Fig. 5: UmCAPE-La peptide is released from UmPR-1La by CatB3.
From: Ustilago maydis PR-1-like protein has evolved two distinct domains for dual virulence activities
a, b E64 blocks the cleavage of PR-1La. The SG200_PR-1La culture supernatant (labeled as UmPR-1LaHA) was incubated: (a) with the apoplastic fluid (AF) proteins from SA-inoculated maize, along with varying amounts of DMSO-dissolved E64 or an equivalent volume of DMSO, (b) with CatB3 in the presence of either 5 µl of E64 (final conc. of 0.5 mM) or DMSO. A representative blot from one of two independent experiments is shown. c The catalytic-site mutants of CatB3 fail to cleave UmPR-1La. The immunoblot showed the results before and after 2-hr incubations of SG200_PR-1La culture supernatant with CatB3 or its mutants. CatB3C121A: the alanine substitution at Cys121. CatB3C121A H276A: the alanine substitutions at Cys121 and His276. A representative blot from one of two independent experiments is shown. The loadings were verified using separated silver-stained PAGEs. d CatB3 does not cleave plant PR-1s. Maize PRB1-3 and tomato PR1b (C-terminal His-tagged proteins), and maize CatB3 (no tag) were purified from the apoplast of N. benthamiana. Full-length PR-1LaHis proteins was purified from E. coli. The CatB3-digested proteins were separated on SDS-PAGE and stained with Coomassie-blue or analyzed by immunoblotting. Asterisks: the CatB3 protein bands. Black arrowheads: plant PR-1 proteins. Black open arrows: PR-1La proteins. Red open arrows: truncated PR-1La proteins. A representative blot from one of three independent experiments is shown. e Detection of UmCAPE-La peptides through targeted LC-MS/MS analysis. The SG200_PR-1La culture supernatant or recombinant PR-1La protein was treated with CatB3, as described in Fig. 5c, d, before subjected to LC-MS/MS analysis. The quantity of short tryptic peptides (PPGNYIGK) was measured in both the -CatB3 and +CatB3 samples using the peak area of fragment ions at specific retention times shown in Supplementary Fig. 6b. A cartoon illustrates the fragment ions of the peptide. Fragment ion b5 extends from the N-terminus, and y6 and y7 ions extend from the C-terminus, while y7++ is a doubly charged ion. The term “intensity” refers to the amplitude of the free induction decay signal. Consistent findings were observed in two independent CatB3-cleavage experiments using recombinant PR-1LaHis and UmPR-1LaHA. Source data are provided as a Source Data file.
