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.