Materials and Reagents

Petri dishes (6 cm and 9 cm diameters, ASONE)
Lens paper (Fisher Scientific)
Filter paper (Whatman)
Medical gauze (Angyang Medical)
Absorbent paper (Kimberly-Clark)
1.5 ml tubes (Eppendorf)
Tips (10 μl, 200 μl and 1,000 μl, Axygen)
Blades (Dexter Russell Cutlery, catalog number: 73-C )
Glass slides (Fisher Scientific, catalog number: 12-550-343 )
Coverslips (Fisher Scientific, catalog number: 12-547 )
Pots (10 cm in diameter and 15 cm in height)
Cultivated land soil
Magnaporthe oryzae strains
Note: The M. oryzae strains are maintained on dried filter paper pieces stock in -80 °C for long-term storage.
Barley seeds (Hordeum vulgare, cv E9)
Sterilized water (Milli-Q)
Boiled oatmeal filtrate
Tomato juice
Agar (Sigma-Aldrich, catalog number: 17209 )
Tween 20 (Sigma-Aldrich, catalog number: P9416 )
Oatmeal tomato agar (OTA) solid media (see Recipes)
0.025% Tween 20 (see Recipes)

Equipment

Scissor, tweezers, spreader
Pipettes (Eppendorf, catalog numbers: 3124000016 [0.5-10 µl], 3124000032 [20-200 µl], and 3124000075 [100-1,000 µl])
Incubation chamber (28 °C) for fungal growth and barley germination (Biolab Scientific, model: BIFG-101 )
Hemocytometer (Marienfeld, catalog number: 0650030 )
Greenhouse capable of temperature and humidity control for growing barley
Optical microscope (Olympus, model: CX23 )
Fluorescence microscope (Leica Microsystems, model: Leica DM2500 )
Juice extractor
Autoclave

Procedure

Preparation of M. oryzae (Figure 1)

Figure 1. Preparation procedure for conidia suspension of M. oryzae
1. Inoculate the M. oryzae strains into solid OTA (Recipe 1) plates (6 cm diameter) and incubate for one week in an illumination incubator at 28 °C.
2. Add 2 ml sterile distilled water containing 0.025% Tween 20 (Recipe 2) to each OTA plate and scrape with a spreader to harvest conidia. Use two layers of sterile lens paper to filter and remove hyphal fragments. Upon filtering, brief vortex by pipetting to re-suspend conidia.
  Note: Tween 20 will help to increase water hydrophobic property, therefore promote the formation of the water drops without collapse.
3. Determine the conidia concentration by using a hemocytometer under an optical microscope, and adjust the final concentration to 1 x 10⁵ per ml of 0.025% Tween 20 sterile water.
Preparation of barley leaves
1. Put barley seeds (around 50 seeds) into a Petri dish with an appropriate volume of sterile water and place the Petri dish in an incubation chamber at 28 °C for 12 h.
2. Remove the water, and wash the seeds for several times with sterile water, then hung up in one layer gauze to dry for 6 h.
3. Put the seed into the Petri dish (9 cm diameter), and immerse the seeds with an appropriate volume of sterile water, then cover several layers of wet gauze. Place the Petri dish in an incubation chamber for 12-18 h at 28 °C.
4. After the seeds germinate, transfer them into pots (10 cm in diameter and 15 cm in height) containing 80% volume of cultivated land soil (20-30 seeds per pot). Place the pots in a greenhouse for growth at room temperature. Water the pot every two days to keep the soil wet. After one week, the barley leaves can be used for inoculation (Figure 2).
  
  Figure 2. Procedure for inoculation of M. oryzae on barley leaves
Inoculation of barley with M. oryzae (Figure 2)
1. Cut the barley leaves from the base with a scissor, then put them (back side up) in a Petri dish (9 cm diameter), which have been covered with two layers of water immersed absorbent paper. Fix the leaf base and tip with water immersed absorbent paper, and keep leaf flat (Video 1).
  Note: When fix the leaves base and tip with water immersed absorbent paper, keep the leaves dry and don’t touch the leaves with fingers or any other materials.
  
