Carboxin resistance transformation of the homobasidiomycete fungus Pleurotus ostreatus

Abstract

A novel selection marker gene for transformation of the white-rot basidiomycete Pleurotus ostreatus was developed by introducing a point mutation in a gene which encodes the iron-sulfur protein (Ip) subunit of succinate dehydrogenase. The mutant gene, CbxR, encodes a modified Ip subunit with an amino-acid substitution (His239 to Leu) and confers resistance to the systemic fungicide, carboxin. The DNA sequence was integrated ectopically in the chromosome of the transformants. This is the first report of a homologous marker gene which is available for the molecular breeding of an edible mushroom.

Transformation of the Mycorrhizal Basidiomycetes,Suillus grevilleiandS. bovinus, by Particle Bombardment

“…The transformation frequency was 1.2 and 0.6 transformants/mg DNA from S. grevillei and S. bovinus respectively. The transformation frequency of a monokaryotic strain of Pleurotus ostreatus to carboxin resistance 7) was similar to our results. In contrast, Aboul-Soud et al 28) found that the transformation efficiency of Fusarium oxysporum was 45 transformants per mg of plasmid DNA after particle bombardment, but they used conidia obtained from F. oxysporum instead of mycelium.…”

“…Pleurotus ostreatus [5][6][7] and Agaricus bisporus 8) have been transformed by PEG/CaCl 2 . Protoplasts of Agrocybe aegerita prototrophs have been transformed to prototrophy by electroporation with transformation frequencies ranging from 1 to 26 transformants/mg of DNA.…”

The White-Rot FungusPleurotus ostreatusTransformant Overproduced Intracellular cAMP and Laccase

“…The plasmid vectors were co-transformed with pTM1 into P. ostreatus PC9 as described in previous reports. 20,23) Full-length insertion of the expression cassettes into the transformant genomes was confirmed by genomic PCR, and the same transcript levels in all the recombinant genes in all the transformants were indicated by performing semiquantitative RT-PCR (data not shown). Four transformants for each of the expression cassettes were obtained and representative data are presented in this report.…”

“…P. ostreatus can be utilized as a host in reproducible-efficient transformation systems. [20][21][22][23][24] In the present study, we created P. ostreatus transformants expressing dominant-activated G genes to overproduce intracellular cAMP and to analyze the relationship between the intracellular cAMP concentration and Lac production in this fungus. Our findings might prove useful in developing a Lac high-producting system with P. ostreatus.…”

“…Sequences of the open reading frames (containing the stop codon) were cloned by performing PCR and inserted into the pIp-DEST-GFP vector under the sdi1 promoter 27) by Gateway Ò LR clonase (Life Technologies). The pIp-DEST-GFP vector was constructed by replacing the Cbx R open reading frame in pTM1 20) with a Gateway Ò reading frame cassette (Life Technologies, carlsbad, CA) and the green fluorescence protein gene. To create dominant active mutants, we introduced point mutations in the plasmids by the substituting glutamine (Q) which is considered to be an importnat animo acid for the GTPase activity of G 26) (Q218 of protein ID 65367, Q204 of protein ID 115090, Q208 of protein ID 117454, and Q207 of protein ID 130194), with arginine (R).…”