The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR/Cas9 and Cre/loxP systems
Colletotrichum orbiculare CRISPR/CAS9Molecular Biology

Strains, culture conditions and infection assays

C. orbiculare strain 104-T (MAFF240422) was used as the WT strain in this study. lig4 strain was previously established by disrupting LIG4 gene in 104-T strain (47). Fungal strains were incubated on PDA medium (Nissui) or minimal medium (1.6 g/L yeast nitrogen base without amino acids (BD), 2 g/L asparagine (Wako), 1 g/L ammonium nitrate (Nacalai tesque), 15 g/L agar (Nacalai tesque)) containing 2 % indicated sugars at 24 °C under dark conditions. Selective agents were used at a final concentration of 100 μg/mL for hygromycin (Wako), 25 μg/mL for bialaphos (Wako), or 100 μM for 2’-deoxy-5-fluorouridine (FdU) (Tokyo Chemical Industry). For infection assays, true leaves from 3- or 4-week-old N. bentehmiana or cotyledons of 1-week-old C. sativa were used. Five μL of 5.0 x 105 spores was dropped on detached leaves, and incubated in sealed dishes with wet paper for keeping humidity at 24 °C under 16 h light / 8 h dark cycle.

Plasmid construction

The detailed information of plasmids used in this study are described in Fig. S2. For pChPtef026 vector, DNA fragments of the C. higginsianum U6 snRNA gene promoter with gRNA scaffold, the A. melanogenum translation elongation factor 1-alpha (tef) gene promoter, the fungal codon-optimized Cas9 gene, and the Agaricus bisporus heat shock protein 26 kDa (HSP26) gene terminator, were synthesized by GenScript. Synthesized gene fragments were assembled and clone into the Apa_I/_Eco_RV sites of pDONR221(Thermo Fisher Scientific) by using T4 DNA ligase (NEB) or In-Fusion HD-Cloning kit (Clontech). For inserting a gRNA fragment, annealed oligoDNA was ligated into _Bsa_I-digested pChPtef026 vector using T4 DNA ligase. For pChPtef026 Cre vector, fungal codon-optimized _Cre gene, synthesized by FASMAC, was inserted to the Nco_I/_Sac_I sites of pChPtef026 vector to replace _Cas9 with Cre. For pCB1636 pks1 vector, 1 kbp upstream sequence from the start codon of PKS1 and 1 kbp downstream sequence from stop codon of PKS1 were assembled into the Xho_I/_Sal_I sites and the _Cla_I/_Hin_dIII sites of pCB 1636 vector (2), respectively, by SLiCE reaction (48). For pCB1636 lox HPT-TK, DNA fragments of _lox66, TrpC promoter, fungal codon-optimized hygromycin phosphotransferase (HPT) gene, fungal codon-optimized thymidine kinase (TK) gene and lox71 were synthesized by FASMAC. These fragments were assembled to the Sal_I/_Cla_I sites of pCB1636 vector by SLiCE reaction. For pCB636 _pks1 lox HPT-TK, 1 kbp upstream sequence from the start codon of PKS1 and 1 kbp downstream sequence from stop codon of PKS1 were assembled into the Xho_I/_Sal_I sites and the _Cla_I/_Hin_dIII sites of pCB1636 lox HPT-TK vector, respectively, by SLiCE reaction. For pCB _pks1 clox HPT-TK, a Coxyl1 promoter fragment was amplified from genome of C. orbiculare. Cre, Coxyl1 promoter and AbHSP26 terminator were assembled into the Nhe_I/_Asc_I sites of pCB1636 _pks1 lox HPT-TK vector. For pCB1636 pks1 clox Bar-TK, HPT gene of pCB pks1 clox HPT-TK vector was replaced with bialaphos resistance (Bar) gene by PCR. For gene disruption of transporter genes, 1 or 0.5 kbp fragments of upstream from start codon and 1 or 0.5 kbp fragments of downstream from stop codon were amplified from genome of C. orbiculare. Self-excision Cre/loxP cassette was amplified from pCB1636 pks1 clox HPT-TK vector or pCB1636 pks1 clox Bar-TK vector. Three fragments (two homologous arms and one marker cassette) were fused by PCR (49). A fused DNA fragment was inserted into the _Xho_I/_Hin_dII sites of pCB1636 vector. All DNA fragments were amplified by KOD one (TOYOBO).

