Figure 8.

In Planta Phosphorylation of Maize SGT1.

(A) Fragmentation spectrum assigned to the phosphorylated form of the peptide EDVANMDNTPPVVEPPSKPK (Mascot score 126). Loss of H₃PO₄ is denoted by –P, loss of water is marked by short horizontal arrows, whereas a longer arrow symbolizes pairs of detected signals corresponding to y_n and y_n-H₃PO₄. The majority of signals of the tandem mass spectrometry spectra are assigned to the above species. The presence of several y_n>11-H₃PO₄ and b₉-H₃PO₄ ions accompanied by y₁₅, y₁₆, and y₁₇ pinpoints threonine at position 9 as the unequivocal phosphorylation site within the peptide.

(B) Peptide sequence with assigned y, b, y-H₂O, b-H₂O, y-H₃PO₄, and b-H₃PO₄ ions present.

(C) Recombinant maize SGT1 produced in E. coli was incubated in the buffer containing [γ-³²P]ATP, and total proteins were extracted from maize seedlings or tassels infected by various U. maydis strains. The samples were fractionated by SDS-PAGE and analyzed with a phosphor imager. Columns 1 to 3, extracts from seedling leaves 6 DPI with U. maydis wild-type SG200 (1), SG200∆see1 (2), or mock-inoculated (3). Columns 4 to 6, extracts from tassel base 9 DPI with U. maydis wild-type SG200 (4), U. maydis-overexpressing Ppit2:see1 (single-copy integration; 5), or U. maydis-overexpressing Ppit2:see1 (multiple-copy integration; 6). Representative data of four independent biological replicates are shown. CBB, Coomassie Brilliant Blue.