Working with plant material

2.2.1.1 Plant growth conditions and propagation

For infection experiments Arabidopsis thaliana or Nicotiana benthamiana plants were grown in perforated plastic pots (17.5 cm · 13 cm · 5.5 cm). In order to ensure optimal aeration a perforated plastic pot was placed into a second perforated plastic pot containing empty 2 ml eppendorf tubes as spacer. The upper pot was filled with 200 ml clay granules (Seramis GmbH, Mogendorf, Germany) and 800 ml of a sand-soil mixture in a ratio of 1:1. Subsequently, the sand-soil mixture was watered with 250 ml H2O supplemented with 0.1 % (v/v) Wuxal^® liquid fertilizer (Manna, Ammerbuch-Pfäffingen, Germany) and ca. 60 single seeds were placed with a toothpick on the surface at a steady distance. Finally, seeds were stored for 2 days at 4 °C for vernalisation and cultivated in a short day growth chamber (8 h light at 22 °C; 16 h dark at 18 °C; 65 % relative humidity).

For seed setting plants were grown in square plastic pots (8 cm) filled with soil in a long day growth chamber (16 h light at 22 °C; 8 h dark at 18 °C; 65 % relative humidity) and seeds collected 12-16 week after germination. In order to collect seed, inflorescence stems were covered with a paper bag before silique maturation and opening. Soil grown plants were watered every 2-3 days with tap water.

In vitro A. thaliana seedlings were grown on angled agar plates, in order to allow root growth on the agar surface. For this purpose one side of a square petri dish (10 cm · 10 cm) was placed on its lid and filled with ½ MS-agar. A. thalianaseeds were surface sterilised as described in 2.2.1.2. After the agar solidified, 10 seeds per plate were placed in a row on the upper, thin side of the agar plate. Agar plates were sealed with Millipore tape (Merck, Darmstadt, Germany), stored for 2 days at 4 °C for vernalisation and cultivated in a Percival^® growth chamber (CLF Plant Climatics, Wertingen, Germany) under short day conditions (10 h light at 22° C, 14 h dark at 20° C, 65 % relative humidity). If used forVerticillium root infection,in vitro seedlings were cultivated in a short day growth chamber (8 h light at 22 °C; 16 h dark at 18 °C; 65 % relative humidity).

2.2.1.2 Seed sterilisation

In order to avoid contamination of growth chambers by insect of theThysanoptera genus, seeds of soil grown plants were cold sterilised. Seeds were sealed in airtight plastic bags and stored for 48 h at -20 °C. Afterwards, seeds were kept in the sealed plastic bags at room temperature until they warmed up.

In vitro grownA. thaliana seedlings were surface sterilised with ethanol under a sterile bench.

Seeds were transferred into 1.5 ml microcentrifuge tubes, covered with 1 ml 70 % ethanol and incubated at room temperature for 10 min under vigorous shaking. Thereafter, 70 % ethanol was exchanged with 96 % ethanol. Subsequently, seeds were shaken for additional 5 min.

Finally, ethanol was removed and seeds washed briefly with H2O by pipetting up and down.

Seeds were then transferred onto a sterile filter paper and allowed to dry.

2.2.1.3 Agrobacterium-mediated stable transformation of A. thaliana by floral dipping

40 ml DYT-medium containing selective antibiotics were inoculated with Agrobacterium tumefaciens. Bacterial cultures were grown over night in a shaker at 28° C and 190 rpm. The 40 ml over night culture was used to inoculate a 400 ml DYT-medium containing selective antibiotics. This culture was incubated over night under aforementioned conditions. Next morning, cultures were spun at 4000 rpm for 15 min and the pellet resuspended in in 5 % (w/v) sucrose. Before floral dipping, 0.05 % (v/v) Sylvet-77 was added to Agrobacterium cell suspension, to reduce the surface tension.A. thaliana plants were grown in trays under long day conditions until bolting. The first inflorescence stems were removed in order to induce development of additional inflorescence stems. Inflorescences were submerged and gently shaken in the Agrobacterium cell suspension. After dipping, residual Agrobacterium cell suspension was removed with a paper towel, trays were covered with plastic lids and stored in the dark overnight. Finally, plants were transferred into a long day growth chamber to allow development of seeds.

2.2.1.4 BASTA

selection of stably transformed A. thaliana

T1seeds of stably transformed A. thaliana were sown in square pots (8 cm) and grown for 1 week under short day conditions. Subsequently, seedlings were sprayed with the herbicide BASTA^® (200 g/L glufosinate ammonium solution, Bayer CropScience AG, Monheim, Germany) diluted 1:1000 in H2O. Afterwards, BASTA^®was applied every 2 days for 2-3 times, until non-transformed seedlings became senescent. Finally, non-transformed, senescent seedlings were removed with forceps and stably transformed A. thaliana T1kept for further analyses and seed setting.

2.2.1.5 Infiltration of A. thaliana with abscisic acid

Abscisic acid (ABA) was dissolved in methanol and stored as a 10 mM stock at -20° C. 50 µM ABA was prepared fresh for each experiment by diluting the ABA stock in 5 mM KCl/MES buffer. For mock treatment, the same amount of methanol without ABA was added to 5 mM KCl/MES buffer. For vacuum infiltration, 8 leaf discs (∅ 0.55 cm) were collected from fully expanded leaves of 10-11 week old A. thaliana plants. Leaf discs were transferred into a 10 ml disposable syringe (Servoprax GmbH, Wesel, Germany) filled with 2 ml of 50 µM ABA or KCl/MES buffer containing methanol. Air was released and syringe sealed with a closing cone (B. Braun Melsungen AG, Melsungen , Germany). The syringe was compressed for several times and thus vacuum applied until all leaf discs became entirely translucent. Infiltrated leaf discs were incubated over night. Next morning, leaf discs were dried with a paper towel, subsequently transferred into microcentrifuge tubes, frozen in liquid nitrogen and stored at -80 °C.

A. thaliana seedlings were incubated in 50 µM ABA. For this purpose, wells of a 24-well plate were filled with 2 ml of 50 µM ABA or KCl/MES buffer containing methanol. 5-6 in vitro grown seedlings per well were submerged in the respective solution. If used for RNA extraction, after 3 or 24 h of incubation, seedlings were briefly washed in H2O, dried with a paper towel and transferred into 2 ml microcentrifuge tubes containing 2 stainless steel beads (∅ 0.4 cm).

Seedlings were frozen in liquid nitrogen and stored at -80 °C. Seedlings, which were used in confocal microscopy, were briefly washed in H2O and directly mounted on microscopy slides.

2.2.1.6 Confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) was performed using a Leica SP5-DM6000 microscope (Leica, Wetzlar, Germany), an argon ion laser as excitation source and the Leica LAS AF software (v.2.6.7266.0). Excitation wavelength of 514 nm was used for Venus and emission detected at 518 to 570 nm. For GFP an excitation wavelength of 488 nm was used and emission detected at 489 to 510 nm. Detection of autofluorescence was performed at 699 to 751 nm.