Supporting Information Legend
Figure S1. Nt-Rac5G15V interaction with Nt-RhoGAP1: effects of YFP fragment swapping on BiFC
efficiency. (a) Single confocal sections are shown through normally growing tobacco pollen tubes,
which were treated and imaged essentially as described in the legend of figure 2a to compare BiFC
signals obtained by coexpression of nYFP:RFP:Rac5G15V and cYFP:RhoGAP1, or of cYFP:RFP:Rac5G15V
and nYFP:RhoGAP1. All indicated YFP fragments or fusion proteins were expressed under the control
of the 2x35S promoter. Expression of ER targeted mRFP1 was driven by a regular CaMV 35S
promoter. Scale bar: 10 μm. (b) Statistical analysis of membrane association of BiFC fluorescence at
the flanks of the tip of pollen tubes treated and imaged as described in (a). The Y-axis indicates the
intensity ratio between plasma membrane associated and adjacent cytoplasmic fluorescence 5-10
μm away from the extreme apex. Each data point represents a total of at least 13 pollen tubes.
Measurements contributing to each data point were performed in 3 or more independent
experiments. Error bars: 95% confidence interval. c) Statistical analysis of membrane associated [PM]
and cytoplasmic [CP] BiFC (YFP) fluorescence, as well as of ER derived mRFP1 fluorescence [ER],
emitted from pollen tubes treated and imaged as described in (a). Error bars: 95% confidence
interval. ** P <0.01: pairwise t-test (pair indicated by bracket).
Figure S2. Nt-Rac5G15V interaction with Nt-RhoGAP1: effects of an mRFP1 linker N-terminally
attached to Nt-Rac5G15V on BiFC efficiency. (a) Single confocal sections are shown through normally
growing tobacco pollen tubes, which were treated and imaged essentially as described in the legend
of figure 2a to analyze effects of an mRFP1 linker between the cYFP fragment and Nt-Rac5G15V
(cYFP:RFP:Rac5G15V) on the BiFC signal generated by pollen tubes coexpressing nYFP:RhoGAP1. All
indicated YFP fragments or fusion proteins were expressed under the control of the 2x35S promoter.
Expression of ER targeted mRFP1 was driven by a regular CaMV 35S promoter. Scale bar: 10 μm. (b)
Statistical analysis of membrane association of BiFC fluorescence at the flanks of the tip of pollen
tubes treated and imaged as described in (a). The Y-axis indicates the intensity ratio between plasma
membrane associated and adjacent cytoplasmic fluorescence 5-10 μm away from the extreme apex.
Each data point represents a total of at least 10 pollen tubes. Measurements contributing to each
data point were performed in 3 or more independent experiments. Error bars: 95% confidence
interval. *** P <0.001: pairwise t-test (pair indicated by bracket). c) Statistical analysis of membrane
associated [PM] and cytoplasmic [CP] BiFC (YFP) fluorescence, as well as of ER derived mRFP1
fluorescence [ER], emitted from pollen tubes treated and imaged as described in (a). Error bars: 95%
confidence interval. ** P <0.01, *** P <0.001: pairwise t-test (pairs indicated by brackets).
Figure S3. Nt-Rac5T20N interaction with Nt-RhoGAP1: effects of an mRFP1 linker N-terminally
attached to Nt-Rac5G15V on BiFC efficiency. (a) Single confocal sections are shown through normally
growing tobacco pollen tubes, which were treated and imaged essentially as described in the legend
of figure 2a to analyze effects of an mRFP1 linker between the cYFP fragment and Nt-Rac5T20N
(cYFP:RFP:Rac5T20N) on the BiFC signal generated by pollen tubes coexpressing nYFP:RhoGAP1. All
indicated YFP fragments or fusion proteins were expressed under the control of the 2x35S promoter.
Expression of ER targeted mRFP1 was driven by a regular CaMV 35S promoter. Scale bar: 10 μm. (b)
Statistical analysis of membrane association of BiFC fluorescence at the flanks of the tip of pollen
tubes treated and imaged as described in (a). The Y-axis indicates the intensity ratio between plasma
membrane associated and adjacent cytoplasmic fluorescence 5-10 μm away from the extreme apex.
Each data point represents a total of at least 17 pollen tubes. Measurements contributing to each
data point were performed in 3 or more independent experiments. Error bars: 95% confidence
interval. *** P <0.001: pairwise t-test (pair indicated by bracket). c) Statistical analysis of membrane
associated [PM] and cytoplasmic [CP] BiFC (YFP) fluorescence, as well as of ER derived mRFP1
fluorescence [ER], emitted from pollen tubes treated and imaged as described in (a). Error bars: 95%
confidence interval. ** P <0.01: pairwise t-test (pair indicated by bracket).
Figure S4. Time-series imaging to measure growth rates of pollen tubes expressing YFP fused to wild
type or mutant Nt-Rac5, or to Nt-RhoGAP1. Medial confocal optical sections through individual
pollen tubes expressing the indicated YFP fusion proteins under the control of the 2x35S promoter
were serially recorded at two time points (displayed in lower left corner of each image). The increase
in pollen tube length during the interval between these two time points is shown in the upper right
corner of each image pair. In all cases, the intracellular distribution of analyzed YFP fusion proteins
remained essentially unchanged during serial imaging. Scale bars: 10 μm.
Figure S5. Measurement of the intensities of membrane-associated and cytoplasmic YFP
fluorescence, as well as of mRFP1 fluorescence within the ER. Upper panel: at a specified distance
from the apex (5 μm in the example shown), lines (yellow) were drawn vertically to the long axis of
analyzed pollen tubes on two separate images taken in the YFP and in the mRFP1 channel. Lower
panel: from intensity plots along these lines, maximal YFP fluorescence intensities at the plasma
membrane (average of the two values obtained at membrane intersections: black arrows), as wells
as average cytoplasmic YFP fluorescence intensities (green dotted line) and average mRFP1
fluorescence intensities within the ER (red dotted line) were read out. Subsequently, the ratio
between maximal membrane associated and average cytoplasmic YFP fluorescence was computed.
Scale bar: 5 μm.
Table S1. Constructs used in this study