Digital gene expression profile analysis of the WT and Δdcl2 mutant.

Conidia of the WT and the Δdcl2 mutant were inoculated into SDAY medium at 1 × 10⁷ conidia/ml. Fungal samples were collected after 1 day (mycelial growth [MY]) and 2.5 days (conidiogenesis [CO]). Total RNA was extracted using TRIzol reagent (Invitrogen, New York, NY, USA), according to the manufacturer's instructions. Total RNA (5 μg) was prepared for Illumina RNA-Seq, as we described previously (53). Briefly, poly(A) mRNA from the total RNA was enriched using oligo(dT) beads. Following purification, fragmentation buffer was added to break mRNA into small pieces. Using these cleaved RNA fragments as the template, the first cDNA strand was synthesized by reverse transcription with random hexamer primers. The second-strand cDNA was synthesized using buffer, dinucleoside triphosphates (dNTPs), RNase H, and DNA polymerase I, and the sequencing library was constructed according to the manufacturer's instructions (Illumina, San Diego, CA, USA). The libraries were sequenced on the Illumina HiSeq 2000 apparatus (Beijing Genomics Institute, Shenzhen, China) to produce 50-bp paired-end reads. Prior to mapping to the M. robertsii reference genome and annotated genes, adaptor tags, unknown, or low-quality tags were filtered to get clean tags. To compare levels of gene expression between the WT and a mutant, the number of raw clean tags in each library was normalized to the number of reads per kilobase per million reads (RPKM) to normalize gene expression levels. DEGs were identified using a false-discovery rate (FDR) of ≤0.001 and a fold change of ≥2 as the threshold. To further characterize the biological functions and metabolic pathways of DEGs, the DEGs were subjected to a GO functional analysis (Blast2GO; BioBam Bioinformatics) (54). A GO enrichment analysis of functional significance uses a hypergeometric test to map all DEGs to terms in the GO database, looking for significantly enriched GO terms among the genes differentially expressed from those in the background genome.

To validate the DGE results, 18 candidate genes were analyzed by qRT-PCR. The selected genes and primers are listed in Table 2. RNA samples used for qRT-PCR were the same as those used for DGE sequencing. First-strand cDNA synthesis and qRT-PCR were performed as described above. Three technical replicates of qRT-PCR were performed for each biological replicate.