Working with bacteria and fungi

2.2.2.1 Glycerol stocks of Verticillium spore cultures

Spores were harvested as described in 2.2.2.2 from a Verticillium liquid culture under sterile conditions. Spore suspension was not adjusted to a specific concentration. 750 µl of Verticillium spore suspension were mixed with 250 µl of 60 % (v/v) glycerol and mixed by pipetting up and down. Immediately, spore suspension was frozen in liquid nitrogen and stored at -80 °C.

2.2.2.2 Cultivation of Verticillium

180 ml Simulated Xylem Sap (SXM) supplemented with 250 mg/ml cefotaxime and 100 mg/ml ampicillin were inoculated with Verticilliumspore suspension from a glycerol stock (a frozen piece of∅ 0.5 cm). The culture was grown in the dark in Baffled Erlenmeyer flasks for 1 week at 22 °C and 90 rpm on a rotary shaker Subsequently, spores were harvested by filtering the culture through a filter paper. The filtrate was spun down for 15 min at 4 °C and 4000 rpm.

Afterwards, the supernatant was discarded and the pellet was washed with 30 ml sterile H2O.

Finally, the pellet was resuspended in 5 ml sterile H2O and spore concentration was determined with a Thoma counting chamber, if spores were used for infection experiments.

injured by twirling in order to facilitate fungal penetration. Plant roots were incubated for 45 min in Verticillium spore suspension (titer of 1 · 10⁶ spores/ml) or mock treated with H2O.

Finally, infected plants were transferred into single round pots (Ø 80 mm, steam-sterilized soil) and further cultivated under short day conditions.

2.2.2.4 Verticillium infection of in vitro grown A. thaliana

A. thaliana were grownin vitro on ½ MS agar as described in section 2.2.1.1 for 2 ½ weeks.

Subsequently, plants were transferred to angled 1 % (w/v) agarose plates and incubated for 1 day in a short day growth chamber. During and after infection, A. thaliana plants remained on 1 % agarose plates. Due to a lack of nutrients, 1 % agarose avoids saprophytic growth of Verticillium on plates and supports infection of the A. thaliana root system. Root system ofin vitro grown plants was spray inoculated with Verticillium spore suspension at a titer of 1 · 10⁵ spores/ml or mock treated with H2O. After infection, agarose plates were sealed with Millipore tape and lower part of plates was covered with aluminium foil, leaving photosynthetically active plant tissues accessible to light. Plants were transferred into a short day growth chamber. Finally, A. thaliana root system was either subjected to confocal laser scanning microscopy 2 days post infection (dpi) or harvested at 4 dpi and subjected to RNA extraction.

2.2.2.5 Botrytis cinerea drop inoculation of A. thlaiana

For infection experiments a spore stock of the B. cinerea strain B05.10, provided by M. Wiermer (University of Göttingen, Germany), was used. Spore stocks containing a titer of 2.5 ∙ 10⁶ spores/ml were stored at -80° C and thawed on ice before use. Spore stocks were diluted in ¼ Potato Dextrose Broth (PDB, Sigma-Aldrich, St. Louis, USA) to a titer of 5 ∙ 10⁴ spores/ml. In order to allow germination, spore suspension was incubated for 4 h at room temperature. Fully expanded leaves of 5-week-old A. thaliana were inoculated with 6 µl droplets ofB. cinerea spore suspension. Plants were propagated in a Percival^® growth chamber under short day conditions in fully sealed table top greenhouses, in order to ensure high humidity. After 3 days, necrotic lesion diameter was measured using a digital caliper MarCal 16ER (Mahr, Göttingen, Germany).

2.2.2.6 Hyaloperonospora arabidopsidis maintenance and spray inoculation of

A culture of H. arabidopsidis isolate NOCO2 was maintained on soil grown seedlings of its compatible hostA. thaliana Col-0. For maintenance, culture was transferred every 7 days onto new seedlings. For spray inoculation, infected seedlings were harvested at 7 dpi into 50 ml Falcon tubes and vortexed in H2O. Subsequently, plant material was separated from the spore suspension by filtering. Concentration of the spore suspension was determined using a Thoma counting chamber and adjusted to 5 ∙ 10⁴spores/ml. 2-week-old seedlings grown under short day conditions were spray inoculated with H. arabidopsidis spore suspension. Finally, plants were transferred into a Percival^® growth chamber (CLF Plant Climatics, Wertingen, Germany) under short day conditions (10 h light at 18° C, 14 h dark at 18° C, 65 % relative humidity) and propagated in tightly sealed table top greenhouses to ensure high humidity.

