Host for highly efficient gene targeting in filamentous fungi

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To investigate gene function, most desirable method is gene targeting. However, gene targeting frequency is extremely low in many organisms. To raise the frequency, some trials have been carried out, but conclusive technique was not found.Double strand DNA breaks are repaired by two different recombination mechanisms: homologous recombination and nonhomologous end joining. From our study in Neurospora crassa, we speculated that homologous recombination frequency is increased if function of nonhomologous end joining is blocked. We disrupted ku70 and ku80 homolog genes of Neurospora and used them as a host in transformation experiments. In this study, high homologous integration of DNA was observed (Ninomiya, et. al. 2004).To develop more convenient host, we disrupted other Neurospora genes involving in nonhomologous end joining; Ligase IV and XRCC4 homolog genes. These mutants showed high targeting frequency even if homologous length of introduced DNA is short. We also tested targeting frequency in MRX-defective strains. MRX is complex of Mre11-Rad50-Xrs2 and functions in double strand breaks repair. Many transformants were from homologous integration, though transformation frequency was low.To know whether this system works in other organisms, Aspergillus oryzae was selected for test. Mutant of LigD ( human Lig4 homolog) was constructed in A. oryzae. Targeting experiments using ligD mutant as a host indicated high homologous integration.In many other fungi, similar experiments have been coducted by researchers all over the world and they confirmed high targeting efficiency by using ku mutants. as we presented.However, our trials in basidiomycetes did not reached to final step since there were unexpected problems in transformation, selection marker, so on. We will continue these experiments for construction of more convenient targeting system.