Figure 5.

SBT-mediated processing of recombinant CLE40 depends on P4 and T5. A and B, Site-directed P4A and T5A precursor mutants were expressed in E. coli and digested with cell wall extracts from N. benthamiana leaves expressing SBT1.7 or SBT4.13. The peptide fraction was isolated by ultrafiltration (10-kD MMCO). Peptides identified by MS are shown; matCLE40 is highlighted in red. C to E, proCLE40 is processed by Arabidopsis SBTs in a P4-dependent manner. C, Erlenmeyer flask with Arabidopsis seedlings grown in submerged culture for the collection of cell wall exudates. D, Processing of proCLE40 by Arabidopsis proteases is sensitive to EPI1a inhibition. Recombinant proCLE40 was digested with cell wall exudates of Arabidopsis wild type (wt) and of seedlings expressing EPI1a under the control of the indicated promoters. The red and blue asterisks show full-length proCLE40 and a processed form after SDS-PAGE and Coomassie blue staining, respectively. The dashed line indicates the untreated proCLE40. E, MS analysis of peptides generated from recombinant proCLE40 and the site-directed P4A mutant by Arabidopsis cell wall exudates. The analysis was performed as for A and B. matCLE40 is shown in red.