Figure 6: ARLPK1 interacts with AKIK1 that is also a negative regulator of disease resistance. | Nature Communications

Figure 6: ARLPK1 interacts with AKIK1 that is also a negative regulator of disease resistance.

From: miRNA863-3p sequentially targets negative immune regulator ARLPKs and positive regulator SERRATE upon bacterial infection

Figure 6

(a) AKIK1-FLAG co-IP with ARLPK1-YFP but not with ARLPK2-YFP. Total proteins (input) from N. benthamiana extracts were immunoprecipitated with ANTI-FLAG M2 affinity gel, and AKIK1-FLAG and ARLPK1-YFP proteins were detected by western blot using anti-FLAG and anti-YFP antibodies, respectively. Similar results were obtained from three biological replicates. (b) The akik1-1 and akik1-2 mutants are more resistance to bacterial Pst (EV) and Pst (avrRpt2), as compared with WT plants. Bacterial inoculum concentration: 5 × 105 c.f.u. per ml. Bacterial growth was measured 3 d.p.i. Error bars represent s.d. for at least 15 leaf discs. Similar results were obtained from three biological replicates. **P value <0.01 (Student’s t-test). (c) PR1 protein levels in Pst (EV)- and Pst (avrRpt2)-infected Col-0 WT and akik1-1 mutant were detected by western blot using an anti-PR1 antibody. Bacterial inoculum concentration: 1 × 107 c.f.u. per ml. Leaf tissue was collected 0 and 12 h.p.i. α-Tubulin was used as a loading control. Similar results were obtained from two biological replicates. (d) Relative expression levels of FRK1 in flg22-treated Col-0 WT, arlpk1-1 arlpk2-1 and akik1-1 mutants were detected by real-time RT–PCR. Samples were collected 0 and 30 min after treatment. 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). (e) The levels of phosphorylated MPK3 and MPK6 in flg22-treated Col-0 WT, arlpk1-1 arlpk2-1 and akik1-1 mutants were detected by western blot using monoclonal rabbit phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204)(D13.14.4E) XP antibody. Leaf tissue was collected 0, 15 and 30 min after treatment. α-Tubulin was used as a loading control. Similar results were obtained from three biological replicates. (f) Recombinant MBP-tagged RIPK kinase domain and GST-tagged AKIK1 kinase domain were subjected to a radioactive kinase assay and kinase activity was detected using autoradiography. Bottom panel: SDS–PAGE gel stained with coomassie brilliant blue (CBB) demonstrating protein abundance. Similar results were obtained from two biological replicates.

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