Emwa1 during the compatible and incompatible interactions, respectively, the number of penetrating spores relative to the total numbers of spores per leaf was investigated for both isolates. In these experiments, data were collected from three independent experiments. Each experiment included ten leaves from ten discrete plants. Furthermore, to understand the growth dynamic difference between both Emwa1 and Noco2, the number of haustoria in the epidermal, as well as the mesophyll cell layers, at ten different penetration sites for Emwa1 and Noco2 in the epidermis and the mesophyll at 1 and 2 dpi was scored. The data from three different experiments using COLORFUL-SA, -JA and -JA/ET were pooled. Finally, images and movies were made to show the cell death symptoms induced by Emwa1 infection but not Noco2 at 3 dpi.
55