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).