- Cotransform the desired constructs inU. maydisas described
in Subheading3.2 (seeNote 14). - Perform the microscopic analysis as described above and gen-
erate kymographs (Subheading3.4,steps 8– 15 ; Fig.4b). - To find the best suited setup, compare the different systems in
respect to the number of detectable moving mRNAs (mRNP
complexes), the velocity of moving signals and the covered
distance (Fig.4c–e).
Fig. 4Analysis of RNA live imaging ofcdc3comparing different systems. (a) Schematic representation of the
secondary structures of boxB, PP7, and MS2 RNA stem-loops. (b) Kymographs of labeledcdc3mRNA with
three different RBPs (λN*, PCP, and MCP) fused to triple GFP and containing an NLS (inverted images).
Arrowheadsindicate moving mRNPs. Hyphae were analyzed 8 h post induction. (c) Number of processively
moving RBP-FP signals per 100μm of hyphae containing different systems for RNA live imaging (error bars
represent s.e.m.; 30 cells were analyzed in three independent experiments). (d) Velocity of observed signals in
hyphae containing different RNA live imaging systems (total directed signals; error bars represent s.e.m., 30
cells were analyzed in three independent experiments; one way ANOVA,α¼0.05). (e) Range of movement of
signals in hyphae containing different RNA live imaging systems (n¼3, data points and median are indicated)
RNA Live Imaging inU. maydis 331