Genome Assembly of the Fungus Cochliobolus miyabeanus, and Transcriptome Analysis during Early Stages of Infection on American Wildrice (Zizania palustris L.)

Abstract

The fungus Cochliobolus miyabeanus causes severe leaf spot disease on rice (Oryza sativa) and two North American specialty crops, American wildrice (Zizania palustris) and switchgrass (Panicum virgatum). Despite the importance of C. miyabeanus as a disease-causing agent in wildrice, little is known about either the mechanisms of pathogenicity or host defense responses. To start bridging these gaps, the genome of C. miyabeanus strain TG12bL2 was shotgun sequenced using Illumina technology. The genome assembly consists of 31.79 Mbp in 2,378 scaffolds with an N50 = 74,921. It contains 11,000 predicted genes of which 94.5% were annotated. Approximately 10% of total gene number is expected to be secreted. The C. miyabeanus genome is rich in carbohydrate active enzymes, and harbors 187 small secreted peptides (SSPs) and some fungal effector homologs. Detoxification systems were represented by a variety of enzymes that could offer protection against plant defense compounds. The non-ribosomal peptide synthetases and polyketide synthases (PKS) present were common to other Cochliobolus species. Additionally, the fungal transcriptome was analyzed at 48 hours after inoculation in planta. A total of 10,674 genes were found to be expressed, some of which are known to be involved in pathogenicity or response to host defenses including hydrophobins, cutinase, cell wall degrading enzymes, enzymes related to reactive oxygen species scavenging, PKS, detoxification systems, SSPs, and a known fungal effector. This work will facilitate future research on C. miyabeanus pathogen-associated molecular patterns and effectors, and in the identification of their corresponding wildrice defense mechanisms.

Fig 1. Schematic representation of hypothetical degradation of wildrice cell wall polysaccharides by CAZymes.

A. Degradation of cellulose by cellulolytic enzymes: endo-β-1,4-glucanase, cellobiohydrolase, β-glucosidase, and lytic polysaccharide monooxygenase; B. Degradation of hemicellulose polysaccharides and substituted residues by hemicellulolytic enzymes. Xylan: endoxylanase, β-xylosidase, acetylxylan esterase, feruloyl esterase, α-L-arabinofuranosidase, α-glucuronidase; Xyloglucan: endo-β-1,4-endoglucanase, α-L-fucosidase, β-galactosidase, α-xylosidase; Galactomannan: endo-β-1,4-mannanase, β-mannoside, α-galactosidase. C. Degradation of pectin polysaccharides and substituted moieties by pectinolytic enzymes. Homogalacturonan: endo- and exopolygalacturonase, pectin and pectate lyase, pectin acetylesterase, pectin methylesterase; Xylogalacturonan: endo- and exogalacturonase, β-xylosidase; Rhamnogalacturonan: rhamnogalacturonase, rhamnogalacturonan lyase, rhamnogalacturonan acetylesterase, α-L-rhamnosidase, β-1,4-galactosidase, β-1,4-galactanase, β-glucuronidase, α-L-arabinofuranosidase, α-L-arabinanase, β-1,6-galactosidase, β-1,3-galactanase α-L-fucosidase. The glycan symbol nomenclature used was according to [85].

Fig 2. Predicted families within the major facilitator superfamily transporters in the CmTG12bL2 genome.

SP: Sugar Porter (2.A.1.1), DHA1: Drug:H⁺ Antiporter-1 (12 Spanner) (2.A.1.2), ACS: Anion:Cation Symporter (2.A.1.14), DHA2: Drug:H⁺ Antiporter-2 (14 Spanner) (2.A.1.3), MCP: Monocarboxylate Porter (2.A.1.13), V-BAAT: Vacuolar Basic Amino Acid Transporter (2.A.1.48), NAG-T: N-Acetylglucosamine Transporter (2.A.1.58), SIT: Siderophore-Iron Transporter (2.A.1.16), FHS: Fucose: H⁺ Symporter(2.A.1.7), PHS: Phosphate: H⁺ Symporter (2.A.1.9), VNT: Vesicular Neurotransmitter Transporter (2.A.1.22), UMF1: Unknown Major Facilitator-1 (2.A.1.24), D-cP: Domain-containing Protein (2.A.1.40), LAT3: L-Amino Acid Transporter-3 (2.A.1.44), SHS: Sialate:H⁺ Symporter (2.A.1.12), AAHS: Aromatic Acid:H⁺ Symporter (2.A.1.15), OCT: Organic Cation Transporter (2.A.1.19), PAT: Peptide-Acetyl-Coenzyme A (2.A.1.25), FLVR: Feline Leukemia Virus Subgroup C Receptor (2.A.1.28), OPA: Organophosphate:P_i Antiporter (2.A.1.4), Pht: Proteobacterial Intraphagosomal Amino Acid (2.A.1.53), NNP: Nitrate/Nitrite Porter (2.A.1.8).

Fig 3. Predicted families of ABC transporters in the CmTG12bL2 genome.

Black bars: ABC2 superfamily that includes: PDR, Pleiotropic Drug Resistance; Gld, Gliding Motility ABC transporter; EPP, Eye Pigment Precursor transporter; ThiW, Putative Thiamine Uptake transporter; ThiP, Thiamine Precursor; EVE, Ethyl Viologen Exporter. Gray bars: ABC1 superfamily with MDR, MultiDrug Resistance exporter; DCT, Drug Conjugate Transporter; HMT, Heavy Metal Transport; P-FAT, Peroxysomal Fatty Acyl-CoA Transporter; MPE, Mitochondrial Peptide Exporter; STE, α-Factor Sex Pheromone Exporter; CPR, Cholesterol/Phospholipid/Retinal (CPR) Flippase Family. White bar: Bact, Bacterial-like transporters.