The tyrosine phosphorylation of the lectin receptor-like kinase LORE regulates plant immunity (2019)
Figure 5

Figure 5. LORE phosphorylates PBL34 at T306/T310 for immune responses

A. PBL34 was phosphorylated by LORE in vitro. Purified recombinant proteins were used for in vitro kinase assays. The kinase dead mutant MBP-PBL34* (PBL K180 was substituted to E, predicted by https://www.uniprot.org/uniprot/), was used as the substrate of GST-LORE-CD or its kinase dead variant, GST-LORE-CD* (LOREK516E). MBP served as a negative control. Incorporation of isotope labeled phosphates was visualized by 32P autoradiography. The CBB staining showing protein abundance.

B. Mutation of PBL34 T306/T310 significantly reduced its phosphorylation by LORE. Recombinant kinase dead PBL34* and PBL34*-2A (T306/T310 was mutated to A) were incubated with LORE-CD. Phosphorylation of PBL34 was visualized by 32P autoradiography. Protein abundance was revealed by CBB staining.

C. Mutation at Y600 diminished trans-phosphorylation activity of LORE. Recombinant LORE-CD and LOREY600F-CD were used for the trans-phosphorylation assays. Phosphorylation of PBL34 was visualized by 32P autoradiography. Protein abundance was revealed by CBB staining.

D. PBL34 phosphorylation is induced by 3-OH-C10:0. The protoplast of Col-0 was transformed with 35S:PBL34-FLAG and 35S:PBL34-2A-FLAG, respectively. Sixteen hours later, the protoplasts were treated with1 μM 3-OH-C10:0. Phosphorylated proteins were immunoprecipitated and detected by anti-pS/T antibody. The experiment was repeated three times with similar results.

E. 3-OH-C10:0-induced PBL34 phosphorylation depends on LORE. The 35S:PBL34-FLAG was introduced to Col-0 and lore, respectively. Four-week-old homozygous transgenic plants were treated with 1 μM 3-OH-C10:0. Phosphorylated proteins were immunoprecipitated and detected by anti-pS/T antibody. The experiment was repeated three times with similar results.

F. PBL34 dissociated from LORE upon 3-OH-C10:0 activation. The 35S:LORE-FLAG plants were transformed with native promoter driven PBL34 (pPBL34:PBL34-HA). 100 nM flg22 or 1 μM 3-OH-C10:0 was sprayed onto plant leaves, and the leaves were sampled for assays15 min later. The experiment was repeated three times with similar results.

G. Phosphorylation of PBL34K180E did not disassociate from LORE. The 35S:LORE-FLAG protoplasts were transfected with 35S:PBL34K180E-HA. 100nM flg22 or 1 μM 3-OH-C10:0 was used to treat the protoplasts. The protoplasts were sampled 15 min later. The experiment was repeated three times with similar results.

H. PBL34 rescues pbl34/35/36 triple mutant ROS burst in response to 3-OH-C10:0. Treatment was same as in (E). pPBL34:PBL34-HA was introduced to pbl34/35/36 triple mutant, and the homozygous transgenic line was used for ROS burst assays. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).

I. PTI marker gene expression in PBL34 complementation lines. The PTI marker genes FRK1 and NHL10 expression was examined by RT-qPCR in Col-0, pbl34/35/36, and CM-PBL34-2A lines after 3-OH-C10:0 treatments for 4 hrs. Values are means ± SD (n=6; ANOVA; p < 0.01).

J. PBL34 phosphorylation at T306/T310 is essential for disease resistance. pbl34/35/36 triple mutants were complemented with PBL34 or PBL34-2A driven by its native promoter. The homozygous plants were pretreated with 3-OH-C10:0(C10) 1 d before pathogen inoculation. The plants were inoculated with 2×105 cfu/ml Pst DC3000, and the bacterial titer was analyzed at 3 dpi. Mock is solvent control. Letters indicate the difference from Col-0. Values are means ± SD (n=6; ANOVA; p < 0.01).