Cloning, preparation of oocyte constructs and cRNA synthesis
The AtPIP2;1 (At3g53420) coding sequence was cloned using high-fidelity
Phusion® polymerase (New England Biolabs, USA) from Arabidopsis root
cDNA into a Gateway-enabled pCR8/GW/TOPO entry vector (Life
Technologies) before being transferred into the pGEMHE (DEST) vector
using LR clonase II (Invitrogen). Primers were designed to generate
site-directed single and double point phosphomimetic mutations in
AtPIP2;1 (Table S1) using AtPIP2;1 in pGEMHE as a template. All the
constructs in pGEMHE were linearized using restriction enzyme NheI-HF
(New England Biolabs, USA) before cRNA was synthesized using mMESSAGE
mMACHINE® T7 Transcription kit (Thermo Fisher Scientific, Australia) as
previously described Qiu et al., (2016). The concentration and
quality of cRNA was determined by NanoDrop and gel electrophoresis.
Preparation of Xenopus
laevis oocytes
X. laevis oocytes were harvested and stored following Byrtet al., (2017). Oocytes were injected with 46 nL of RNAse-free
water using a micro-injector (Nanoinject II, automatic nanolitre
injector, Drummond Scientific) with either no cRNA or 23 ng cRNA. Post
injection and prior to experiments oocytes were stored at 18 °C in a Low
Na+ Ringer’s solution (62 mM NaCl, 36 mM KCl, 5 mM
MgCl2, 0.6 mM CaCl2, 5 mM Hepes, 5%
(v/v) horse serum and antibiotics (0.05mg mL-1tetracycline, 100 units mL-1 penicillin/0.1 mg
mL-1 streptomycin)), pH 7.6 for 24-36 h. Expression of
AtPIP2;1 within each oocyte batch was confirmed via burst test following
Byrt et al., (2017).