Establishment of a selection marker recycling system for sequential transformation of the plant‐pathogenic fungus Colletotrichum orbiculare

Abstract

Summary Genome sequencing of pathogenic fungi has revealed the presence of various effectors that aid pathogen invasion by the manipulation of plant immunity. Effectors are often individually dispensable because of duplication and functional redundancy as a result of the arms race between host plants and pathogens. To study effectors that have functional redundancy, multiple gene disruption is often required. However, the number of selection markers that can be used for gene targeting is limited. Here, we established a marker recycling system that allows the use of the same selection marker in successive transformations in the model fungal pathogen Colletotrichum orbiculare , a causal agent of anthracnose disease in plants belonging to the Cucurbitaceae. We identified two C. orbiculare homologues of yeast URA3/pyrG , designated as URA3A and URA3B , which can be used as selection markers on medium with no uridine. The gene can then be removed from the genome via homologous recombination when the fungus is grown in the presence of 5‐fluoroorotic acid (5‐FOA), a chemical that is converted into a toxin by URA3 activity. The ura3a/b double mutants showed auxotrophy for uridine and insensitivity to 5‐FOA. Using the ura3a/b mutants, transformation with the URA3B marker and its removal were successfully applied to disrupt the virulence‐related gene, PKS1 . The pks1 mutants showed a reduction in virulence, demonstrating that the method can be used to study virulence‐related genes in C. orbiculare . The establishment of a URA3 ‐based marker recycling system in plant‐pathogenic fungi enables the genetic analysis of multiple genes that have redundant functions, including effector genes.

The ConservedColletotrichumspp. Effector CEC3 Induces Nuclear Expansion and Cell Death in Plants

“…Therefore, Colletotrichum fungi may deploy a layer of effectors with restricted virulence effects and with functional redundancy that would result in weaker selection pressure. For a better understanding of the collective virulence effect of core effectors, further work is required, for example by using multiple knock-out mutants with a selection marker recycling system (Kumakura et al, 2019).…”

An efficient marker recycling system for sequential gene deletion in a deep sea-derived fungus Acremonium sp. HDN16-126

“…Importantly, the pyrG-A1 marker was successfully recycled from the chromosome, which allows the use of the same selection marker in successive transformations. The pyrG-A1 -based marker recycling system showed several advantages over other gene deletion systems, such as sit-directed recombination methods including Flp-FRT and Cre-loxP recombinations, which have been widely used in prokaryotes and eukaryotes [22] . Those tools could easily conduct the excision of target DNA between two repeated specific sites, while for a legacy, one of the repeated sites is left on the chromosome, which makes it an unstable factor as the site specific sequences accumulated in the genome.…”

“…Indeed, the pyrG / URA3 -based counter-selection system has been successfully applied in Saccharomyces cerevisiae [ 15 ], Aspergillus spp . [16] , [35] , Candida albicans [ 18 ], Neurospora crassa [ 19 ], Talaromyces versatilis [ 20 ], Mucor circinelloides [ 21 ] and Colletotrichum orbiculare [22] . However, this system has never been applied to the genus of Acremonium species.…”

Nonribosomal peptides in fungal cell factories: from genome mining to optimized heterologous production

“…Beside NRPS genes, numerous genes such as those encoding a PPTase, tailoring enzymes, or transporter-associated genes, are required to successfully produce a bioactive NRP or NRP derivative. The cloning of many genes can imply the synthesis of several plasmids which is laborious, costly, and problematic in case only few selection markers are available for the chosen host (Kumakura et al, 2019;Li et al, 2017). Several solutions particularly well-suited to NRP heterologous production have been proposed to alleviate this issue.…”