Fig 1.
Motifs in ribosomal protein gene promoters.
(A) distribution of PhRiboBox upstream of ribosomal protein genes. A logoplot of the motif is indicated in the box. (B) same as panel A except showing CCAAT element.
Table 1.
Over-represented Gene Ontology terms in genes containing PhRiboBox.
Table 2.
Selected genes with PhRiboBox with GO:0003676 (nucleic acid binding).
Fig 2.
Transcription of P. infestans genes encoding ribosomal proteins.
The heat map shows the per-gene normalized expression, based on microarrays, of the genes in nonsporulating hyphae (HY), sporangia (SP), sporangia chilled to induce their cleavage into zoospores (CL), motile zoospores (ZO), and germinated cysts forming appressoria (GC). The genes are ordered based on their expression stability, with the most invariant at the top. The bar graph indicates relative expression (FPKM) in hyphae based on RNA-seq. The three genes marked by asterisks are the donors of promoters for the expression studies described later in Results. Genes PITG_09552 and PITG_09555 are nearly identical in sequence and not distinguished well by the microarray. For comparison, shown at the bottom of the chart is PITG_00505, which encodes an ubiquitin-conjugating enzyme; a prior study of putative housekeeping genes ranked its Phytophthora parasitica ortholog at the top of genes having the most consistent mRNA levels during growth and development [42].
Fig 3.
GUS expression driven by five ribosomal protein promoters in stable transformants.
The box plots for each gene reflect the distribution of activities from a minimum of ten independent transformants for each construct, which employed 500-nt promoter fragments from P. infestans (Pi) or P. capsici (Pc) genes. Expression driven by the PiRPL23 promoter (PiL23) is not shown due to its instability.
Fig 4.
Stability of GUS expression in transformants.
The bars represent the percentage of clones still expressing GUS after 24 months. Indicated below each bar is the promoter name and the number of transformants analyzed.
Fig 5.
Expression of native PiRPL10 and PiRPS9 genes in transformants bearing transgenes with RPL10 and RPS9 promoters.
mRNA levels were determined by RT-qPCR of cultures grown in rye-sucrose broth, and are expressed relative to the level in untransformed strain 1306 (WT). Error bars reflect variation in three biological replicates.
Fig 6.
Effect of PcRPS9 promoter size on GUS expression.
(A) Expression vectors based on the 500, 420, and 325-nt versions of the PcRPS9 promoter. (B) Expression driven by different versions of the PcRPS9 promoter in P. infestans. Each bar represents values from independent transformants, based on the average of two biological replicates. NC is an empty-vector control.
Fig 7.
Comparison of PcRPS9 and ham34 promoter strength.
Transformants of P. infestans were obtained in parallel experiments using plasmids containing the two promoters fused to the GUS gene. Specific activities of the transformants were then determined. Also shown are historic data for GUS driven by ham34 and hsp70, which was taken from reference 45. The middle line in the box plot represents the median expression level.
Fig 8.
In planta expression of PcRPS9 promoter.
Detached tomato leaflets were inoculated with a P. infestans transformant expressing a fusion between GUS and the promoter from PcRPS9, and stained histochemically after 4 days. (A, B) edges of lesions in which P. infestans was growing within the leaflet. The direction of growth is from right to left. Little staining is observed in the older part of the lesion, since the hyphae there have become vacuolated. (C) region of leaflet where sporulation was starting, showing staining of hyphae emerging on the plant surface.