Category Archives: Protocols

Protocols, Methods, Recipes, Tips

Protocol to produce and quantify conidial anastomosis tubes (CAT) fusion in Colletotrichum lindemuthianum

Francine H. Ishikawa and Gabriela Roca

francine.hiromi@univasf.edu.br

G.Roca@tu-braunschweig.de

Background

Conidial anastomosis tubes (CATs) are specialized cell-type that allows fusion between conidia. Colletotrichum lindemuthianum naturally undergoes CAT fusion on the bean plant leaf surface (Ishikawa et al., 2010a) and within asexual fruiting bodies in anthracnose lesions on its host (Roca et al., 2003). We have being using the following protocol to produce and analyse CAT fusion in vitro with robust results (Ishikawa et al., 2010b) (Fig 1). CAT fusion in this pathogen was found to exhibit significant differences to the model system Neurospora crassa (Ishikawa et al., 2010b). In contrast to N. crassa, CAT fusion in C. lindemuthianum is inhibited by nutrients (it only occurs in water) and the process takes considerably longer, conditions that are not commonly used experimentally.

Fig 1. CAT fusion of LV115 strain of C. lindemuthianum in water.  The conidia were harvested from 16 day old bean pod agar cultures. (A) After 24 hrs of incubation (B) After 72 hrs. Arrows indicated fusions between conidia/conidial germlings via CATs. Note the appressorium formation (asterisk). Bar: 10µm.

Protocol

1)           Grow Colletotrichum lindemuthianum for sporulation. We use glass tubes containing autoclaved French bean pods submerged in 2% water agar (bean pod agar medium). Incubated in the dark at 22ºC.

2)           Collect conidia from 10-16 day old tube cultures. CAT fusion is strain dependant, so we suggest to use 15 days old spores to start and the culture age could be adapted depending of the results obtained. Our previous results showed that older conidia fuse more frequently and quicker than younger conidia, however, timing of CAT fusion is strain dependent – (see Ishikawa et al. 2010b for more). Suspend them in water. First fill an Ependorf tube with 1 ml of sterilized water, then collect the conidia (pink mass) and mix in the water. Note the time. Your measurement of CAT fusion will be 24 hours after this point.

3)           Rapidly shake the Eppendorf tubes on vortex for a few seconds to produce a suspension of dissociated conidia.

4)           Measure the concentration of conidia in your suspension.

5)           Diluted your suspension to a concentration of 2 x 106 conidia/ml (conidial work suspension). Ensure that you have at least 600 µl of suspension.

6)           Place 200 µl of conidial suspension into 3 separate compartments of a slide culture chamber (we use an eight-well slide culture chamber from Nalge Nunc International, Rochester, NY). You will therefore have 3 x 200 µl samples to analyse.

7)           Place the culture chamber in an incubator in the dark at 22ºC until the first measurement to be made after 24 h incubation. C. lindemuthianum germination and CAT formation is slow, thus the next measurement point could be after 12-24 h. If the objective is to follow all process of CAT fusion, you may need to start between 24-48 h of incubation . Higher number of fusions (chain of fused conidia) is observed after 72 h of incubation (Fig. 1 B).

8)           Examine the samples at room temperature by using brightfield or differential interference contrast optics with a 60x objective . We need an inverted microscope because our cover glass slide are on the bottom. If you have and upright microscope you would need to adapt the slide sample preparation

9)           Quantify CAT fusion as the percentage of conidia or conidial germlings involved in fusion. For each strain and time point, use 3 samples (the 3 chambers you filled with conidial suspension) and quantify 100 conidia in each sample (n=300). You can record 10-15 images per sample condition for subsequent analysis. Repeat these three times (i.e. another days but use always the same conidial age). Estimate mean and standard deviation (SD).

10)        Incubate the samples at 22°C between measurements. Be very careful not to shake samples when transferring them between the incubator and microscope as this may perturb CAT fusion.

11)        Produce a graph of conidial germination and CAT fusion at different time points.

References

Ishikawa F.H., Barcelos Q.L., Alves E.A., Camargo Jr. O.A., Souza E.A., 2010a. Symptoms and pre-penetration events associated with the infection of common bean by the anamorph and teleomorph of Glomerella cingulata f. sp. phaseoli. Journal of Phytopathology 158: 270-277.

Ishikawa F. H., Souza E. A., Read N. D, Roca M..G., 2010b. Live-cell imaging of conidial fusion in the bean pathogen Colletotrichum lindemuthianum. Fungal Biology, 114: 2-9.

Roca M.G., Davide L.C., Mendes-Costa M.C., Wheals A., 2003. Conidial anastomosis tubes in Colletotrichum. Fungal Genetics and Biology 40:138-145.

Protoplast Preparation

Here is a protocol for preparation of protoplasts. I first used it for C. graminicola while I was a postdoc in Lisa Vaillancourt’s lab at the University of Kentucky, but we’ve made a few modifications to it since then.

FCG LAB, 2005

1. Harvest conidia from 2 or more plates that are 3-4 weeks old, center inoculated. Expect about 1×108 conidia per plate.
2. Wash conidia once with sterile water. Resuspend the last time in Fries medium.
3. Count with hemacytometer.
4. Incubate in Fries medium for 48 hours with shaking at 30C. I use a 100 mL volume culture in a 250 mL Erlenmyer flask and shake at 150 to 175 rpm. Apparently if the culture receives too much aeration the culture will be induced to vegetative growth rather than the production of oval conidia.
5. Filter through Nytex membrane.
6. Count with a hemacytometer. Expect about 20X to 25X the number of starting falcate conidia.
7. Pellet in 50 mL centrifuge tubes in a bench-top centrifuge, 3000 rpm, 5 min at room temp. Resuspend in enzyme solution to a conidium concentration of 1.5×108/mL.
8. Incubate at 30C with slow shaking for 4-5 hours.
9. Pellet in a bench-top centrifuge, 3000 rpm, 5 min, 4C. Resuspend in 40 mL STC, ice cold. Keep the protoplasts on ice for the rest of the procedure. Count with a hemacytometer.
10. Pellet as in the previous step. Resuspend in STC medium to a concentration of 108/mL.
11. Divide into aliquots and freeze at -80C. I usually use 500 uL aliquots, which is enough for 5 transformations.

Enzyme solution
Glucanex*, 100 mg/mL dissolved in 0.7M NaCl.
Filter through a 0.2 uM filter before use. The solution will have a high solids content so only 6-7 mL of this solution can be passed through a 0.2 uM syringe filter.

*in the United States:
Novo Nordisk Biochem NA
State Road 1003 Box 576
Franklington NC 27525
Phone 800-879-6686
(note – this is no longer available directly from Novo Nordisk. Lysing Enzyme from Sigma (Catalog # L1412) is reported to be the same product.

STC 50 Ml

1.2 M Sorbitol (10.9 g)
10mM Tris (500 uL of a 1 M stock)
50mM CaCl2 (2.5 mL of a 1 M stock)
Adjust to pH 7.5