Isolation of Amatoxin-Resistant Lines of Chlamydomonas reinhardtiil

The inhibitory activities of amatoxins on the growth of Chiamydomonas reinhardtii have been determined using a convenient assay based upon incubation in multiwell tissue culture plates followed by turbidimetric estimates of growth on a multiwell plate reader. Values for the inhibitory dosage at which growth is 50% of untreated culture (ID.) of 5.4, 6.6, and 5.6 micromolar were obtained for ci-amanitin, O-methyl-a-amanitin, and amaninamide, respectively. Treatment of liquid cultures with 1 microgram per milliliter N-methyl-N'-nitro-N-nitrosoguanidine followed by growth in agar pour tubes containing 25 micromolar a-amanitin led to the selection of several lines demonstrating varying resistance to amanitin inhibition, with ID.0 values from 36 micromolar to greater than 200 micromolar. Two lines completely resistant to inhibition by 200 micromolar a-amanitin provided partially purified RNA polymerase activities that were 160-fold and 5600-fold more resistant to inhibition than the analogous enzyme activity from the wild-type strain. These studies provide evidence that Chlamydomonas reinhardtii does not contain significant activity capable of inactivating a-amanitin and that this amatoxin may be used to select for RNA polymerase mutants. Significant progress has recently been made with respect to our understanding of the structures and functions of eucaryotic DNA-dependent RNA polymerases (1, 4). Three classes of enzyme, i.e. I, II, and III, have been defined for the respective transcription of genes coding for ribosomal, messenger, and tRNA. While a number of properties have served to distinguish each class, the most definitive now being subunit structures and amino acid and/or DNA sequences, the unique sensitivity of the RNA polymerase II to inhibition by the fungal peptide AMA2 has been an important factor assisting in the purification and characterization of this enzyme from a number of sources. The relative specificity of AMA for the inhibition of transcription of mRNA in animals and plants appears to derive from its high affinity for I Supported by SAES Project on Genetic Mechanisms in Plant Improvement (Project No. FLA-MCS-02183-BI) from the University of Florida Experiment Station of the Institute of Food and Agricultural Science. Journal Series No. 8379. 2 Abbreviations: AMA, a-amanitin; meAMA, 6'0-methyl-a-amanitin; TAP, tris-acetate-phosphate medium; PMSF, phenylmethylsulfonyl fluoride; BME, 8-mercaptoethanol; MNNG, N-methyl-N'-ditro-N-nitrosoguanidine; deAMA, 6'-deoxy-a-amanitin or amaninamide; ID50, inhibitory dosage at which growth is 50% of untreated culture; IC-50, inhibitory concentration at which 50% of the enzyme activity remains; WT, wild type. RNA polymerase II (KD of 10-9-10-8M). This specificity has been of particular value in the selection and isolation of RNA polymerase II mutants as amanitin-resistant mutants and has permitted the identification and/or cloning and sequencing of RNA polymerase II genes from several animal sources (1). Plant systems have not been amenable to the use of amatoxins for the selection of RNA polymerase II mutants, although all of the RNA polymerase II activities which have been purified from plants show a sensitivity to inhibition by a-amanitin similar to the enzyme from animal sources (3, 6, 12). Both carrot (15, 19) and tobacco (19) cultures are relatively resistant to inhibition by AMA as a result of amanitin-inactivating activities, and meAMA was required to select for amatoxin resistant lines. A careful examination of several amatoxin-resistant lines of Daucus carota has indicated the basis for their resistance to be other than the presence of amatoxin resistant RNA polymerase activities (16). A report conflicting with this (27) indicated AMA-resistant clones of D. carota have been isolated. With the objective of further investigating the interaction of amatoxins with plant RNA polymerases, we have initiated studies on DNA-dependent RNA polymerases from the green alga Chlamydomonas reinhardtii. A number of features recommend this organism as a model for photosynthetic eucaryotes, including (a) growth as single cells in liquid or solid media of defined composition, (b) vegetative growth as a haploid with the potential for meiosis following zygote formation, (c) extensive genetic characterization and the existence of numerous markers to facilitate gene mapping (7). Transformation systems have been developed for this organism based upon selection of resistance to aminoglycoside antibiotic inhibition (9) and arginine prototrophy (21). In this paper, we have evaluated the sensitivity of cultures of C. reinhardtii to inhibition by different amatoxins, determined the sensitivities of partially purified RNA polymerase activities to inhibition by AMA, and selected AMA-resistant lines. Two of these lines have been shown to contain AMAresistant RNA polymerase activities. MATERIAL AND METHODS Culture Conditions. Chlamydomonas reinhardtii strain CC125, WT, mating type plus, maintained in the Duke University culture collection, was a gift from Dr. Elizabeth Harris of Duke University. All media were sterilized by autoclaving for 20 min at 1.05 kg/cm2. Incubations of all cultures were at 25°C under continuous light of approximately 3000 lux generated by 20 W softwhite fluorescent bulbs. Cells were grown on TAP medium prepared according to protocols provided by E. Harris. Plating media consisted of the above TAP medium containing 1.5% Difco bacto agar supplemented with AMA where appropriate. Liquid cultures were shaken on a New Brunswick G2 gyrotory shaker