Use of Plants and Fungal Spores

3.1.1 Plant Cultivation

To insure the availability of suitable host plants for the experiments carried out during this thesis, Broad Bean seeds (Vicia faba cv. Con amore, Nickerson Zwaan, Edemissen, D) were planted in steam-treated soil and cultivated in a climate chamber (Lichtthermostatschrank, Laborgerätebau E. Schutt, Göttingen, D) for 21 days (21°C; 230 µEm^-2S^-1) with a 16 h photoperiod. Thereafter they were transferred to the greenhouse and subjected to a varying number of day/night cycles (16 h light / 8 h darkness).

3.1.2 Inoculation and Harvesting Methods

To generate the necessary amount of infected Vicia faba leaves, 21-day-old Broad Bean plants were spray-inoculated with Uromyces fabae uredospores (race I2) (Deising et al. 1991).

The suspension was prepared by mixing 500 ml of deionized water with 350 mg fat-reduced milk powder and 250 mg uredospores. The suspension was evenly sprayed onto the plants. In case only individual leaves needed to be inoculated, the uredospores were applied with a brush.

The plants were then kept in the dark for 24 h at 100% humidity and allowed to dry for at least 12 h under ambient conditions without extra illumination. Thereafter the plants were subjected to a varying number of day/night cycles (16 h light / 8 h darkness). New shoots and buds were removed at least every three days. This prevented the plants from shedding their infected leaves. Those leaves required for further processing were subsequently harvested using a razor blade.

3.1.3 Isolation of Haustoria from Uromyces fabae

Isolation of haustoria from Uromyces fabae was performed according to Hahn and Mendgen (1992). Five to seven days after infection, 25 g of heavily infected leaves without visible uredospores on the leaf surface were gently washed in cold, deionized water to remove spores and mycelium from the surface. Because most of the haustoria were used for RNA preparation, the following steps were carried out in the cold storage room with pre-cooled solutions and, if possible, on ice.

After transfer to the cold storage room, leaves were first homogenized with 160 ml Homogenization buffer using a Warring blender (max. rpm, 15 sec.). To remove the debris, the leaf homogenate was filtered through nylon tissue with 20 µm pore size and rinsed once more with 20 ml Homogenization buffer. The filtrate was then centrifuged for 5 min (4°C, 5’000 g), and the resulting pellet, consisting mainly of chloroplasts and haustoria, was brought into suspension with 8 ml Suspension Buffer resulting in a concentration of approximately 1 to 1.5 x 10⁹ haustoria/ml.

Accumulation of haustoria was carried out with Concanavalin A bound to a Sepharose 6MB column (Hahn and Mendgen 1992). To prepare the column, 5 g Sepharose 6 MB Megabeads (Amersham Bioscience Europe GmbH, Freiburg, D) were washed with 1 mM HCl for 15 min, before being rinsed with Coupling Buffer. 12 ml of this gel were mixed with 75 mg Concanavalin A in 12 ml Coupling Buffer and put on a roller over night at 4°C. Afterwards, the Sepharose gel was washed thoroughly with Coupling buffer and the remaining active groups were blocked with Blocking Buffer (see Table 3-2) for 2 h.

After the column had been prepared, haustoria (and chloroplast) suspension was applied until it was dark green. The haustoria were then allowed to bind to Concanavalin A for about 10 minutes. The column was washed with Suspension Buffer until clear and the binding step was repeated with new haustoria and chloroplast suspension. After the entire suspension had been applied to the column and the excess chloroplasts had been washed away, the matrix was vigorously dispersed with 2 ml Suspension Buffer to remove the haustoria from the Sepharose beads and to bring them into suspension. The haustoria in the Suspension buffer were then transferred to an Eppendorf tube and centrifuged for 5 min at 12’000 rpm and 4°C. The supernatant was discarded, while the pellet was either processed directly or frozen in liquid nitrogen and stored at –70°C.

Table 3-1 Homogenization- and Suspension Buffer for Haustoria Isolation (Hahn and Mendgen 1992)

Homogenization Buffer: Suspension Buffer:

300.0 mM Sorbitol 300.0 mM Sorbitol

20.0 mM 3-morpholinopropansulfonic acid, pH 7.2

10.0 mM 3-morpholinopropansulfonic acid, pH 7.2

0.1% BSA 0.2% BSA

0.2% β-Mercaptoethanol 1.0 mM KCl

0.2% PEG 6000 1.0 mM CaCl2

1.0 mM MnCl2

Table 3-2 Coupling and Blocking Buffer for Haustoria Isolation (Hahn and Mendgen 1992)

Coupling Buffer pH 8.6 Blocking Buffer pH 8.3

0.1 M NaHCO3 0.1 M NaHCO3

0.5 M NaCl 0.5 M Glycine

3.1.4 Preparation of Uromyces fabae in vitro Structures Germ tube preparation:

Germ tubes are U. fabae infection structures that can be easily isolation in a pure fraction.

After 4 g Uredospores of U. fabae had been washed for 30 min in 1.5 l of deionized water with 0.01% Tween20, they were allowed to grow in the dark in 4 l of deionized water with 0.01% Tween20 for 3 to 5 h. In order to ensure a permanent oxygen supply, the spores were mixed at high speed using a drilling machine with an attached flexible tube instead of a drill bit (Hahn and Mendgen 1997). The spores were checked for germination under a microscope and if germination was at least 80%, the spores and germ tubes were vacuum filtered through two layers of filter paper in a Buchner funnel. The nearly dry spores were then collected for further use.

Preparation of other infection structures (germ tube to haustorial mother cell):

It is possible to produce Uromyces fabae infection structures on artificial surfaces (Deising et al. 1991). This approach is useful for isolating specific infection structures (such as appressoria, penetration hyphae, substomatal vesicle, infection hyphae and haustorial mother cells). It must be noted that with this approach, individual infection structures are not isolated in pure fractions but in fractions were one of the infection structure stages is predominant.

In preparation, Polyethylene foil of the type Polystar® 300 x 0.10 mm (Rische + Herfurth GmbH, Hamburg, D) were heated to 35°C for a duration of 8 weeks and stored under ambient conditions for several months to allow for the dissipation of volatile additives. The thus prepared Polyethylene foil was then introduced into the base of an inoculation tower, where 400 mg of uredospores were subsequently dispersed using compressed air. After the spores were allowed to settle on the PE foil for 20 min, the foil was moistened with about 4 ml H2O and incubated in a dark room (19°C, 100% humidity) for a defined period (4 h to 24 h). The subsequent formation and presence of the specific infection structures (appressoria, penetration hyphae, substomatal vesicle, infection hyphae and haustorial mother cells) was verified and quantified using light microscopy.

3.1.5 Bean Leaf Infiltration Experiments

Infiltration experiments were carried out to study what effect the introduction of antibodies against the studied PIGps into the plant has on the development of the infection. For this purpose, 3 to 4 week old broad bean plants (Vicia faba) in different stages of U. fabae infection were used. A solution containing antibodies against the PIGps studied during this thesis in a dilution of 1/10 and 1/100 was injected into one leaf per plant using a syringe with a fine needle. After about 12 h under ambient conditions without extra illumination, the plants were transferred to a green house. Thus treated plants were subjected to a varying number of day/night cycles (16 h light / 8 h darkness) before being studied and compared for variation in infection development.