Figure 2

Alternatively polyadenylated and spliced isoforms of ERF4 are rapidly and transiently induced by flg22 treatment.

(A) Quantitative RT-PCR (RT-qPCR) validation of ATRIKEN28815 expression in Col-0 (filled circle) and fls2 (open circle) seedlings following treatment with 1 μM flg22. Data show the mean and standard error of two biological replicates. Asterisks indicate differences between Col-0 and fls2 (P < 0.05). (B) i) Schematic diagram of a 7.5-kB genomic fragment, indicating annotated ERF4 (ERF4-R), ATRIKEN28815 probe and flanking gene locations. Full-length cDNAs corresponding to ERF4-A and ERF4-IR are indicated below the annotated genes. Thick arrowheads denote exons, thicker lines denote UTRs and thin lines denote introns. ii) Primers used for transcriptional analyses. Conventional RT-PCR primers used in figure (D) are indicated by arrowheads, RT-qPCR amplicons used in (E) are indicated by thick lines and thin lines indicate the intron present in the ERF4-A amplicon. (C) The predicted ERF4-A coding region lacks the EAR motif present in both ERF4-R and ERF4-IR. (D) Conventional RT-PCR using primers P1 and P3 (top panel; 35 cycles) or P1 and P2 (bottom panel; 30 cycles) shows that the distally polyadenylated intron-retaining isoform ERF4-IR is rapidly induced within 15 min, followed by induction of the distally polyadenylated spliced isoform ERF4-A after 30 min. (E) RT-qPCR analyses of ERF4 isoforms in Col-0 or fls2 seedlings following treatment with water (0 time point) or 1 μM flg22. Asterisks indicate differences between Col-0 and fls2 (P < 0.05). Respective amplicons are shown in (B).