Annotation and analysis of the mitochondrial genome of Coniothyrium glycines, causal agent of red leaf blotch of soybean, reveals an abundance of homing endonucleases

Abstract

Coniothyrium glycines, the causal agent of soybean red leaf blotch, is a USDA APHIS-listed Plant Pathogen Select Agent and potential threat to US agriculture. Sequencing of the C. glycines mt genome revealed a circular 98,533-bp molecule with a mean GC content of 29.01%. It contains twelve of the mitochondrial genes typically involved in oxidative phosphorylation (atp6, cob, cox1-3, nad1-6, and nad4L), one for a ribosomal protein (rps3), four for hypothetical proteins, one for each of the small and large subunit ribosomal RNAs (rns and rnl) and a set of 30 tRNAs. Genes were encoded on both DNA strands with cox1 and cox2 occurring as adjacent genes having no intergenic spacers. Likewise, nad2 and nad3 are adjacent with no intergenic spacers and nad5 is immediately followed by nad4L with an overlap of one base. Thirty-two introns, comprising 54.1% of the total mt genome, were identified within eight protein-coding genes and the rnl. Eighteen of the introns contained putative intronic ORFs with either LAGLIDADG or GIY-YIG homing endonuclease motifs, and an additional eleven introns showed evidence of truncated or degenerate endonuclease motifs. One intron possessed a degenerate N-acetyl-transferase domain. C. glycines shares some conservation of gene order with other members of the Pleosporales, most notably nad6-rnl-atp6 and associated conserved tRNA clusters. Phylogenetic analysis of the twelve shared protein coding genes agrees with commonly accepted fungal taxonomy. C. glycines represents the second largest mt genome from a member of the Pleosporales sequenced to date. This research provides the first genomic information on C. glycines, which may provide targets for rapid diagnostic assays and population studies.

Fig 1. Circular mapping of the mitochondrial genome of Coniothyrium glycines.

Black blocks, grey blocks, hatched blocks, stipled blocks, and bars show, respectively, protein-coding, orfs, rRNA, introns, and tRNA genes. Arrows indicate the direction of transcription.

Fig 2. Mitochondrial genome rearrangements among Dothidiomycetes.

Asterisk (*) indicates reverse direction of transcription. Each gene is assigned a separate color. Gene order was obtained from GenBank: Bipolaris cookei (MF784482), Didymella pinodes (NC_029396), Parastagonospora nodorum (NC_009746), Pithomyces chartarum (KY792993), Shiraia bambusicola (NC_026869), Stemphylium lycopersici (KX453765), Zasmidium cellare (NC_030334), and Zymoseptoria tritici (NC_010222).

Fig 3. Phylogenetic tree constructed from unambiguously aligned portions of concatenated protein-coding sequences of twelve protein-coding genes shared in common among 25 fungal mt genomes.

Topology shown was inferred with PhyML 3.0 using LG as the evolutionary model. Sequences were obtained from GenBank: Arthroderma otae (NC_012832); Aspergillus niger (NC_007445); Beauveria bassiana (NC_010652); Botryotinia fuckeliana (KC832409); Cladophialophora bantiana (NC_030600); Didymella pinodes (NC_029396): Epichloe typhina (NC_032063); Glarea lozoyensis (KF169905); Hypocrea jecorina (NC_003388); Lecanicillium saksenae (NC_028330); Metarhizium anisopliae (NC_008068); Parastagonospora nodorum (NC_009746); Peltigera dolichorrhiza (NC_031804); Penicillium polonicum (NC_030172); Pseudogymnoascus pannorum (NC_027422); Pyronema omphalodes (NC_029745); Sclerotinia borealis (NC_025200); Shiraia bambusicola (NC_026869); Talaromyces marneffei (NC_005256); Trichophyton rubrum (NC_012824); Verticillium dahliae (NC_008248); Zasmidium cellare (NC_030334); Zymoseptoria tritici (NC_010222); Phialocephala subalpina (NC_015789).