Methods S1. Method for whole mount immunolocalization
The 5- to 7-day-old Arabidopsis seedlings were fixed in 95% pentane for 10 min and
washed in PBST (PBS1X + 0.05% Triton-X100) (3 x 5 min) prior to overnight fixation
at 4°C in 1.5% formaldehyde (v/v) and 0.5% glutaraldehyde (v/v) in PBST. The
following day, the fixation step was pursued for an additional hour on ice under vacuum
and then washed with PBST (3 x 5 min). The following steps were performed in the
InsituPro VSi robot (Intavis AG, http://www.intavis.com) except if indicated otherwise.
Cell walls were digested with 0.05% pectolyase and 0.05% cellulase in 0.4 M
Mannitol/PBST for 1 h at 30°C and then by 2.5% driselase in deionized water for 45
min at 37°C. Samples were washed with PBST (3 x 5 min) and then removed from the
robot for the incubation step in methanol at -20°C for 10 min. Prior to the primary
antibody incubation, samples were subjected to a series of washing steps as follows:
PBS (1 x 10 min), 1% BSA/50mM glycine/PBS (1 x 30 min), 50 mM glycine/PBS (1 x
5 min), 0.5% Triton-X100/50 mM glycine/PBS (1 x 1-2 h), 50 mM glycine/PBS (1 x 5
min) and PBS (1 x 5 min). Samples were subsequently incubated in the primary
antibody in 1% BSA/50 mM glycine/PBS (overnight at 30°C). After washing in 50 mM
glycine/PBS (3 x 10 min), the samples were incubated in the secondary antibody in 1%
BSA/50mM glycine/PBS for 3 h at 37°C in the dark (aluminum foil) under mild
agitation. Finally, the samples were washed in PBS (3 x 5 min) and transferred into
VECTASHIELD®
mounting medium (https://www.vectorlabs.com) and then examined
with confocal imaging microscopy.
Methods S2. Method for qPCR.
Four genes were considered as reference genes: APT1 (At1g27450), TIP41
(At4g34270), EF1α (At5g60390) and UBQ10 (At4g05320). The expression of these
four genes was measured from the cDNA obtained from each sample in each
experiment. The geNorm algorithm (Vandesompele et al., 2002) was used to identify
the most stably expressed under the specific experimental conditions.
qPCR reactions were performed in triplicate in a 96-well white plate (for the
LightCycler® 480 System) or a 96-well transparent plate (for the Bio-Rad machines)
using SYBR Green Master mix (Roche). The following qPCR program was applied for
all the machines: initial denaturation at 95°C for 10 min, followed by forty-five cycles
of 95°C for 15 sec, 58°C or 60°C for 20 sec, 72°C for 30 sec. Melting curves were
derived after each amplification by increasing the temperature in 0.5°C increments from
60°C to 95°C. The Cq values for each sample were acquired by using the appropriate
software (MyIQTM software 1.0, or LC480 Software 1.5). The specificity of the
amplification was also assessed for each gene by dissociation curve analysis. A unique
peak on the dissociation curve was confirmed for each gene. The Cq values were
carefully checked and in some cases, one value was discarded because it differed from
the other two replicates by > 0.5.
For each permutation of the PCR machine and primer pair used (Table S1 for
primer sequences and Table S2 for annealing temperatures), amplification efficiencies
were calculated using a 10-fold serial dilution series of cDNA. The primer pair
amplification efficiency varied between 75% and 104% (see Table S2 for details for
each machine).
The relative expression level for each sample was calculated according to the
Pfaffl method (Pfaffl, 2001) using the following formula: average Et∆Cq(A-B)/average
Er∆Cq(A-B), where Et is the amplification efficiency of the target gene primers, Er is the
reference gene primer efficiency, A stands for the sample for which the quantification is
relative to, and B is the other sample. Average values were obtained for three biological
replicates and the results were presented on a logarithmic scale normalized to the wildtype values, which were set to 1 or non-normalized.
Pfaffl, M.W. (2001). A new mathematical model for relative quantification in real-time
RT-PCR. Nucleic Acids Res. 29.
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A.,
and F., S. (2002). Accurate normalization of real-time quantitative RT-PCR
data by geometric averaging of multiple internal control genes. Genome Biol. 3.