  Video 1. Put barley leaves into the Petri dish and fix with water immersed absorbent paper
2. Drop the conidia suspension onto barley leaf beside the vein using a pipette (0.5-10 μl range) (Video 2).
  Note: Keep each drop with a diameter of around 2-3 μm and without collapse, if the drop is too large or collapsed, it may tend to the formation of aerial mycelium, but not appressorium. Keep the tips away from the epidermis could avoid collapse. Re-suspending conidia by shaking up and down of the tubes every time before absorbing the conidia suspension.
  
  Video 2. Drop conidia suspension onto the barley leaves
3. Put on the lid, cover the Petri dishes with wetted absorbent papers, and place them into a sealed moist box. Then put the box in a dark incubator at 28 °C.
Infection process observation (Figure 3)
1. Takeout the barley leaves with a tweezer at different time points after inoculation. Use a blade to cross cut the leaf from up to down, at the tip side or base side. Keep the lower epidermis still connected, and subsequently tear down the lower epidermis (Figure 3A, Video 3).
  Note: During this process, try best not to touch the water drops, which could break the infection structures. Recommend observing penetration pegs and primary infection hyphae at 18 hpi, and secondary infection hyphae at 24-30 hpi.
  
  Video 3. Tear down the barley lower epidermis and put onto a glass slide
2. Put the lower epidermis onto a glass slide, add some water to immerse the epidermis, then cover it with a coverslip.
3. Observe the infection processes of M. oryzae in the cells of epidermis under a microscope, take images and calculate formation ratios of different infection structures (Figure 3A). For each time, more than 50 conidia should be calculated.
4. Observe the subcellular localization of GFP-tagged proteins in infection structures under a fluorescence microscope (Figure 3B).
  Note: During observing, remember to add water when the sample is dried.
  
  Figure 3. Procedure for observing of different samples. A. Procedure of observing the infection process. B. Observation of protein subcellular localization. DIC, differential interference contrast; GFP, green fluorescent protein. Scale bars = 20 μm.

Data analysis

All information about data processing, statistical tests, replicates and independent experiments were already included in the original research paper (Chen et al., [2014], N-glycosylation of effector proteins by an α-1,3-mannosyltransferase is required for the rice blast fungus to evade host innate immunity. Plant Cell 26(3):1360-1376. doi: 10.1105/tpc.114.123588).

Notes

Plant healthy is very important for the success of the infection assay. Barley plant should grow under adequate sunshine, and avoid soil drought. Otherwise, the barley epidermis can’t be well infected with M. oryzae.
Accurately addition of 0.025% Tween 20 into the sterile water is important for successful inoculation. The tips should be cut off before absorbing the reagent. Absorb the Tween 20 slowly and the adhering reagent on tips should be removed.
To prevent the drops from collapse during inoculation steps, the Petri dish should be moved as slowly as possible.

Recipes

Oatmeal Tomato Agar (OTA) solid media (1 L)
40 g boiled oatmeal filtrate
150 ml tomato juice
20 g agar
1. Boil the oatmeal in 800 ml distilled water for 30 min, then filtrate the mixture by double-layer gauze
2. Extract tomato juice by an extractor, then also filtrate the mixture by double gauze. Mix 150 ml tomato juices with boiled oatmeal filtrate, then add water to 1 L
3. Add 20 g agar, and autoclave for 40 min
0.025% Tween 20
Add 250 μl Tween 20 in 800 ml sterilized distilled water and adjust the volume to 1 L

Acknowledgments

The protocol was adapted from Chen et al. (2014). This work was supported by grants from the National Natural Science Foundation of China (31571952, 31601585). The author declares no conflict of interests.

References

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