Confocal microscopy analysis
For the analysis of the subcellular localization, A. tumefaciensGV3101 carrying plasmids with expression constructs were co-infiltrated
with pFlub vector (RFP-peroxisome tagged marker) into leaves of
4-week-old N. benthamiana CB157 (nucleus mRFP marker) and CB172
(ER mRFP marker) reporter lines using 1 ml needleless syringes. Two days
after infiltration, leaves were excised and prepared for confocal
microscopy. GFP and mRFP fluorescence was examined under Nikon A1R
confocal microscope with a water-dipping objective, Nikon X 40/ 1.0W.
GFP was excited at 488 nm from an argon laser and its emissions were
detected between 500 and 530 nm. mRFP was excited at 561 nm from a diode
laser, and its emissions were collected between 600 and 630 nm.
Western blot
analysis
Leaves from N. benthamiana leaves infiltrated with A.
tumefaciens GV3101 carrying plasmids with expression constructs were
excised, ground and proteins extracted as previously described (Gilroy
et al., 2011, Yang et al., 2016). Western blotting was performed as
previously described (Qin et al., 2018). Detection of GFP was performed
using a polyclonal rabbit anti-GFP antibody (1:4,000 dilution) and
secondary anti-mouse antibody (IG HRP 1:10,000) according to the
manufacturer’s instructions. ECL development kit (Amersham) detection
was used according to the manufacturer’s instructions.
Transformation ofArabidopsis thaliana stable overexpression transgenic
lines
Arabidopsis overexpression plants were transformed using A.
tumefaciens GV3101 carrying plasmids with expression constructs using
the flower dipping method (Clough and Bent, 1998). Selection of
Arabidopsis transformants and homozygous lines selection were performed
as described (Luna et al., 2014). Resistance was tested against B.
cinerea as described before. Two independent homozygous overexpression
lines were obtained per construct.
Pathosystem
statistics
Statistical analysis of induced resistance and growth
phenotypes were performed as described (Luna et al., 2016). Data
analysis was performed using SPSS Statistics 23 and GenStat® 18th
Edition (VSN International, Hemel Hempstead, UK). Statistical analysis
of resistance phenotypes in Arabidopsis overexpression lines was done by
ANOVA with ‘construct’ as a single treatment factor at 10 levels: Col-0
(wild-type treatment); two empty vector lines ‘EV 3.1’ and ‘EV 4.1’;
‘SlACRE75 1.1’ and ‘SlACRE75 2.1’; ‘SlACRE180 1.2’ and ‘SlACRE180 3.1;
‘NbACRE180 1.1’ and ‘NbACRE180 2.1’; and ‘NbACRE75 1.1’. The replicate
units were individual plants of which there were 8-16 for each
construct. Measurements of four lesions were recorded for each plant.
Random effects were modelled as plant + plant × lesion to capture the
plant-to-plant and within-plant variation. As part of the ANOVA,
specific planned (non-orthogonal) contrasts were included to test for
significant differences between the mean for each construct line
compared to
Col-0.