Fig 2: GUS levels in transgenic tobacco induced by SA and MeJA, alone or in combination.(A and B) WB analysis of GUS induced by SA and MeJA, alone or in combination. Four- to six-leaf tobacco seedlings were sprayed with SA and MeJA alone or in combination. After treatment for (A) 12 and (B) 48 h, leaves were collected for protein extraction. A total of 200 µg of protein was detected with rabbit monoclonal antibody to GUS. In all cases, actin antibodies were used for the loading control. Blots in (A) and (B) are representative of three biological replicates that showed similar results.(C and D) GUS amount was quantified based on the band intensity, standardized to the actin signals. Results shown in the graphs (C and D) represent three biological replicates. Error bars indicate SD. Statistically significant differences were determined by Student’s t-test (*P < 0.05; **P < 0.01) compared with SA or MeJA alone. SA, sprayed with 1 mM SA; MeJA, sprayed with 0.5 mM MeJA; SA + MeJA, sprayed with 1 mM SA plus 0.5 mM MeJA; Mock, deionized water as a mock control for SA treatment or 0.1% ethyl alcohol as a mock control for MeJA and SA + MeJA treatment.

Fig 3: Induction of GUS activity in transgenic tobacco and Arabidopsis upon infection with biotrophic and necrotrophic pathogens.(A) GUS staining of leaves from representative PR-1a::GUS-28, SJ-609::GUS-131, and PDF1.2::GUS-65 transgenic tobacco lines infected with the biotrophic pathogen E. cichoracearum or the necrotrophic pathogen A. alternata. White arrows indicate the site of A. alternata inoculation. To withdraw the pathogen challenge, fungicide (fenaminosulf) was sprayed onto infected leaves 72 h after inoculation. GUS staining was performed 72 h after withdrawal of the challenge. Ten transgenic tobacco lines per construct were used for analysis of GUS activity.(B) GUS staining of leaves from representative PR-1a::GUS-22, SJ-609::GUS-52, and PDF1.2::GUS-15 transgenic Arabidopsis lines infected with the biotrophic pathogen E. cichoracearum or the necrotrophic pathogen Alternaria brassicae. Five transgenic Arabidopsis lines per construct were used for analysis of GUS activity.(C)GUS expression patterns of representative PR-1a::GUS-22, PDF1.2::GUS-15, and SJ-609::GUS-52 transgenic Arabidopsis lines induced by E. cichoracearum (above) and A. alternata (below). Values are the means of three technical replicates with SD. Similar results were obtained for three repeated experiments. E. cichoracearum and A. alternata were sprayed as 107 spore/ml spore suspensions.(D and E)GUS expression in PR-1a::GUS-22, PDF1.2::GUS-15, and SJ-609::GUS-52 transgenic Arabidopsis lines with the indicated treatments. Mock, deionized water as a mock control for SA treatment (E) or 0.1% ethyl alcohol as a mock control for MeJA treatment (D); MeJA, sprayed with 0.5 mM MeJA; Ery, inoculated with E. cichoracearum; Ery + MeJA, inoculated with E. cichoracearum, then sprayed with 0.5 mM MeJA 24 h later; SA, sprayed with 1 mM SA; Alt, inoculated with A. alternata; Alt + SA, inoculated with A. alternata, then sprayed with 1 mM SA 24 h later. Results in (D and E) are averages of three biological replicates with SD. Lowercase and capital letters above bars indicate significant differences according to Duncan’s multiple comparison procedure at the 0.05 (P < 0.05) or 0.01 level (P < 0.01), respectively. GUS expression levels in (C), (D), and (E) were quantified by qRT-PCR of cDNA prepared from RNA extracted from treated or infected leaves. AtACT2 served as the reference gene. GUS and AtACT2 primers for qRT-PCR are shown in Supplemental Table 9.