MoEnd3 phosphorylation requires MoArk1.

(A) MoEnd3:GFP proteins treated with phosphatase Inhibitor or phosphatase were separated by Mn²⁺-Phos-tag SDS-PAGE and normal SDS-PAGE respectively, and were probed with GFP antibody. (B) MoEnd3 phosphopeptide (PASLRASFERNKI) in the strain expressing MoARK1 was identified by mass spectrometer analysis and the phosphorylated site was Ser-222. (C) MoEnd3:GFP protein was extracted from the strain expressing MoArk1 and not expressing MoArk1, respectively. MoEnd3^S222A:GFP protein was extracted from the strain expressing MoArk1. Then these proteins were separated by Mn²⁺-Phos-tag SDS-PAGE and normal SDS-PAGE, respectively, and probed with GFP antibody. (D) Hyphae were examined by fluorescence microscopy following 5 min FM4-64 staining. The selected regions where fluorescence was measured by ImageJ software were labeled by ellipse frame. Bars = 5 μm. (E) The bar chart shows mean fluorescence intensity at the hyphal tip region calculated using ImageJ software. At least 15 hyphae were measured for each strain. Asterisks represent significant differences (P < 0.01). (F) Pmk1 phosphorylation level was detected by applying phosphor-Pmk1 antibody. Endogenous Pmk1 level was detected by the Pmk1 antibody. (G) Pathogenicity assay on rice with the MoEnd3 phosphorylation site mutants. Photographs were taken following 7 days of inoculation.