Fig 1.
Flowchart of steps taken for R. solani AG1-IA RNA-seq sample preparation and data analysis.
Hyphae of R. solani, grown on ¼ strength PDA (control) or on soybean unifoliate leaves were harvested and processed for RNA sequencing using the Illumina HiSeq 2000 platform. Data were analyzed using standard analytical pipelines for gene annotation and differential expression analysis. Data were further compared using heatmap analysis and gene ontology terms, and affected pathways examined using Kyoto encyclopedia of genes and genomes (KEGG).
Fig 2.
Rhizoctonia solani-soybean interactions at onset and 24 h.p.o. of necrosis.
In vitro controls on PDA harvested at onset (A) and 24 h.p.o. (B) of necrosis. Microscope images of hyphae on nitrocellulose membranes in vitro at onset (C) and 24 h.p.o. (D) of necrosis showing normal growth and lack of hyphal aggregates and infection structures. Soybean leaf samples infected with R. solani AG1-IA at onset (E) and 24 h.p.o. (F) of necrosis. Arrows indicate the onset of necrotic lesions approximately 36 h post-inoculation. Microscope images of hyphae on nitrocellulose membranes from soybean leaves at onset (G) and 24 h.p.o. (H) of necrosis showing infection cushion structures (arrows). Note that hyphal aggregation and infection cushion initials occurred only in R. solani samples grown on membranes overlaid on leaves (G, H) and not on PDA (C, D).
Fig 3.
Overview of analysis of Rhizoctonia solani differentially expressed genes at onset and 24 h.p.o. of necrosis in soybean.
(A) PCA score plot of R. solani-soybean interactions (solid inverted triangles and circles) and controls (open inverted triangles and circles) at onset of necrosis (inverted triangles) and 24 h.p.o. of necrosis (circles). (B) Heatmap and hierarchical cluster analysis of R. solani-soybean interactions at onset and 24 h.p.o. of necrosis. Dendrograms were constructed using the Ward method [42]. (C) Venn diagram of transcriptionally active genes (at least 2 reads per sample in 2/3 biological replicates for any treatment) and the treatments in which they were detected (n = 3 per treatment). (D) Venn diagram of the 1,888 differentially expressed genes and the treatments in which they were detected (n = 3 per treatment). A number of zero indicates that no genes were found to be unique to the specific comparison.
Table 1.
Gene ontology slim terms over- and under-represented in Rhizoctonia solani during soybean infection.a
Fig 4.
KEGG pathway annotations common between Rhizoctonia solani-soybean interaction time points.
KEGG pathway annotations common between R. solani-soybean interactions at onset (grey) and 24 hours-post onset (red) of necrosis for differentially expressed genes. (A) KEGG pathways commonly containing up-regulated DEGs, (B) KEGG pathways commonly containing down-regulated DEGs (n = 3 per treatment). Numbers represent the number of differentially expressed genes with fold change values +/- 3 detected for each KEGG pathway.
Fig 5.
Rhizoctonia solani transcript abundance fold changes in response to infection of soybean.
Transcript abundances were quantified using qRT-PCR (blue) or RNA-seq (red) in R. solani cultures infecting soybean compared to controls in vitro at onset of necrosis or 24 h.p.o. of necrosis. (A) Up-regulation of transcripts during infection of soybean. (B) Down-regulation of transcripts during infection of soybean. Stars represent fold changes that were statistically (P<0.01 for RNA-seq or P<0.05 for qRT-PCR) and biologically (fold change ≤ -3 or ≥ 3 for RNA-seq or ≤ -1.5 or ≥ 1.5 for qRT-PCR) significant (n = 3 per treatment). Numbers below gene names represent Spearman’s correlation coefficients with stars representing significance thresholds of: *P ≤0.05, **P ≤0.01, ***P ≤0.001 or ****P ≤0.0001. ABC, ABC transporter; AMY, ALPHA-AMYLASE; BGLUC, BETA-GLUCOSIDASE; CDC, CHITIN DEACETYLASE; FDH, FORMATE DEHYDROGENASE; GCS, GLYCOGEN SYNTHASE; GST, GLUTATHIONE-S-TRANSFERASE; LAC, LACCASE PRECURSOR; NOX, NADH OXIDASE; P450, CYTOCHROME P450 MONOXYGENASE PC-3; PDX, PYRIDOXAL-DEPENDENT DECARBOXYLASE; SOD, Cu/Zn SUPEROXIDE DISMUTASE; THI, THIAMINE BIOSYNTHESIS.