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.
Department of Biotechnologies, Aventis CropScience, Lyon, France.
F
F
Villalba
M H
MH
Lebrun
A
A
Hua-Van
M J
MJ
Daboussi
M C
MC
Grosjean-Cournoyer
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