Architecture of the Sporulation-Specific Cdc14 Promoter from the Oomycete Phytophthora infestans

Alignment of Cdc14 promoters from four species of Phytophthora. Sequences are from P. infestans, P. sojae, P. ramorum, and P. capsici (top to bottom), are numbered with respect to the major transcription start site in P. infestans (bent arrow at + 1), and terminate at the ATG start codon. M1 to M7 represent the conserved blocks described in Results, and triangles are drawn below the four CTYAAC motifs. The original alignments were performed using 750-nt promoters, but to save space only the more conserved regions (from −155 to +91 in P. infestans) are shown.

Site-directed mutagenesis of conserved blocks in the Cdc14 promoter. (A) Schematic of promoter-GUS fusion, with the M1 to M7 and TSS blocks indicated by rectangles. Solid and open rectangles represent native and mutagenized sequences, respectively, and the bent arrow indicates position +1. To save space, unmutagenized 5′ regions of the rest of the 868-nt promoters are not shown. In pInrSwap, the TSS block was replaced by an equivalent region from Piexo1. All eight plasmids confer sporulation-induced expression in transformants. (B) RNA blots obtained from 1.2% agarose gels were hybridized with a probe for GUS, using RNA from sporangia (S1 and S2) or hyphae (H1 and H2) of two representative transformants containing each plasmid. Ethidium bromide-stained images are shown as loading controls.

5′ deletion analysis of the Cdc14 promoter. (A) Schematic of promoter constructs, with M1 to M7 and TSS blocks indicated by rectangles above the center line and CTYAAC motifs represented by inverted triangles below the line. Plasmid names indicate the size of the promoter upstream of the TSS (bent arrow; position +1), i.e., p335 contains DNA up to nucleotide −335. Whether plasmids confer sporulation-induced expression is indicated by plus or minus signs. (B) RNA from sporangia (S) or hyphae (H) of representative transformants containing each plasmid, electrophoresed on 1.2% gels. Filters were probed for GUS, stripped, and then hybridized with the Cdc14 open reading frame as a control. Hyphae from p81- and p75-containing transformants were also negative for GUS RNA (not shown). Ethidium bromide-stained images are shown as controls.

Use of a chimeric promoter to prove function of the −67 to −90 region. (A) Schematic of plasmids, showing the TSS (rectangle) from the NIFS gene and CTYAAC motifs (inverted triangles, with filled and open triangles representing wild-type and mutagenized sequences). Whether plasmids confer sporulation-induced expression is indicated by plus or minus signs. (B) Blot of RNA from sporangia (S) or hyphae (H) of representative transformants containing p74Nif, performed under high-resolution (1.0% agarose) conditions. Filters were hybridized with a probe for GUS, stripped, and hybridized with the Cdc14 open reading frame as a control. The ethidium bromide-stained image is shown as a control. (C) The same experiment as shown in panel B, except that transformants contained p24Cdc::74Nif. The promoter in p17Cdc::74Nif showed no activity based on blot analysis of RNA from hyphae and sporangia (not shown).

EMSA using labeled probe representing the −94 to −62 region of Cdc14 and nuclear protein from sporangia. Labels denote a specific protein-DNA complex (SP), nonspecific complex (NS), and free probe (FP). Lane 1, no extract; lane 2, extract and probe; lanes 3 to 14, reaction mixtures with increasing amounts of unlabeled competitor DNA (1×, 10×, 100×, 200×). Competitors are the DNA used as the labeled probe (self), DNA derived from another gene (nonself), and the −94 to −62 region in which the CTYAAC motifs were changed to other bases (mutant self).