RNA Detection

linearized constructs can be transformed intoU. maydisproto-

plasts. A detailed visual protocol showing the procedure is available

[32]. In brief:

1. Inoculate a preculture in 3 mL CM-glc and incubate it on a
   rotating wheel for 24 h at 28C.


2. Inoculate a main culture in 50 mL CM-glc and allow growth
   until exponential phase (OD 600 of around 0.8).


3. Pellet cells for 5 min, 1500gand wash the pellet in 25 mL
   SCS.


4. Resuspend the pellet in 2 mL protoplasting solution and incu-
   bate cells for 5–20 min at room temperature (RT). Check
   protoplasting process under the microscope and stop process
   when 30–40% of the cells are round or resemble pinheads.


5. Wash 3in 10 mL cold SCS, centrifuge at 1000gfor 5 min.
   Keep protoplasts on ice.


6. Wash once in 10 mL cold STC and resuspend the pellet in 1 mL
   cold STC. Make 100μL aliquots in prechilled tubes and freeze
   them at 80 C until further use.


7. For transformation prepare two-layered selection plates
   (12 mL for each layer). Bottom layer contains RegLight agar
   with antibiotic, e.g., hygromycin (double amount of the usual
   concentration; 400μg/mL), the upper layer contains only
   RegLight agar.


8. Thaw protoplast on ice and add 1μL heparin (15 mg/mL) and
   1 μg linearized plasmid. Incubate for 10 min on ice.


9. Add 500μL STC/PEG to the transformation tube. Incubate
   for 15 min on ice.


10. Distribute cells on two transformation plates.


11. Incubate plates at 28C for 5–10 days.


12. To confirm correct transformants genomic DNA of potentially
    positive candidates are extracted and homologous integration
    events need to be verified by Southern blot experiments [32,
    33 , 35].

3.3 Live Cell Imaging
of a Highly Motile RNA-
Binding Protein

RBPs are key components orchestrating mRNA transport. There-

fore, analyzing the localization of motile RBPs like Rrm4 fromU.

maydiscan give a first insight into the transport machinery of

mRNAs.

1. To visualize the subcellular localization of Rrm4, fuse the
   coding sequence of GFP C-terminally to Rrm4 using standard
   cloning techniques [33, 34](seeNote 2).


2. Do not alter the endogenous promoter to avoid overexpression
   artifacts. This is achieved by designing the integration event
   downstream of the coding sequence.

RNA Live Imaging inU. maydis 325