Integrated omics study of lipid droplets from Plasmodiophora brassicae

Abstract

Plasmodiophora brassicae causes clubroot disease in cruciferous. In this report, lipid droplets were observed in the resting spores of P. brassicae. 295 lipid droplet-associated proteins were identified and categorized into nine groups. Transcriptome analysis of these proteins during three different zoosporic stages revealed differences in gene expression pattern. GO enrichment analysis revealed that these proteins associated with lipid droplets were mainly linked to biosynthesis and metabolism. GC-MS analysis revealed that lipid droplets contain seven types of free fatty acids: saturated fatty acids C16:0 and C18:0, and unsaturated fatty acids C18:1Δ9, C18:1Δ11, C18:2, C20:4 and C20:5. P. brassicae accumulated a large amount of triacylglycerols (TAGs). We systematically analyzed the putative proteins involved in TAG biosynthesis and its metabolic pathway. KEGG pathway analysis defined 3390 genes, including 167 genes involved in lipid metabolism. Transcriptome analysis revealed that 162 candidate enzymes involved in lipid metabolism were differential expressed. Our omics studies are the first to investigate the lipid droplet organelles in P. brassicae, providing a reference resource to study protist lipid droplets.

Figure 1. The observation of B. rapa galls infected with P. brassicae.

(A) Transmission electron microscopy (TEM) of resting spores of P. brassicae in clubroots. (B) TEM of resting spores of P. brassicae. (C) TEM of secondary plasmodia of P. brassicae. RS = resting spores, white arrow; LD = lipid droplets, red arrow; PC = plant cell; PCW = plant cell wall; NS = nucleus; CM = cell membrane; MT = mitochondria. SP = second plasmodia, white arrow. Scale bar A-B = 1 μm. (D) Fluorescent Nile red staining for P. brassicae resting spores under a 100× oil objective. (E) Nile red staining for primary zoospores of P. brassicae. The primary zoospores are visualized after staining with nile red in Arabidopsis roots 2 days after infection, when they reached the surface of a root hair. They penetrated the cell wall and forms primary plasmodia in the root hairs (F). (G) Nile red staining for P. brassicae zoosporangia, which were visualized after staining with nile red in Arabidopsis roots 12 days after infection. H and I, Nile red staining of P. brassicae secondary plasmodia and resting spores in Arabidopsis roots at the root cortex cell. Arabidopsis roots infected with P. brassicae for 15 days were sectioned in the transverse plane (H). Arabidopsis roots infected with P. brassicae for 21 days (I). Scale bar = 5 μm.

Figure 2. Isolation of lipid droplets from P. brassicae resting spores.

(A) Spore suspensions are disrupted under high pressure and then centrifuged in SW40 tubes to remove nuclei, cell debris and unbroken cells. The LDs floated to the top of the gradient. (B) Lipid droplets stained with nile red, as viewed by fluorescence microscopy. Scale bar = 10 μm. (C) Isolated lipid droplets imaged by TEM after negative staining (scale bar = 0.5 μm). (D) TLC analysis of the total lipid extracted from isolated lipid droplets. (E) The size distribution of the purified lipid droplets. Values were generated using a Malvern Zetasizer Nanoseries (Malvern, England) equipped with a 633 nm laser. (F) Lipid droplets-associated proteins were extracted from P. brassicae, separated by 10% SDS-PAGE, and visualized by Coomassie blue staining. Arrows indicate the positions at which the gel was sliced.

Figure 3. Functional and expressional analysis of lipid droplets-associated proteins.

(A) 295 lipid droplet-associated proteins were identified in P. brassicae and categorized into nine groups based on genome searches and the KEGG and NCBI databases. (B) A heat map showing the expression of all 295 genes. (C) A histogram showing the percentage of lipid droplet–associated genes with dramatic differences in expression between the three life stages.

Figure 4. GC-MS analysis of lipid droplet free fatty acid content and expression heat maps of genes associated with unsaturated fatty acid biosynthesis, arachidonic acid metabolism, and fatty acid metabolism in P. brassicae.

(A) GC-MS analysis of the free fatty acid methyl ester composition of lipid droplets. Note: 1. C16:0; 2. C18:0; 3. C18:1Δ9; 4. C18:1Δ11; 5. C18:2Δ9, 12; 6. C20:4Δ5, 8, 11, 14; 7. C20:5Δ5, 8, 11, 14, 17; 8. 2, 4-bis (1,1-dimethylethyl)-phenol; 9. Benzenepropanoic acid-methyl ester; and 10. C₃₂H_66. (B) Gene expression of genes in KEGG categories involved in unsaturated fatty acid biosynthesis, arachidonic acid metabolism and fatty acid metabolism. A colored bar indicating the normalized reads per LOG2 (RPKM) accompanies the expression profile.

Figure 5. Measurement of TAG contents and an expression analysis of gene families involved in TAG biosynthesis and degradation.

(A) Measurements of the TAG contents of multiple species. TAG content was assessed using a tissue glyceride assay kit from APPLYGEN. Protein content was assessed using a Total Protein Assay Kit from Thermo Scientific. Data are means from three independent experiments; error bars show the SD. p < 0.01 (spss). Plasmodiophora brassicae: P. brassicae, Saccharomyces cerevisiae: S. cerevisiae, Trichoderma viride: T. viride, Chlamydomonas reinhardtii: C. reinhardtii, Escherichia coli: E. coli, and Pseudomonas syringae: P. syringae. (B) TAG biosynthesis, storage and degradation pathways are divided into 4 biochemical stages and 11 reactions. (C) A heatmap of the enzymes involved in each reaction. (D) EC numbers of enzymes involved in (B).

Figure 6. KEGG categories and gene expression of genes involved in lipid metabolism.

(A) KEGG categories of P. brassicae lipid metabolism genes. (B) The expression levels of genes involved in lipid metabolism during different developmental stages. The levels were measured using the geometric mean of the TPM values of all genes in the corresponding group. To calculate the geometric mean of the TPM values, all TPM values of 0 were replaced by 0.001. (C) Gene expression in KEGG categories related to lipid metabolism. A colored bar indicating the normalized reads per LOG2 (RPKM) accompanies the expression profile. (D) A histogram showing enzyme number and changes in expression during the three life stages.