Fungal transformation

PEG-mediated fungal transformation, which was previously described (2), was applied to introduce plasmids or PCR fragments to C. orbiculare protoplasts. Protoplasts were prepared by degrading cell wall via driselase (Sigma) and lysing enzyme (Sigma). Ten μg of plasmids or PCR fragments was used for transformation. Transformants were selected on PDA with 0.6 M glucose containing appropriate anti-biotics for 5-7 days. HR efficiency of pks1 mutants was calculated at this time without PCR-based genotyping. Transformants were further transferred onto PDA medium with appropriate anti-biotics for several days. For genotyping PCR, mycelium was picked into 100 μL TE buffer and microwaved for 5 min twice. After centrifugation, supernatant was used as a template. For removing the selective marker cassette of self-excision Cre/loxP system, KO strains were transferred onto PDA medium containing 2 % xylose for 5 days. Conidia were harvested and streaked onto PDA media containing FdU. After a few days, single colonies were picked and transferred onto PDA medium containing FdU. For checking a removal of the selective marker cassette, genotyping of transformants were performed by the above-described method. In addition, transformants were transferred onto PDA medium containing appropriate selective agents to check whether they lost the resistance against the anti-biotics.

Measurement of sugar content in infected leaves

C. orbiculare-infected leaves were frozen and ground in liquid nitrogen, and homogenized with 1 ml of extraction buffer (methanol:water:chloroform = 5:2:2) with 10 μg ribitol (Wako) as an internal control. Mixtures were incubated for 30 min at 37 °C. After centrifugation, 900 μl of the supernatant was transferred to a new tube, and 400 μl of water was added. After centrifugation, the upper phase was transferred to new tube. These samples were evaporated using a spin dryer at 50 °C, and subsequently freeze-dried. Samples were sonicated in 30 μl of methoxyamine (Sigma) (20 mg/ml dissolved in pyridine (Wako)), and incubated for 90 min at 30 °C. Subsequently, 30 μl of MSTFA + 1% TMCS (Thermo Fisher Scientific) was added and incubation was continued for 30 min at 37 °C. After centrifugation, the supernatants were subjected to gas chromatography-mass spectrometry analysis. Each sample (1 μl) was separated on a gas chromatograph (7820A; Agilent Technologies) combined with a mass spectrometric detector (5977B; Agilent Technologies). For quantitative determination of metabolites, peaks that originated from selected ion chromatograms (quinate 345, cellobiose 361, ribitol 319) were used.

Article TitleThe establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR/Cas9 and Cre/loxP systems


Phytopathogenic fungi belonging to the Colletotrichum genus cause devastating damage for many plant species. Among them, Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to efficiently generate multiple knockout mutants of C. orbiculare. We first found that CRISPR/Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR/Cas9 vector, more than 90 % of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre/loxP marker recycling system for C. orbiculare because limited availability of desired selective markers hampers sequential gene disruption. In this system, integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling reuse of the same selective marker for the next transformation. Using our CRISPR/Cas9 and Cre/loxP systems, we attempted to identify functional sugar transporters in C. orbiculare. Multiple disruptions of putative quinate transporter genes restrict fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. Our analyses revealed that quinate acquisition is dispensable during fungal infection because this mutant displayed normal virulence to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants established in this study, we inferred that repetitive rounds of gene disruption using CRISPR/Cas9 and Cre/loxP systems do not cause negative effects for fungal virulence and growth. Therefore, these systems will be powerful tools to perform systematic gene targeting approach for C. orbiculare.

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