Efficient transformation of Pleurotus eryngii with a safe selective marker mutated from the Pesdi1 gene

Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporiopsis subvermispora

“…This may come from the disability of its gene product to associate and form an active complex with other endogenous subunits of the SDH complex II in the host. Therefore, it is recommended to make a homologous mutant sdi1 to develop a carboxin-resistant marker system in each mushroom, as seen in L. edodes (Irie et al 2003), A. aegerita (Herzog et al 2019), G. lucidum (Xu et al 2012), and P. eryngii (Shang et al 2018).…”

“…The development of a genetic transformation system in filamentous basidiomycetes was first reported in model species like Schizophyllum commune (Munoz-Rivas et al 1986) and Coprinopsis cinerea (Binninger et al 1987), followed by commercially important mushrooms such as Lentinula edodes (Sato et al 1998), Pleurotus ostreatus (Peng et al 1992; Honda et al 2000), Agaricus bisporus (Van de Rhee et al 1996), Flammulina velutipes (Kuo et al 2004), and Ganoderma lucidum (Sun et al 2001). Recently, a transformation system was developed in Agrocybe aegerita (Herzog et al 2019) and Pleurotus eryngii (Shang et al 2018). However, transformation remains a challenge in many basidiomycete fungi, possibly because of the absence of a compatible marker gene, the incompatibility of many gene expression signals, the low efficiency of DNA fragment introduction into the host chromosome, and/or the inefficient protoplast production/regeneration rate.…”

Establishment of an Efficient Polyethylene Glycol (PEG)-Mediated Transformation System in Pleurotus eryngii var. ferulae Using Comprehensive Optimization and Multiple Endogenous Promoters

“…According to the test of fungal sensitivity, cbx resistance was used to screen positive transformants. Referring to a previous study [25], cbx is a 1,4-oxathiin derivative, which blocks the growth of basidiomycetes by inhibiting the activity of the succinate dehydrogenase B subunit (SDHB). The mutant SDHB with an amino acid substitution (His239 to Leu) confers resistance to cbx.…”

“…Then, the tissue was rinsed three times with 0.6 M mannitol. The mycelia were incubated for 1.0 h in 15 mL of 10 mg/mL lywallzyme (Guangdong Institute of Microbiology, Guangdong, China) in 0.4 M mannitol at 30 • C. Optimized conditions include osmotic pressure stabilizers (mannitol, sorbitol, MgSO 4 , sucrose, or KCl), osmotic pressure stabilizer concentration (0.2, 0.4, 0.6, 0.8, and 1.0 mol/L), osmotic pressure stabilizer (10,15,20,25, and 30 mg/mL), reaction time (1, 2, 3, 4, and 5 h), and reaction temperature (22,26,30,34, and 38 • C). Protoplasts were separated by filtration through a 40 µm cell strainer, collected by centrifugation at 3500 g for 10 min at 4 • C, washed twice with 15 mL STC buffer (0.6 M sorbitol, 10 mM CaCl 2 , and 10 mM Tris-HCl pH 7.5), resuspended in STC buffer, and stored at 4 • C until use.…”

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

“…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).…”