Tagging of genes involved in multidrug resistance in Aspergillus nidulans

Abstract

We have used a plasmid containing the Neurospora crassa pyr4 gene to transform an Aspergillus nidulans pyrG89 mutant strain in the presence of Bam-HI, and isolated multidrug-sensitive mutants among the transformants. Using this approach, we hoped to identify genes whose products are important for drug resistance by analyzing gene disruptions that alter the drug sensitivity of the cell. About 1300 transformants isolated following transformation were screened for sensitivity to drugs or various stress agents with different and/or the same mechanism of action. Seventy-seven of these transformants showed sensitivity to at least one drug, while fourteen transformants showed a complex phenotype of sensitivity to different drugs. The pyr4 marker was shown to be tightly linked to the mutant phenotype in only 36% of the pleiotropic mutants analyzed in sexual crosses. Genetic crosses between our multidrug-sensitive transformants and cycloheximide-sensitive and imazalil-resistant mutants of A nidulans were performed to determine whether mutations were present at the same loci. We have shown that the gene imaD that confers resistance to imazalil may also be involved in cycloheximide and hygromycin sensitivity, since this mutation is allelic to scyB (mutant scy290). In addition, the cross between the transformant R223 and the imazalil-resistant mutant ima535 showed that both mutations are in the same complementation group, suggesting that the gene imaG could also be involved in cycloheximide and itraconazole sensitivity.

Benomyl resistance of Colletotrichum acutatum is caused by enhanced expression of β-tubulin 1 gene regulated by putative leucine zipper protein CaBEN1

“…Transformation-mediated insertional mutagenesis provides an effective approach to identify essential genes required for fungal characters (Balhadère et al, 1999;Bö lker et al, 1995;De Souza et al, 2000;Epstein et al, 1998;Lu et al, 1994;Seong et al, 2005;Shi and Leung, 1995;Sweigard et al, 1998). However, C. acutatum is less sensitive to various antifungal agents, including hygromycin B, benomyl and bialaphos, used for transformation of other fungi (Chung et al, 2002).…”

“…The selection of tagged mutants provides a straightforward approach for identifying essential genes required for fungal development (Epstein et al, 1998;Shi and Leung, 1995), toxin production (Lu et al, 1994;Tanaka et al, 1999), pathogenicity (Balhadère et al, 1999;Bö lker et al, 1995;Seong et al, 2005;Sweigard et al, 1998;Thon et al, 2000;Yakoby et al, 2001), and drug resistance (De Souza et al, 2000). However, because C. acutatum is less sensitive to various antifungal agents including hygromycin B, benomyl and blasticidin S, which were used in fungal transformation (Chung et al, 2002), only a few molecular biological studies on C. acutatum have been based on genetic transformation.…”

Functional Genomics Approaches for the Identification and Validation of Antifungal Drug Targets

“…[107] Here, insertion of the mutagenic plasmid into the genome can result in activation of transcription of the target gene. More recently, De Souza et al [108] reported on a REMI-based system to look at effects of integration on multi-drug resistance in A. nidulans.…”

REMI in Molecular Fungal Biology

“…REMI has been used successfully for this reason in a wide variety of ascomycetes and basidiomycetes (Table 26.1 ). This technique revealed genes involved in fungal pathogenicity, the production of mycotoxins and secondary metabolites, heterologous protein expression, drug resistance, development, and cellular processes (Bölker et al 1995 ;Sweigard et al 1998 ;Balhadère et al 1999 ;Thon et al 2000 ;Yaver et al 2000 ;Kimura et al 2001 ;Yakoby et al 2001 ;Chung et al 2002 ;Fujimoto et al 2002 ;Busch et al 2003 ;Chen et al 2005 ;Seong et al 2005 ; Shim et al 2006 ;Shim and Woloshuk 2001 ;De Souza et al 2000 ;Bowyer et al 2012 ;Choi et al 2008 ;Cummings et al 1999 ;Makino and Kamada 2004 ;Muraguchi et al 2008 ;Nakazawa et al 2010 ;Stromhaug et al 2001 ;Mukaiyama et al 2002 ;Larsen et al 2013 ). Mucor circinelloides is the only zygomycete in which REMI has been used (Papp et al 2013 ).…”