Global transcriptome analysis of Sclerotinia sclerotiorum following infection of resistant and susceptible soybean lines

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. Overall design: Four week old soybean plants of both a susceptible (S) and resistant (R) lines were infected with S. sclerotiorum by petiole inoculation using a 1 ml pipette tip agar plug of actively growing fungal hyphae. Plant tissue was sampled by cutting horizontally above and below (1.5 cm) the node of the inoculated petiole with a clean straight-edge razor. Tissue samples were then immediately frozen in liquid nitrogen prior to RNA extraction. The experimental design was completely randomized and consisted of three biological replicates for each of the treatments. For each of the experimental conditions, stem segments (~3 cm, first internode) from 2 different plants were pooled together to form one of three biological replicates. Total RNA was extracted from soybean stem tissues and infecting S. sclerotiorum using a modified TRIzol™ Reagent protocol (Invitrogen Corp., Carlsbad, CA, USA). Further, samples were cleaned using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RNA concentration and purity was determined by Nanodrop (Thermo Fisher Scientific, Wilmington, DE) and sample quality was assessed using an Agilent Bioanalyzer 2100 and an RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA). Three biological replicates were generated per treatment. Library preparation was performed at the University of Wisconsin – Madison Biotechnology Centre (Madison, WI, USA). Individually indexed libraries were prepared using the TruSeq RNA Sample Preparation v2 kit according to the manufacturer's instructions (Illumina, San Diego CA, USA). Library concentrations were quantified with the Qubit HS DNA kit (Thermo Fisher Scientific, Wilmington, DE). The size and quality of the libraries were evaluated with an Agilent Bioanalyzer 2100 and an Agilent DNA 1000 kit (Agilent Technologies, Santa Clara, CA) and the libraries were sequenced using Illumina HiSeq2500 (1X100bp) (Illumina, San Diego CA, USA). Illumina raw read data quality was verified with FastQC (http://www.bioinformatics.babraham.ac. uk/projects/fastqc/). The soybean and S.sclereotiorum genomes were collected from Phytozome v12.1 (https://phytozome.jgi.doe.gov/pz/portal.html#! bulk?org=Org_Gmax) and the Broad institute (https://www.broadinstitute.org/fungal-genome-initiative/sclerotinia-sclerotiorum-genome-project), respectively. Raw sequence reads were mapped to both genomes using the Subjunc aligner from Subread. Alignments were compared to the gene annotation GFF files for both organisms (Soybean: Gmax_275_Wm82.a2.v1.gene.gff3, S. sclereotiorum: sclerotinia_sclerotiorum_2_transcripts.gtf) and raw counts for each gene were generated using the feature Counts tool from Subread. The raw counts data were normalized with the trimmed mean of means (TMM) normalization method using edgeR package. The normalized gene counts were transformed to log2 scale using the voom method from the R Limma package, then used to generate differential expression (log2FC) values [97, 98]. DEGs (log2FC >1 or <-1) were generated for S. sclereotiorum genes from the comparison of inoculated soybeans at different timepoints to the culture control (FDR<0.05), between early (24 and 48 hpi) and late (96 hpi) infection (FDR<0.05), and between the timepoints of resistant (R) and susceptible (S) line infections (p-value < 0.01).