achieved by integration at the endogenous locus using standard
techniques adapted from model fungi [32–34]. In addition, the
corresponding RBP fused to a fluorescence protein needs to be
coexpressed. For this, we recommend ectopic expression at defined
loci inU. maydis[35]. To ensure high quality RNA live imaging,
the expression level of the RBP has to be adjusted by using suitable
promoters. To achieve a high signal-to-noise ratio during RNA live
imaging, the RBP can be fused to an NLS to remove cytoplasmic
background fluorescence (seeSubheading3.5).3.1 Cloning of the
Reporter Constructs
Mainly three systems are used to visualize mRNAs in living cells:
The MS2, PP7 andλN system (Fig.1c). All three systems were
applied for the visualization ofcdc3mRNA inU. maydis.- To visualizecdc3 mRNA in vivo two plasmids have to be
generated. The first one encodes the RBP coupled to a fluores-
cence protein. The other plasmid contains cognate stem-loops,
integrated into the 3^0 UTR of the gene of interest (GOI). - To construct the MS2 and PP7 containing plasmids for RNA
live imaging the already availablecdc3-16boxB/λN system was
used [13, 18]. As an example, the construction of the PP7
system is described in the following steps. - The plasmid “pmax pona 12xTRICK 24xMS2SL” was used for
the amplification of 12 PP7 stem-loops. To amplify the PP7
stem-loops the PCR oligonucleotides PP7loops-SacII-Fwd
and PP7loops-SacII-Rev can be used. For cloningSacII restric-
tion sites were integrated upstream and downstream of the
stem-loops. The resulting PCR product was inserted into the
30 UTR of cdc3 using the plasmid “Potef-mCherry-cdc3-
16boxB-3^0 UTR”. Here, 16 copies of the boxB stem-loops
were replaced with 12 copies of the PP7 stem-loops using
SacII restriction sites (seeNote 1). - The PP7 RNA-binding coat protein was taken from the plas-
mid “phage ubc nls ha pcp gfp”. For amplification of the PP7
coat protein PCR oligonucleotides PP7CP-BamHI-Fwd and
PP7CP-BamHI-Rev can be used. The resulting PCR products
were integrated into plasmid “Potef-λN-3xGfp-NLS” by
replacing theλN protein usingBamHI restriction sites. - The MS2 loops and the MS2 RNA-binding protein were
integrated in the same way as described for the PP7 system,
using the “pST4 TET CMV intron renilla 24xPP7 24xMS2”
and “phage UBC NLS-HA-2xMCP-tagRFP” plasmids as tem-
plates of the MS2 loop array and the MS2 coat protein,
respectively.
3.2 Generation
ofU. maydisStrains
To generateU. maydisstrains the desired constructs are integrated
in the genome by homologous recombination. The homologous
flanking regions should be about 1 kb in size [33, 34]. To this end,324 Sabrina Zander et al.