Fig 1.
Generalised sterol biosynthesis pathway.
Dashed arrows indicate that multiple enzymatic steps are occurring. The end sterols β-sitosterol, stigmasterol, campesterol, fucosterol and cholesterol have a common sterol nucleus structure (R1, shown in the box) differing only at the side chain, while the core of ergosterol (R2, shown in the box) has an additional point of desaturation in the sterol core.
Fig 2.
GC-MS analysis of S. parasitica sterol composition.
Samples analysed were grown for 3 days in Machlis, YM, or Peptone media. GC-MS analyses were performed in triplicate. The dominant sterol was desmosterol for mycelia grown in Machlis (red trace in the GC chromatogram) and YM media (black trace), while 24-methylenecholesterol dominates in the mycelium grown in Peptone medium (blue trace). RT: Retention time.
Table 1.
Gene sequences putatively involved in the MVA and sterol biosynthesis pathways of S. parasitica.
Fig 3.
Gene expression analysis by qPCR of putative S.parasitica sterol related genes.
Expression levels of each gene were standardised by comparison with the levels of expression of 3 housekeeping genes, and normalised to expression levels during growth on the defined Machlis medium. Genes are identified by their Gene ID. SPRG_11783: oxidosqualene cyclase (lanosterol synthase). SPRG_09493: CYP51 sterol 14α-demethylase. SPRG_00418: Δ14 sterol reductase. SPRG_01623: Δ4 methyl sterol oxidase. SPRG_05001: Δ 24 sterol methyltransferase. SPRG_13330: Δ8 sterol isomerase. SPRG_18544: Δ5 sterol desaturase. SPRG_01085: Δ7 sterol reductase. SPRG_04988: Δ24 sterol reductase. Three replicate samples were analysed in each case. Error bars represent one standard deviation from the mean.
Table 2.
Gene sequences putatively involved in the mevalonate (MVA) pathway of P. infestans, or with roles in sterol modification.
Fig 4.
Gene expression analysis by qPCR of putative P. infestans sterol related genes.
Expression levels of genes with possible roles in sterol modification were studied during growth in media supplemented with a range of different sterols. Transcript abundance was standardised by comparison with 3 housekeeping genes, and normalised to expression levels during growth on the host sterol β-sitosterol. Genes are identified by their Gene ID. PITG_21426: Δ5 sterol desaturase. PITG_13128: putative Δ7 sterol reductase. PITG_12495: HMG-CoA synthase. Three replicate experiments were performed in each case. Error bars represent one standard deviation from the mean.
Fig 5.
In silico reconstruction of the sterol synthesis pathway of S. parasitica.
Synthesis steps are indicated by the gene (SPRG_xxxxx) predicted to encode the responsible enzyme. An asterisk (*) highlights those sterols which were previously extracted from Saprolegniales species. All sterols highlighted in grey boxes were identified in S. parasitica mycelium during this study. Zymosterol is shown larger than other sterols to highlight its role as a branching point between multiple synthetic routes.
Fig 6.
Potential cross-talk between the three major pathways of sterol biosynthesis in Saprolegnia parasitica.
The flow-chart shows how the enzymes encoded by SPRG_05001 (Δ24 sterol methyltransferase) and SPRG_04988 (Δ24 sterol reductase) may be capable of acting on multiple different sterols, creating cross-talk between the three synthetic routes described in Fig 5. Solid arrows show the main synthesis routes depicted in Fig 5, while the dashed arrows indicate predicted points of cross-talk between these routes.