Computational protocol: The Fusarium graminearum Histone Acetyltransferases Are Important for Morphogenesis, DON Biosynthesis, and Pathogenicity

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Protocol publication

[…] To identify putative histone acetyltransferase genes in F. graminearum, a BLASTp search with GCN5, RTT109, SAS2, and SAS3 of S. cerevisiae, F. oxysporum, A. nidulans, N. crassa, S. pombe, and C. albicans were performed in the F. graminearum genome database. The phylogenetic tree analysis was conducted using the neighbor-joining method in MEGA 5.0. Bootstrap analysis was conducted using 1,000 replicates in MEGA 5.0. [...] To generate constructs for disruption of each of the four putative F. graminearum HATs gene, their flanking regions were amplified by PCR using wide-type PH-1 genomic DNA, extracted with the E.Z.N.A.® Fungal DNA Mini Kit (Omega Bio-tek, Inc. Orlando, USA), as the template. Primers for amplification of the upstream (primers UF+UR) and downstream (primers DF+DR) flanking regions are listed in (Table ). Flanking sequences were ligated into plasmid pKH-KO (that carries the hygromycin resistance gene HPH). For the construction of gene complementation vectors, sequences, which include promoter, gene, and terminator regions are amplified by PCR using primers F+/R+ (Table ), and were ligated into plasmid pKN (which carries the G418 resistance gene NEO) with the seamless assembly cloning kit (Clone Smarter Techology, USA). The gene deletion and complementation plasmids are digested and subsequent, transformed to PH-1 using the protocols described previously (Turgeon et al., ). Hygromycin B (AMRESCO USA) and geneticin (AMRESCO USA) were added to the final concentrations of 200 and 100 μg/mL, respectively, for transformant selection. Putative gene deletion and complementation mutants were identified by PCR assays with the relevant primers (Table ), and were further validated by genome sequencing performed on the HiSeq X Ten sequencing system. Reads were aligned to reference genome by Burrows-Wheeler Aligner (Li and Durbin, ). Sam alignment files were converted to bam format, sorted, and indexed with samtools (Li and Durbin, ) and then visualized by Integrative Genomics Viewer (IGV) (Thorvaldsdóttir et al., ). [...] For RNA-seq analysis, the ΔFgSAS3, ΔFgGCN5, and PH-1 strains were cultured in TBI medium for 3 days. Total RNA was isolated from the lyophilized mycelium as described above and mRNA was isolated using a Poly(A)Purist MAG kit (Ambion). DNA was removed by treatment with RNase-free DNAase (Qiagen), followed by column clean-up according to manufacturer's instructions, then Illumina TruSeq RNA Sample Preparation kits were used to make RNA-seq libraries. cDNA was sequenced with the HiSeq X Ten sequencing system. The RNA-seq reads (150 bp) were mapped to the genome of the F. graminearum strain PH-1 (http://fungi.ensembl.org/) using hisat2 (Kim et al., ). Sam-formatted alignment files were converted to bam format, sorted, and indexed with samtools (Li and Durbin, ). Identification of differentially expressed genes (DEGs) from RNA-seq data was conducted by using cufflinks (Trapnell et al., ). Detection of genes differentially enriched was analyzed with TBtools (https://github.com/CJ-Chen/TBtools). Primary metabolism-associated genes were download from The Fungal and Oomycete Genomics Resource Database (http://fungidb.org/) with GO term “0044238, primary metabolic process.” Secondary metabolism gene clusters were predicted by Sieber et al. (). Genes associated with sexual reproduction were from two previous reports (Hallen et al., ; Kim et al., ). Virulence-related genes were found from PHI-base database (http://www.phi-base.org/index.jsp). […]

Pipeline specifications

Software tools BLASTP, MEGA, BWA, SAMtools, IGV, HISAT2, Cufflinks, TBtools
Databases FungiDB PHI-base
Applications Phylogenetics, RNA-seq analysis
Organisms Fusarium graminearum, Triticum aestivum, Hordeum vulgare, Aspergillus fumigatus, Solanum lycopersicum