Figure 2: miR863-3p targets and silences ARLPK1 and ARLPK2 and SE by two modes of action.

(a) Nucleotide sequences of wt and mutated (m) versions of ARLPK1, ARLPK2 and SE aligned against the miR863-3p sequence. (b) Relative expression levels of ARLPK1, ARLPK2 and SE transcripts in mock- and Pst (EV), Pst (ΔhrcC) and Pst (avrRpt2)-infected Col-0 WT plants were detected by real-time RT–PCR. Bacterial inoculum concentration: 1 × 10⁷ c.f.u. per ml. Leaf tissue was collected 14 h.p.i. Relative mRNA levels in mock-infected plants were set at 1. Error bars indicate s.d. from three technical replicates. Similar results were obtained from three biological replicates. **P value <0.01 (Student’s t-test). (c) Agrobacterium-mediated transient co-expression assay with WT (wtARLPK1 and wtARLPK2) or mutated kinases (mARLPK1 and mARLPK2) and MIR863. mRNA levels of the wt and mutant kinases were detected by RT–PCR. rRNA was used as a loading control. miR863-3p levels were detected by northern blot analysis. U6 was used as a loading control. Similar results were obtained from two biological replicates. (d) Cleavage sites of ARLPK1 and ARLPK2 mRNAs were revealed by mapping cloned RLM-5′-RACE products using Pst (avrRpt2)-infected WT Arabidopsis plants. Arrows indicate positions and proportions of clones mapping to the sites. (e) Agrobacterium-mediated transient co-expression assay with GFP-tagged wt (GFP-wtSE-3′-UTR) or mutated (GFP-mSE-3′-UTR) 3′-UTR SE fragment and MIR863. wtSE-GFP and mSE-GFP mRNA were detected by RT–PCR using primers specific to the SE 3′-UTR. Actin was used as a loading control. GFP protein levels were detected by western blot. α-Tubulin was used as a loading control. miR863-3p levels were detected by northern blot analysis. U6 was used as a loading control. Similar results were obtained from two biological replicates. (f) SE protein levels in mock-, Pst (avrRpt2)-, Pst (ΔhrcC)- and Pst (EV)-infected Col-0 WT plants were detected by western blot using an anti-SE antibody. Bacterial inoculum concentration: 1 × 10⁷ c.f.u. per ml. Leaf tissue was collected at 14 h.p.i. α-Tubulin was used as a loading control. Relative abundance (RA) levels are indicated. Similar results were obtained from two biological replicates.