Extended Data Fig. 10: ALR1^S696/698 phosphorylation synergize with Al binding.

(a,b) Pull-down assay of the interaction between BAK1^KD and ALR1 variants (ALR1^CD, ALR1^CD-S2A, ALR1^CD-S2AC4A, ALR1^CD-S2D and ALR1^CD-S2DC4A) under 100 nM Al treatments (in binding buffer). GST-tagged ALR1 variants were used as the baits, GST was used as the control, and TF-tagged BAK1^KD was used as the prey. (c, d) Pull-down assay of the interaction between RbohD^N and ALR1 variants (ALR1^CD, ALR1^CD-S2A, ALR1^CD-S2AC4A, ALR1^CD-S2D and ALR1^CD-S2DC4A). GST-tagged ALR1 variants were used as the baits, GST was used as the control, and TF-tagged RbohD^N was used as the prey. (e, f) Inter-phosphorylation of ALR1^CD variants (ALR1^CD, ALR1^CD-C4A, ALR1^CD-S2A, ALR1^CD-S2AC4A, ALR1^CD-S2D and ALR1^CD-S2DC4A, TF-tagged) and BAK1^KD (GST-tagged) with or without 100 nM Al treatments in vitro. (g, h) Phosphorylation of the recombinant N-terminal of RbohD (RbohD^N) by the ALR1 variants (ALR1^CD, ALR1^CD-C4A, ALR1^CD-S2A, ALR1^CD-S2AC4A, ALR1^CD-S2D and ALR1^CD-S2DC4A) in an pS39-dependent in vitro phosphorylation assay with or without 100 nM Al treatments. CBS indicates coomassie blue staining. All experiments were repeated at least three independent times with similar results.

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