Agrobacterium-mediated insertional mutagenesis of the ochratoxigenic fungus Aspergillus westerdijkiae.
Aspergillus westerdijkiae is a potent ochratoxin A (OTA) producer that has been found in coffee beans. OTA is known to have nephrotoxic effects and carcinogenic potential in animal species. Here we report for the first time the Agrobacterium-mediated transformation for Aspergillus westerdijkiae and the generation of ochratoxin-defective mutants. Conidia were transformed to hygromycin B resistance using strain AGL-1 of Agrobacterium tumefaciens. The obtained transformation frequency was up to 47 transformants per 10(6) target conidia. Among 600 transformants, approximately 5% showed morphological variations. Eight transformants with consistently reduced OTA production were obtained. Two of these transformants did not produce OTA (detection limit: 0.1 microg/kg); the other six mutants produced lower amounts of OTA (1%-32%) compared with the wild-type strain. By using thermal asymmetric interlaced polymerase chain reaction, we successfully identified a putative flavin adenine dinucleotide monooxygenase gene.
Mata
Marcia M
MM
Centro de Ciências Biológicas, Universidade Estadual de Londrina, Paraná, Brazil.
Taniwaki
Marta H
MH
Iamanaka
Beatriz T
BT
Sartori
Daniele
D
Oliveira
André L M
AL
Furlaneto
Márcia C
MC
Fungaro
Maria Helena P
MH
D001230:Aspergillus / Q000235:genetics* / Q000378:metabolism*; D003069:Coffee / Q000382:microbiology; D004271:DNA, Fungal / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000502:physiology*; D009793:Ochratoxins / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D012231:Rhizobium / Q000235:genetics*; D014170:Transformation, Genetic
D003069:Coffee; D004271:DNA, Fungal; D009793:Ochratoxins; C025589:ochratoxin A