Gene regulation of Sclerotinia sclerotiorum during infection of Glycine max: on the road to pathogenesis

Abstract

Background: Sclerotinia sclerotiorum is a broad-host range necrotrophic pathogen which is the causative agent of Sclerotinia stem rot (SSR), and a major disease of soybean (Glycine max). A time course transcriptomic analysis was performed in both compatible and incompatible soybean lines to identify pathogenicity and developmental factors utilized by S. sclerotiorum to achieve pathogenic success.

Results: A comparison of genes expressed during early infection identified the potential importance of toxin efflux and nitrogen metabolism during the early stages of disease establishment. The later stages of infection were characterized by an apparent shift to survival structure formation. Analysis of genes highly upregulated in-planta revealed a temporal regulation of hydrolytic and detoxification enzymes, putative secreted effectors, and secondary metabolite synthesis genes. Redox regulation also appears to play a key role during the course of infection, as suggested by the high expression of genes involved in reactive oxygen species production and scavenging. Finally, distinct differences in early gene expression were noted based on the comparison of S. sclerotiorum infection of resistant and susceptible soybean lines.

Conclusions: Although many potential virulence factors have been noted in the S. sclerotiorum pathosystem, this study serves to highlight soybean specific processes most likely to be critical in successful infection. Functional studies of genes identified in this work are needed to confirm their importance to disease development, and may constitute valuable targets of RNAi approaches to improve resistance to SSR.

Keywords: Effectors; Glycine max; Hydrolytic enzymes; Oxalic acid; Reactive oxygen species; Resistance; Sclerotinia sclerotiorum; Sclerotinia stem rot; Transcriptomics; White Mold.

Fig. 1

Disease symptoms observed following petiole inoculation with an agar plug containing actively growing mycelia of S. sclerotiorum at 24, 48, 96 h post-inoculation (hpi) and 7 days post inoculation (dpi). a Susceptible (S) line. b Resistant (R) line. At 7 dpi in the R line, red coloration at point of inoculation (red node) is prominently visible. The red stem phenotype developed in 90% of the R plants tested (9 out of 10), while none of the S plants showed this phenotype

Fig. 2

Real-time quantitative PCR (RT-qPCR) validation of RNA Sequencing (RNA-Seq) data in the S line infection. Log2 Fold Change (LogFC) values were generated for RT-qPCR samples by comparing the expression of genes at each timepoint of infection vs. the culture control using the 2 − ΔΔCt method. LogFC values were generated for RNA-Seq samples by comparing the average RPKM values of genes at each timepoint of infection vs. the culture control. Data are presented as means ± standard error (SE) from three independent replicates

Fig. 3

Summary heat map showing all differentially expressed genes (DEGs) in the comparison of the 96 hpi timepoint to the average of the 24 and 48 hpi timepoints. Columns represent replicates of each timepoint and rows represent individual genes. The tree above the heatmap demonstrates the hierarchical clustering of the samples. Red is used to represent genes which were up-regulated, and blue is used to represent genes which were down-regulated

Fig. 4

A functional distribution characterization of genes differentially expressed over the course of infection in the S line. a the characterized functional distribution of genes up-regulated during infection (Bonferroni correction < 0.05), b the characterized functional distribution of genes down-regulated during infection (Bonferroni correction < 0.05)

Fig. 5

Venn diagram of genes up-regulated at each timepoint of infection (24, 48, and 96 hpi) when compared to the culture control (C)

Fig. 6

Secondary metabolite cluster identified on chromosome 15 of S. sclerotiorum. The cluster contains three putative Cytochrome P450s (Sscle15g106540, − 30, − 00), three putative PKSs (Sscle15g106520, − 10, − 480), two putative acetyltransferases (Sscle15g106460 and − 50), a putative FAD-dependent monooxygenase (Sscle15g106490), a putative alpha/beta hydrolase (Sscle15g106470), and a predicted protein of unknown function (Sscle15g106440). The gene loci, logFCs (compared to culture), predicted function, and homologue in B. cinerea of each gene are shown. Sscle05g042560 and Sscle05g042570 appear within this gene cluster in B. cinerea, but are found on chromosome 5 of S. sclerotiorum

Fig. 7

Functional distribution of differentially expressed in the R and S lines at 24 (a) and 96 hpi (b). Green regions of each bar represent genes which were up-regulated in the R line. Blue regions of each bar represent genes which were down-regulated in the R line