FIG. 2.
Rua1 is essential for UA biosynthesis. (A) Molecular structure of ustilagic acid (UA). UA consists of cellobiose O-glycosidically linked to the ω-hydroxyl group of 15,16-dihydroxypalmitic acid or 2,15,16-trihydroxypalmitic acid. The cellobiose moiety is esterified with an acetyl group and a short-chain β-hydroxy fatty acid. (B) TLC analysis of extracellular glycolipids produced by wild-type and Δrua1 mutant strains. Deletion of rua1 results in complete loss of UA secretion, while production of MELs is not affected. (C) Total RNA of wild-type and rua1 deletion strains was prepared before (0 h) and 12 h after shifting of cells to nitrogen-limited medium. Under these conditions, transcription of cluster genes is strongly induced in wild-type strains but is completely abolished in rua1 mutant strains. The actin gene is constitutively expressed and serves as a loading control. (D) Rua1 localizes to the nucleus. U. maydis FB2 strains expressing full-length eGFP-Rua1 or the N-terminally truncated version eGFP-Rua1308-757 were analyzed by differential interference contrast (DIC) light microscopy (right column) and epifluorescence (left column) to detect eGFP. Both fusion proteins show strong localization to the nucleus. Expression of the unfused eGFP reporter gene serves as a control (GFP). (E) Genetic organization of the UA biosynthesis gene cluster. For gene designations, see reference 29.