Generation of transformable spheroplasts from mycelia, macroconidia, microconidia and germinating ascospores of Neurospora crassa

For Neurospora to be generally useful in molecular studies it would be desirable to be able to prepare transformable spheroplasts from mycelia and any of the three types of spores produced by this organism. Transformable spheroplasts are currently prepared from germinating macroconidia by digestion with Novozym 234 in the presence of 1.0 M sorbitol (Vollmer and Yanofsky 1986. PNAS 83:4869-4873). This method is efficient, but requires a 3-5 hr germination step. Elimination of the germination step would be a technical advance. In addition, the standard method is usable only with strains that form large numbers of macroconidia. Thus, interesting mutants that are incapable of forming macroconidia cannot be used as recipients in cloning experiments. A procedure for generating spheroplasts from mycelia of N. crassa has been reported (Buxton and Radford 1984. MGG 196:339-344). While large numbers of spheroplasts are released by this procedure, the frequency of transformation is low, and we have experienced difficulty obtaining repeatable results. Since we want to clone genes implicated in the macroconidiation process, we devised a procedure that improves the efficiency of transformation of mycelial spheroplasts. As an alternative approach, we developed an transformation protocol for microconidia. Since aconidial mutations can be introduced into a microcycle microconidiating background such as mcm (Maheshwari 1991. Exp. Mycol. 15:346-350), transformation of microconidia represents a viable option for the cloning of conidiation genes. A procedure for generating competent spheroplasts from germinating ascospores also was developed and provides an additional strategy for cloning conidiation genes. 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/vol39/iss1/14 Generation of transformable spheroplasts from mycelia, macroconidia, microconidia and germinating ascospores of Neurospora crassa J.C. Royer and C.T. Yamashiro Department of Biological Sciences, Stanford University, Stanford, CA 94305 For Neurospora to be generally useful in molecular studies it would be desirable to be able to prepare transformable spheroplasts from mycelia and any of the three types of spores produced by this organism. Transformable spheroplasts are currently prepared from germinating macroconidia by digestion with Novozym 234 in the presence of 1.0 M sorbitol (Vollmer and Yanofsky 1986. PNAS 83:4869-4873). This method is efficient, but requires a 3-5 hr germination step. Elimination of the germination step would be a technical advance. In addition, the standard method is usable only with strains that form large numbers of macroconidia. Thus, interesting mutants that are incapable of forming macroconidia cannot be used as recipients in cloning experiments. A procedure for generating spheroplasts from mycelia of N. crassa has been reported (Buxton and Radford 1984. MGG 196:339-344). While large numbers of spheroplasts are released by this procedure, the frequency of transformation is low, and we have experienced difficulty obtaining repeatable results. Since we want to clone genes implicated in the macroconidiation process, we devised a procedure that improves the efficiency of transformation of mycelial spheroplasts. As an alternative approach, we developed an transformation protocol for microconidia. Since aconidial mutations can be introduced into a microcycle microconidiating background such as mcm (Maheshwari 1991. Exp. Mycol. 15:346350), transformation of microconidia represents a viable option for the cloning of conidiation genes. A procedure for generating competent spheroplasts from germinating ascospores also was developed and provides an additional strategy for cloning conidiation genes. In this paper, we describe protocols which generate transformable spheroplasts from mycelia, macroconidia, microconidia and germinating ascospores. The main alteration from the standard spheroplast formation protocol is the use of MgSO4 (1.0 M) instead of sorbitol (1.0 M) as the osmotic stabilizer. In addition, a higher concentration of Novozym 234 (Novo Laboratories, Wilton CT, lot no. PPM 1530) and a modified culture procedure are utilized for generation of spheroplasts from mycelia. We also report on a modification of the standard spheroplast regeneration procedure which results in more rapid appearance of transformants and a 2-4 fold increase in transformation frequency of spheroplasts from germlings and mycelia.