Transposon impala, a novel tool for gene tagging in the rice blast fungus Magnaporthe grisea.
impala, a Tc1-mariner transposable element from Fusarium oxysporum, was introduced into the rice blast fungus Magnaporthe grisea to develop transposon-based insertional mutagenesis. A construct (pNIL160) containing an autonomous impala copy inserted in the promoter of niaD encoding Aspergillus nidulans nitrate reductase was introduced by transformation into a M. grisea nitrate reductase-deficient mutant. impala excision was monitored by restoration of prototrophy for nitrate. Southern analysis of niaD+ revertants revealed that impala was able to excise and reinsert at new loci in M. grisea. As observed for its host Fusarium oxysporum, impala inserted at a TA site left a typical excision footprint of 5 bp. We have shown that a defective impala copy was inactive in M. grisea, yet it can be activated by a functional impala transposase. A transformant carrying a single copy of pNIL160 was used to generate a collection of 350 revertants. Mutants either altered for their mycelial growth (Rev2) or nonpathogenic (Rev77) were obtained. Complementation of Rev77 with a 3-kb genomic fragment from a wild-type locus was successful, demonstrating the tagging of a pathogenicity gene by impala. This gene, called ORP1, is essential for penetration of host leaves by M. grisea and has no sequence homology to known genes.
Villalba
F
F
Department of Biotechnologies, Aventis CropScience, Lyon, France.
Lebrun
M H
MH
Hua-Van
A
A
Daboussi
M J
MJ
Grosjean-Cournoyer
M C
MC
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004251:DNA Transposable Elements; D004271:DNA, Fungal / Q000032:analysis; D004268:DNA-Binding Proteins / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005670:Fusarium / Q000235:genetics; D020082:Magnaporthe / Q000235:genetics* / Q000472:pathogenicity; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames; D012275:Oryza / Q000382:microbiology*; D029702:Schizosaccharomyces pombe Proteins; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic; D019895:Transposases / Q000235:genetics
D018797:Cell Cycle Proteins; D004251:DNA Transposable Elements; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029702:Schizosaccharomyces pombe Proteins; C097339:orc1 protein, S pombe; D009565:Nitrate Reductases; D050901:Nitrate Reductase; C081454:Tc1 transposase; D019895:Transposases