Two Yeast Plasmids that Confer Nourseothricin-Dihydrogen Sulfate and Hygromycin B Resistance in Neurospora crassa and Cryphonectria parasitica

Abstract

Two plasmids that were previously used with yeast, pRS41N and pRS41H, were found to confer clonNAT and hygromycin B resistance, respectively, in the filamentous fungi Neurospora crassa and Cryphonectria parasitica. These plasmids are suitable for routine cloning and for use in forcing heterokaryons and are available through the FGSC. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol54/iss1/4 12 Fungal Genetics Newsletter Two yeast plasmids that confer nourseothricin-dihydrogen sulfate and hygromycin B resistance in Neurospora crassa and Cryphonectria parasitica. Robert Phillip Smith and Myron L. Smith Nesbitt Biology Building, Department of Biology, Carleton University, Ottawa, Ontario, Canada. Fungal Genetics Newsletter 54:12-13 Two plasmids that were previously used with yeast, pRS41N and pRS41H, were found to confer clonNAT and hygromycin B resistance, respectively, in the filamentous fungi Neurospora crassa and Cryphonectria parasitica. These plasmids are suitable for routine cloning and for use in forcing heterokaryons and are available through the FGSC. Identification of novel resistance markers is important to further our ability to manipulate and characterize genetic elements in filamentous fungi. Hygromycin B (hygB) inhibits protein translation and has been used extensively as a selectable marker in filamentous fungi (Rao et al,, 1985). Alternative, affordable selectable markers are desirable for various applications, including for forcing heterokaryons. Previously, nourseothricin-dihydrogen sulfate (clonNAT) has been used as a selectable marker in fungi (Kück and Hoff, 2006). This aminoglycoside binds to ribosomes and results in inhibition and errors in protein synthesis (Cundliffe, 1989). Here we report that two vectors, pRS41N (clonNAT resistance) and pRS41H (hygB resistance), that were previously characterized in S. cerevisiae but not in filamentous fungi, are appropriate transformation vectors and suitable for forcing heterokaryons in Neurospora crassa and Cryphonectria parasitica. Figure 1. Yeast plasmids pRS41N and pRS41H that confer clonNAT and hygB resistance, respectively, to N. crassa and C. parasitica. Unique restriction sites in the multiple cloning site are indicated. Plasmids pRS41N and pRS41H (Figure 1) were derived from pRS416 as described in Taxis and Knop (2006). Resistance genes in both plasmids are driven by the TEF promoter from Ashbya gossypii, and S. cerevisiae CYC1 and ADH1 terminators are used in pRS41H and pRS41N, respectively. This contrasts to previously characterized vectors such as pCB1004 (Carroll et al., 1994) and pD-NAT1 (Kuck and Hoff, 2006) that use Aspergillus nidulans trpC promoter and terminator. Transformations with pRS41N and pRS41H were done with a PEG-mediated transformation protocol (Smith et al., 2000). Selection of N. crassa, pRS41N transformants was on Vogel’s minimal medium containing 200 ug/ml of clonNAT (Werner Bioagents, Jena, Germany) and yielded ~20 colonies / ug plasmid DNA. Similarly, C. parasitica transformed with pRS41N, was selected on Potato Dextrose Agar (PDA) Published by New Prairie Press, 2017