Efficient gene identification and targeted gene disruption in the wheat blotch fungus Mycosphaerella graminicola using TAGKO.
TAGKO ( transposon- arrayed gene knock out) is a highly efficient method for gene discovery and gene function assignment in the rice blast fungus Magnaporthe grisea. Here, we report the application of genome-wide TAGKO to the wheat blotch fungus Mycosphaerella graminicola, including the successful development of electroporation-based transformation for this fungus. A M. graminicola genomic cosmid library was constructed and a pool of 250 cosmid clones was mutagenized by in vitro transposition. Sequence analysis identified 5,110 unique insertion events in the M. graminicola genome. Eleven transposon-tagged cosmid clones (TAGKO clones) were chosen and transformed into the wild-type strain by electroporation. Ten TAGKO clones out of 11 produced gene-specific mutants at a targeting frequency of 15-28%, significantly higher than that of conventional gene-disruption constructs. The remaining clone failed to produce viable mutants, thereby providing indirect evidence for the identification of an essential gene.
Adachi
Kiichi
K
Paradigm Genetics Inc., 108 Alexander Drive, Research Triangle Park, NC 27709, USA.
Nelson
Grant H
GH
Peoples
Keith A
KA
Frank
Sheryl A
SA
Montenegro-Chamorro
Maria V
MV
DeZwaan
Todd M
TM
Ramamurthy
Lakshman
L
Shuster
Jeffrey R
JR
Hamer
Lisbeth
L
Tanzer
Matthew M
MM
D001203:Ascomycota / Q000235:genetics*; D003001:Cloning, Molecular; D003360:Cosmids / Q000235:genetics; D004251:DNA Transposable Elements; D018274:Electroporation; D018390:Gene Targeting / Q000379:methods; D005818:Genetic Engineering / Q000379:methods*; D009154:Mutation; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic; D014908:Triticum / Q000382:microbiology
D004251:DNA Transposable Elements