Towards a novel efficient T-DNA-based mutagenesis and screening system using green fluorescent protein as a vital reporter in the industrially important fungus Trichoderma reesei.
Agrobacterium-mediated T-DNA transfer has been proven to be an efficient strategy for insertional mutagenesis and elucidation of gene function in filamentous fungi. The implementation of large-scale T-DNA insertional mutagenesis requires the development of high-efficient transformation and high-throughput screening procedures. Here, using green fluorescent protein (GFP) as a vital marker, a highly efficient T-DNA-based mutagenesis and screening system was developed in Trichoderma reesei. The uridine auxotrophic T. reesei M23 as the host was transformed with A. tumefaciens EH105 strain harboring a binary vector pC-OEP, which beared the pyrG gene for primary selection on minimal medium without uridine and the egfp gene for fluorescence-based rapid screening of the mitotically stable transformants. The efficiency of transformation was up to 10-20 transformants per 10(5) target conidia. Microscopic examination revealed strong GFP expression and fluorescence emission in conidia, growing hyphae and mycelia. An effective and convenient screening procedure using 96-well plates and multilabel counter for fluorescence intensity counting was developed to rapidly identify the T-DNA tagged T. reesei mutants. Furthermore, the presence of T-DNA integration at random sites in the genome was confirmed by Southern blot analysis. This report of the T-DNA-based mutagenesis and rapid screening system using GFP as a vital reporter provides a promising strategy to speeding up the genome-scale T-DNA insertional mutagenesis and functional genomics analysis of this cellulolytic fungus T. reesei.
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, 27 Shanda South Road, Jinan, 250100, People's Republic of China.
Yaohua
Y
Zhong
Haina
H
Yu
Xiaoli
X
Wang
Yi
Y
Lu
Tianhong
T
Wang
D004269:DNA, Bacterial / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D016681:Genome, Fungal / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D007218:Industrial Microbiology; D008856:Microscopy, Fluorescence; D016254:Mutagenesis, Insertional / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012231:Rhizobium / Q000235:genetics; D014170:Transformation, Genetic; D014242:Trichoderma / Q000166:cytology / Q000235:genetics*
D004269:DNA, Bacterial; D004271:DNA, Fungal; D011993:Recombinant Fusion Proteins; C036483:T-DNA; D049452:Green Fluorescent Proteins