2.2.2.7 Quantification of Hyaloperonospora arabidopsidis propagation

35-45 seedlings of the analysedA. thaliana genotype were inoculated in 4 biological replicates and propagation of H. arabidopsidis was quantified at 6 dpi. For this purpose spores were harvested from spray inoculated seedlings as described in section 2.2.2.6. In order to obtain the number of spores per g plant tissue the weight of harvested plant material was determined before addition of H2O. Thereafter, 10 µl H2O were added per mg plant tissue. The number of spores was determined using a Thoma counting chamber. Counting was performed 4 times per biological replicate and mean value used for calculation of spore concentration.

2.2.2.8 Pseudomonas syringae vacuum infiltration of A. thaliana

Pseudomonas syringae pv. tomato (Pst) DC3000 ΔAvrPto/AvrPtoB was maintained on selective NYG agar plates containing 50 µg/ml Rifampicin and Kanamycin. Agar plates were Pst DC3000 ΔAvrPto/AvrPtoB was transferred

shaker at 28° C and 190 rpm. Next morning, 4 ml of this culture were used for inoculation of 50 ml of fresh NYG-medium containing selective antibiotics. This culture was incubated under same conditions for 3 h, in order to ensure that bacteria used for vacuum infiltration of A. thaliana were in the log growth phase. Afterwards, bacterial culture was spun down at 4000 rpm for 20 min and resuspended in 50 ml 10 mM MgCl2. OD of the culture was measured and adjusted to an OD600= 0.0002 (1 ∙ 10⁵c.f.u/ml). One square plastic pot containing 5- to 6-week-old A. thaliana was inverted and placed into a desiccator filled with 600 ml bacterial suspension. Vacuum was applied for 1 min 45 sec and maintained for further 15 sec. The desiccator was shaken gently during application of vacuum in order to release air bubbles from plant leaves. Thereafter, plants were washed in a water bath containing tap water.

Non-infiltrated leaves were removed using forceps.

Day 0 (d0) samples were harvested in duplicates from 4 leaves of independent plants. 4 leaf discs (∅ 0.55 cm) were macerated in a 1.5 ml microcentrifuge tube containing 100 µl 10 mM MgCl2, subsequently diluted 1:10 and 50 µl of macerated plant tissue were plated on selective NYG agar plates. Vacuum infiltrated plants were transferred into a short day Percival^® growth chamber. Day 3 (d3) samples were taken in triplicates. 4 leaf discs (∅ 0.55 cm) were macerated in a 1.5 ml microcentrifuge tube containing 100 µl 10 mM MgCl2. A 10^-1 to 10^-7 dilution series was prepared and 10 µl of each dilution pipetted o a selective NYG agar plate using a multichannel pipette. Finally, agar plates were incubated for 2 days at 28° C and Pst DC3000 ΔAvrPto/AvrPtoB c.f.u. were counted.

2.2.2.9 Transformation of chemically competent E. coli

50 µl aliquots One Shot^® TOP 10 chemically competent E. coli cells (Invitrogen^™, Karlsruhe, Germany) were used. Aliquots were stored at -80° C and thawed on ice before use. 10 to 25 ng plasmid DNA were added to theE. coli aliquot and incubated on ice for 30 min. Subsequently, the reaction mix was transferred into a preheated 42° C thermomixer for 45 sec and immediately cooled down on ice for 5 min. Afterwards 800 µl LB medium were added and cells incubated for 1 h at 37 °C on a rotary shaker. The suspension was spun down at 2500 g, the supernatant removed and pellet resuspended in 50 µl LB medium. Finally, E. coli cells were plated on selective LB agar plates and incubated over night at 37° C.

2.2.2.10 Transformation of electro competent A. tumefaciens

40 µl aliquots of electro competentA. tumefaciens cells were diluted 1:3 with dH2O and mixed with 50 ng of plasmid DNA. Subsequently, cells were transferred into a precooled electroporation cuvette. The BioRad Micro Pulser^™electroporation apparatus was set to 25 µF, 2.5 kV and 400 Ω. Immediately after electroporation, cells were transferred on ice and cuvettes filled with 1 ml precooled DYT medium. Cells were resuspended, pipetted into 2 ml microcentrifuge tubes and incubated in a thermomixer for ca. 3 h at 28° C and 600 rpm. Finally, 5 µl and 50 µl aliquots of the transformed cells were plated on selective DYT- agar plates and incubated for 2 days at 28° C.

2.2.3 Biochemical methods