Methods for working with plants and plant material

2.2.1.1 Surface sterilization of Arabidopsis seeds 2.2.1.1.1 Sterilization using chlorine

For seed sterilization using chlorine, the packed seeds were placed in a desiccator. Next 5 ml HCl (37%) were added to 100 ml 12% NaClO in a glass beaker placed in this desiccator. Since the resulting gas is harmful the whole procedure takes place under the fume hood overnight.

2.2.1.1.2 Sterilization using ethanol

Seeds were placed into a 2 ml reaction tube and were washed 3 times with 1 ml 70% ethanol.

After that, 1 ml ethanol (96%) was added and seeds were transferred to a filter paper for drying.

The sterilization procedure was performed in a sterile workbench.

2.2.1.2 Plant growth conditions for tissue culture

Surface sterilized seeds were placed on ½ MS agar plates. Seeds were then stored for 2 d at 4°C to break dormancy and then transferred to a growth cabinet conditions (CLF Plant Climatics, Wertingen, Germany) with short day condition (light for 10 h at 22°C and darkness for 14 h at 20°C). Two weeks after germination the plants were transferred to soil.

Arabidopsis plants used in expression analyses were grown in vitro in 24-well plates. For this, the wells were filled with 2 ml liquid ½ MS medium before adding 3-5 surface sterilized Arabidopsis seeds. The seeds were allowed to germinate and grow for 13 d in a plant growth chamber under short day conditions (see above). Then the medium was replaced with 1.5 ml new ½ MS medium. The next day (day 14) the seedlings were treated with polymeric chitin or medium as control. For this, 0.5 ml ½ MS medium with 4x the final concentration of chitin was added.

2.2.1.3 Plant growth conditions for cultivation on soil

The soil (Frühstorfer Erde, Type T25, Str1, Archut) for plant cultivation was steamed prior to use (80°C for 20 min) to remove of soil-borne pests and pathogens. Seeds were placed directly on

soil and then stored for 2 d at 4°C to break dormancy. After that, the pots were transferred into short-day climate chambers with 8 h of light per day, 22°C/20°C day/night, 65% relative humidity and 120 μmol/m²s light intensity. After 10 d, seedlings were pricked out. Alternatively, seedlings grown in vitro on MS agar plates were transferred to soil after approximately 2 weeks. About 4-6 week-old plants were then used for experiments. 6-8 week-old plants were transferred from short-day to long-day conditions for seed propagation for further 3-4 weeks. For faster propagation, plants were directly grown under long-day conditions (16 h light [~150 μmol/m²s], 22°C, 8 h dark, 20°C, 65% rel. humidity). Climate chambers: JC-ESC 300 chamber system (Johnson Controls, Milwaukee, WI, USA)

2.2.1.4 Crossing Arabidopsis thaliana

In order to generate crosses of different Arabidopsis mutant lines the plants were manually crossed. Therefore, closed flower buds were chosen. First, a shoot was selected and all side branches were removed to prevent confusions with non-crossed flowers. Second, sepals, petals and stamina of the maternal flower were removed until only the carpel was left. The stigma was then pollinated with single stamina from the paternal flower. Finally, the plant was allowed to develop a silique at long-day conditions.

2.2.1.5 Agrobacterium-mediated stable transformation of Arabidopsis

The floral dipping method was used to transform Arabidopsis thaliana. The protocol used is based on the method described by Clough and Bent (1998). Plants were grown under short day conditions for 2-4 weeks and then transferred to long day to induce flowering. The first bolts were clipped to break apical dominance. 2-4 days after clipping the new bolts were ready to be transformed. A single colony of A. tumefaciens cells transformed with the construct of interest (2.2.2.5) was used to inoculate a 25 ml pre-culture of DYT mixed with the appropriate antibiotics. The bacteria were grown at 28°C and 180 rpm in the Certomat^® BS-1 incubator (Sartorius-Stedim Biotech, Göttingen, Germany) for 2 d and the pre-culture was used to inoculate 250 ml DYT with appropriate antibiotics. The culture was grown overnight and then spun down at 4500 g for 30 min at RT. The supernatant was discarded and the remaining pellet was resuspended in 250 ml 5% sucrose solution. Silwet L-77 was added to a concentration of 0.005 – 0.02 % to reduce surface tension. Inflorescences were dipped briefly in the Agrobacterium solution and were then stored at low light conditions under a cover for 16 – 24 h to maintain high humidity. Then, plants were placed into a climate chamber with long-day conditions to set seeds.

Materials and Methods

2.2.1.6 Selection of stably transformed Arabidopsis plants 2.2.1.6.1 Basta^® selection on soil

Selection of stably transformed, Basta^®-resistant Arabidopsis plants was performed using Basta^® solution (200 g/l glufosinate [phosphinothricin ammonium], Bayer CropScience AG, Monheim, Germany). For this purpose, T1 seeds were sown on soil and allowed to germinate covered with a plastic lid. One week after germination, seedlings were sprayed with a 1:1000 diluted Basta^® solution. This was repeated three times in two day intervals. Successfully transformed seedlings were resistant and thus survived the Basta^® treatment. The transformants were picked and transplanted into fresh single pots.

2.2.1.6.2 In vitro selection of Arabidopsis transformants

In vitro selection was carried out to analyze the segregation pattern of transgenic Arabidopsis T2/T3 plants. For this purpose, sterilized seeds were spread onto ½ MS agar plates containing either 25 μg/ml phosphinothricin (PPT), 50 μg/ml kanamycin (Kan) or 50 µg/ml hygromycin (Hyg) as selection markers. Seedlings were grown under short-day conditions until transformed seedlings clearly differed from non-resistant seedlings. Transformants were picked and transferred onto soil for further propagation (see 2.2.1.3).

2.2.1.7 Treatment of Arabidopsis thaliana leaves with elicitors and inhibitors

For assaying phosphorylation of CERK1, LYK5, LYK4 or MAPKs, 100 µg/ml chitin was vacuum-infiltrated into detached leaves of 6-8 week-old plants using a plastic desiccator and incubated for 10 min. For phosphorylation time course experiments, the incubation times are indicated in the figures. For quantitative real-time PCR experiments, chitin was added to the liquid growth medium of 2 week-old in vitro grown seedlings. The final chitin concentrations and incubation times varied and are indicated in the respective figures and legends. Treated plant material was then blotted dry and transferred into a new tube before being frozen in liquid nitrogen. The samples were either directly used for protein extraction or stored at -80°C.

For confocal microscopy, chitin or flg22 (EZBiolab) were vacuum-infiltrated into leaf pieces of preferably 4-6 week-old plants using a syringe. Polymeric chitin was used at a concentration of 100 µg/ml and flg22 at 1 µM. If not otherwise indicated, the incubation time was 60 min. For pharmacological studies, leaf pieces were pre-incubated in inhibitor solution for 30 min and then the inhibitor solution with or without 100 µg/ml chitin was vacuum-infiltrated. For K252a the pre-incubation step was omitted. The endocytic tracer dye FM4-64 (SynaptoRed) was purchased

from Sigma-Aldrich and a 10 mM stock solution was prepared in DMSO. Leaf pieces were incubated in 5 µM FM4-64 for 15 min prior to microscopy or additional treatments. For callose the leaf discs were incubated for 15 min in aniline blue staining solution after elicitor treatment.