Development of a novel strategy for fungal transformation based on a mutant locus conferring carboxin-resistance in Magnaporthe oryzae

Abstract

The accurate manipulation of genomic integration of mutant alleles or fluorescent fusion-protein constructs is necessary for understanding of pathogenic mechanism of Magnaporthe oryzae. Recently, this can be achieved by integrating of exogenous DNA randomly into genome of this pathogen, but ectopic integration may result in alteration of gene expression or gene disruption due to unpredictable position effects and/or disruption of protein-coding regions. In this study, we establish a novel strategy for locus-specific integration of exogenous DNA via carboxin-resistance reconstitution by a point-mutation (H245L) on succinate dehydrogenase subunit Mosdi1. Independent transformants derived from the same reconstitution construct showed consistent fluorescent signal and undiversified phenotypes, including hyphae growth, conidiation and pathogenicity, in M. oryzae. Meanwhile, 96 % of all transformants integrate correctly into the Mosdi1 locus as a single copy. Furthermore, we provide a vector carrying yeast recombination cassette and thus allow assembly of multiple overlapping DNA fragments by yeast in vivo recombination for gene complementation and protein localization assay.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0232-x) contains supplementary material, which is available to authorized users.

The effect of carabrone on mitochondrial respiratory chain complexes inGaeumannomyces graminis

“…; Guo et al . ). Resistance mechanisms mainly involve modification of the primary site of action of the fungicide within the pathogen.…”

“…Many different respiration inhibitors are known with many distinct binding sites on the respiration pathway. Carboxin is a systemic fungicide against most fungi, and it can inhibit the respiratory chain by affecting the enzyme activity of succinate dehydrogenase (Guo et al 2016). The fungicidal activity of all strobilurin compounds highlights their ability to inhibit mitochondrial respiration by binding at the Q o site of cytochrome b, which is part of the cytochrome bc 1 complex (Bartlett et al 2002).…”

“…The fungicides are still effective in many situations. The resistance of carboxin and the strobilurin was discovered in some pathogens (Bartlett et al 2002;Guo et al 2016). Resistance mechanisms mainly involve modification of the primary site of action of the fungicide within the pathogen.…”

A Novel Site-Specific Integration System for Genetic Modification of Aspergillus flavus

“…The pFC-eGFP vector was generated by in vivo recombination in the yeast strain FY834 following published procedures (Guo et al 2016). To facilitate assembly of the DNA fragments in yeast, primers were designed with 30 bp DNA sequences homologous to the upstream and downstream of regions of the adjacent fragments to be combined together.…”

“…A single histidine-to-leucine point mutation in the third cysteine-rich cluster of the SdhB subunit confers resistance to carboxin and was first used as dominant selectable marker in Ustilago maydis (Broomfield and Hargreaves 1992;Keon et al 1991). This valuable selection marker was then used in several other fungi, such as Zymoseptoria tritici (Kilaru et al 2015), Magnaporthe oryzae (Guo et al 2016), Mortierella alpine (Ando et al 2009), Aspergillus oryzae (Shima et al 2009), and a few mushrooms (Herzog et al 2019;Shang et al 2018). Although point mutations in either SdhC or SdhD were also shown to confer carboxin resistance (Ito et al 2004), the SdhB-type mutations exhibited the strongest resistance (Shima et al 2009).…”