Two dominant selectable markers for genetic manipulation in Neurospora crassa

Neurospora crassa is an excellent model fungus for studies on molecular genetics, biochemistry, physiology, and molecular cell biology. Along with the rapid progress of Neurospora research, new tools facilitating more efficient and accurate genetic analysis are in high demand. Here, we tested whether the dominant selective makers widely used in yeasts are applicable in N. crassa. Among them, we found that the strains of N. crassa are sensitive to the aminoglycoside antibiotics, G418 and nourseothricin. 1000 μg/mL of G418 or 50 μg/mL of nourseothricin is sufficient to inhibit Neurospora growth completely. When the neomycin phosphotransferase gene (neo) used in mammalian cells is expressed, N. crassa shows potent resistance to G418. This establishes G418-resistant marker as a dominant selectable marker to use in N. crassa. Similarly, when the nourseothricin acetyltransferase gene (nat) from Streptomyces noursei is induced by qa-2 promoter in the presence of quinic acid (QA), N. crassa shows potent resistance to nourseothricin. When nat is constitutively expressed by full-length or truncated versions of the promoter from the N. crassa cfp gene (NCU02193), or by the trpC promoter of Aspergillus nidulans, the growth of N. crassa in the presence of nourseothricin is proportional to the expression levels of Nat. Finally, these two markers are used to knock-out wc-2 or al-1 gene from the N. crassa genome. The successful development of these two markers in this study expands the toolbox for N. crassa and very likely for other filamentous fungi as well.