Fig 1.
Overview of transcriptome data set with host infection and asexual development in P. sojae.
(A) Schematic diagram of asexual development and infection stages. Asexual stages including Mycelia (MY), Sporangia (SP), Zoospores (ZO), Cyst (CY) and Germinating cysts (GC). Infection stages includes IF1.5h, IF3h, IF6h, IF12h, IF24h, which mean that samples were taken after 1.5, 3, 6, 12, 24 h inoculation onto susceptible soybean leaves. (B) Hierarchical clustering of expression matrix of transcripts in asexual and infection stages. The expression matrix was removed of transcripts with low expressions. (C) Correlation analysis of expression data from ten asexual and infection stages. x-coordinate: Hj index for quantification of transcriptome diversity; y-coordinate: δj index for transcriptomic profile specialization.
Fig 2.
Transcription patterns and functional enrichment of clustered genes.
Normalized expressions of all genes were clustered into six models using STEM. Significant modules are shown in different colors. (A-F) Left: Rader maps display the average expression levels in each expression models. In each module, the five rounded pictures represent the five asexual stages (MY: mycelia; SP: sporangia; ZO: zoospores; CY: Cyst; GC: germinating cysts), and the distance from the center of the map represents the overall mRNA levels of each stage compared with the mean of the five stages. Right: the right side illustrates enriched functional clusters in each module. GOs (Gene Ontology) enrichment analysis of included genes in each expression models. GOs are represented by circles sized according to the number of expressed genes per GO. GOs significantly enriched in genes in a chi-squared test (adjusted p < 0.01 after Bonferroni correction for multiple testing) are colored according to their enrichment score.
Fig 3.
Serine/threonine phosphatase (PP2C) required for sporangia formation and mycelial growth in P. sojae.
(A) Hierarchical clustering of transcription levels of the genes from serine/threonine phosphatase cluster. Domain structure of each gene were displayed on the right panel. (B) Growth characteristics of the wild-type (P6497) and PP2C-knockout mutants (PP2C-Line1 and PP2C-Line2) on V8 agar medium without (Mock) or with different concentrations of NaCl, Sorbitol or H2O2. (C) Rader maps display the relative colony diameters after 4 days of growth in different stresses. Colony diameters were measured in each independent biological experiment after 4 days of growth. And Relative colony diameters were calculated for each treatment relative to growth on V8 agar medium only. Different stress treatments were colored with pale green (Nacl), thistle (H2O2), and sky blue (Sorbitol). (D) Upper panel: The numbers of sporangia in wild-type (P6497) and PP2C-knockout mutants (PP2C-Line1 and PP2C-Line2). The relative numbers of sporangia were labeled under the pictures. Lower panel: The sporangia morphology and cytoplasm cleavage within sporangia were observed in the wild-type (P6497) and PP2C-knockout mutants (PP2C-Line1 and PP2C-Line2).
Fig 4.
A histidine kinase (HK) with phosphotransferase activity necessary for zoospore chemotaxis in P. sojae.
(A) On the left shows that hierarchical clustering of the genes with phosphotransferase activity from fifth module in Fig 2. And on the right show the corresponding domain structures. (B) Analyses of zoospore chemotaxis to root hairs and agar blocks with daidzein. Root tips or agar blocks contains daidzein were placed in glass slides containing equal amounts of zoospore suspensions from the wild-type (P6497) and HK-knockout mutants (HK-Line1 and HK-Line2). Photos were taken after 5 min in root hairs and 20 min in agar blocks, while most of the zoospores got encysted. The directions of germinated cysts were indicated with red triangles. The Germinated cysts were marked with dotted circles. All experiments were repeated three times with similar results.
Fig 5.
A bZIP transcription factor (bZIP32) required for cyst germination in P. sojae.
(A) Hierarchical clustering of mRNA levels of genes involves in transcription activity in the first Module. And also the domain structures of included genes were displayed on the right panel. (B) Observation of germinated tubes at 8 h after zoospores encysted. Cysts from wild-type (WT) and bZIP32 silenced mutants (bZIP32-Line1 and bZIP32-Line2) were incubated at 25°C for 8 h and photographed. Bar, 50 μm. (C) The morphologies of germinated cysts were observed under a microscope, and the ratio of abnormal germinated cysts to the total number of cysts was calculated. All experiments were repeated three times with similar results. **Significant difference at p < 0.01.
Fig 6.
Asexual development-linked genes function in virulence.
(A) Virulence assays of mycelium plugs from wild-type (WT-MY), PP2C-knockout mutants (PP2C-Line1 and PP2C-Line2) or of zoospores from the wild-type (WT-ZO), HK-knockout mutants (HK-Line1 and HK-Line2), and bZIP32-knockout mutants (bZIP32-Line1 and bZIP32-Line2). Etiolated hypocotyls were inoculated with same size of mycelium plugs or equal numbers of zoospores (100 zoospores/5–10 μL) and incubated at 25°C in the dark. (B and C) Relative lesion length (B) and pathogen biomass (C) in inoculated hypocotyls expressed as the ratio of the amounts of Phytophthora sojae DNA to soybean DNA detected at 48 hpi, with the P. sojae/soybean ratio set at 1. All experiments were repeated three times with similar results. **Significant difference at p < 0.01. *Significant difference at p < 0.05. (D) Microscopic observations of infectious ostioles and invasive hyphae in epidermis of soybean hypocotyls at 12 hpi and 24 hpi. DAB staining was performed on the epidermis of seedling hypocotyls. Red arrowheads, infectious ostioles; gray arrowheads, invasive hyphae; bright yellow arrowheads, cell death. Bar, 20 μm. (E) Numbers of infectious ostioles in epidermal cells of soybean hypocotyls at 12 hpi. (F) Percentage of cell death response of inoculated soybean hypocotyls of wild-type (P6497), PP2C-knockout mutants (PP2C-Line1 and PP2C-Line2) and HK-knockout mutants (HK-Line1 and HK-Line2), and bZIP32-knockout mutants (bZIP32-Line1 and bZIP32-Line2) at 24 hpi. In each sample 50 invaded epidermal cells were examined and the experiments were repeated three times.
Fig 7.
Transcriptional profiling identifies novel functional genes of P. sojae associated with asexual development and pathogenesis.
This model proposes a possibility to study phase-specific genes that may play essential roles specifically in one or a few stages in P. sojae lifestyle by means of a combination of an explicit data-analysis pipeline and a molecular genome editing tool. By performing hierarchical clustering, specification, and diversification analyses, we focused on the stages of asexual development that are more remarkable in transcriptional plasticity, leading to transcriptome data being resolved into distinct phase-specific patterns. Next taking good advantages of genome editing in P. sojae, knockout mutants were generated for three candidate stage-specific genes and then found these genes function at specific asexual developmental stages, associated with their stage-specific expressions. These include a histidine kinase functioning in zoospore and cyst stages, a transcription factor in cyst germination, and a serine/threonine phosphatase in mycelia to sporangia stages. These novel functional genes participate in multiple layers of the pathogenicity process, which providing potential targets for precisely controlling diseases.