The 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.

Genome Sequence Resources for Four Phytopathogenic Fungi from the Colletotrichum orbiculare Species Complex

Colletotrichum orbiculare species complex fungi are hemibiotrophic plant pathogens that cause anthracnose of field crops and weeds. Members of this group have genomes that are remarkably expanded relative to other Colletotrichum fungi and compartmentalized into AT-rich, gene-poor and GC-rich, gene-rich regions. Here, we present an updated version of the C. orbiculare genome, as well as draft genomes of three other members from the C. orbiculare species complex: the alfalfa pathogen C. trifolii, the prickly mallow pathogen C. sidae, and the burweed pathogen C. spinosum. The data reported here will be important for comparative genomics analyses to identify factors that play a role in the evolution and maintenance of the expanded, compartmentalized genomes of these fungi, which may contribute to their pathogenicity.