RNA Detection

5. Nitrate minimal medium (NM): 0.3 w/v % KNO 3 , 6.25 v/v %
   salt solution, add deionized water, adjust pH with 5 M KOH to
   7.0, add glucose (glc) or arabinose (ara) solution after auto-
   claving (1% f.c.).


6. Sodium citrate sorbitol solution (SCS): Solution 1: 20 mM tri-
   Na-citrate, 1 M sorbitol; Solution 2: 20 mM citric acid, 1 M
   sorbitol; add solution 2 to solution 1 until pH 5.8 is reached,
   autoclave.


7. Sorbitol, Tris–HCl, CaCl 2 (STC): 1 M sorbitol, 10 mM
   Tris–HCl pH 7.5, 100 mM CaCl 2 , sterile filter.


8. STC/PEG: 40 v/v % PEG 3350 in STC-buffer.


9. RegLight: 1.0 w/v % yeast extract, 0.4 w/v % Bacto peptone,
   0.4 w/v % sucrose, 18.22 w/v % sorbitol, 1.5 w/v % agar, add
   deionized water and autoclave.


10. Selection antibiotics, e.g., hygromycin.


11. Protoplasting solution: 12.5 mg/mL Trichoderma lysing
    enzymes in SCS, filter-sterilize through a 22μm filter, solution
    has to be fresh for optimal enzymatic activity.


12. Glass reaction tubes, baffled flasks 250 and 100 mL, petri
    dishes, rotation wheel, shaker.

2.4 General
Microscopy Equipment
for Live Cell Imaging of
U. maydis [31]

1. Wide-field fluorescence microscope. We use a Zeiss Axio
   Observer.Z1 in combination with a Photometrics CoolSNAP
   HQ2 CCD camera.


2. Laser illumination to excite GFP (488 nm/100 mW) or
   mCherry (561 nm/150 mW).


3. 63NA 1.4 or 100NA 1.3 objectives.


4. Filter sets for GFP: ET 470/40, ET 252/50 m, T495_PXR.


5. Filter sets for mCherry: ET 560/40, ET 630/75 m, T585lp.


6. For dual-color microscopy we use a VS-LMS4 Laser Merge-
   System (Visitron Systems).


7. MetaMorph (Molecular Devices, version 7) is used to control
   the microscopes, to process and analyze the acquired images.


8. Microscope slides with 7626 mm (e.g., Marienfeld).


9. High precision coverslips with 1818 mm,D¼ 170  5 μm
   (e.g., Zeiss).


10. 3 w/v % melted agarose (e.g., Bio-Rad).

3 Methods

An important requirement for the detection of mRNAs in fungal

model systems is the stable integration of heterologous RNA stem-

loops in the 3^0 UTR of the mRNA of interest (Fig.1b). This can be

RNA Live Imaging inU. maydis 323