To make complementation lines, AtPLDα1 (from 3,300 bp upstream of
the start codon to the end of the 3’ UTR) was amplified from wt genomic
DNA with Phusion polymerase (New England Biolabs) and cloned into the
pENTR3C vector (Invitrogen). To create mutants mutated in both HKD
motifs, megaprimers MP334-F and MP663-R were generated first from wt
cDNA using AtPLDa1-F/AtPLDa1-K334R and AtPLDa1-K663R/AtPLDa1-R primers,
respectively. Next, primers MP334-F and MP663-R were used to produce
AtPLDa1-K334R K663R. The part of DNA containing mutations was cut with
HindIII and used to replace corresponding part in wt sequence cloned in
pENTR3C. Both entry clones were recombined into the Gateway binary
vector pGWB601 (Nakamura et al., 2010) using LR Clonase II
(Invitrogen). Final constructs were transformed into Agrobacterium
tumefaciens strain GV3101, which was used to transform plda1-1plants by floral dip (Clough & Bent, 1998). Transformants were selected
by spraying with BASTA. The presence of PLDα1 or mutated PLDα1 in
transgenic lines was confirmed by western blotting using anti-PLDα1
antibodies (Fig. 2a).