KILLER PHENOMENON IN USTZLAGO MAYDZS:

The double-stranded RNA content, the production of inactive killer protein, and the presence of virus-like particles were examined in induced nonkiller mutants and nonkiller progeny from a cross between a killer strain and a sensitive strain. A correlation between the loss of the 0.7 x 106 daltons dsRNA of the Ustilago m y d i s P6 virus and the lack of synthesis of the killer protein was established. In vltro and in vivo complementation between nonkiller strains provide additional support for the suggestion that the 0.7 x 106 daltons dsRNA is related to the killer function. The coding capacity of the various species of dsRNA is discussed in relation to their possible function. NTERSTRAIN inhibition in Ustilago mcrydis, known as the killer phenom'enon, was shown to result from the excretion of a proteinaceous inhibitory substance by some strains of Ustilago (HANKIN and PUHALLA 1971; KOLTIN and DAY 1975). Most strains are sensitive to this substance. Three killer specificities are known and the killer strain with one specificity is sensitive to the killer protein of a different specificity. The three specificities are known as P1, P4 and P6. Each killer is immune to the killer protein it produces. The immunity is inherited as a cytoplasmic character. The excretion of the killer toxin is related to the presence of double-stranded RNA (dsRNA) virus-like particles (VLPs) in the cells of the killer strains (WOOD and BOZARTH 1973; DAY and ANAGNOSTAKIS 1973; KOLTIN and DAY 1976a). Strains without VLPs are sensitive, unless they carry a nuclear gene fo r resistance. Sensitive strains with dsRNA lacking VLPs and sensitive strains carrying VLPs with the entire viral genome were reported recently (KOLTIN and DAY 1976b; KOLTIN 1977). The latter are laboratory-induced nonkiller mutants. The dsRNA recovered from the VLPs or from cells of killer strains is segmented. Three distinct dsRNA patterns are known, each typical of a distinct killer specificity (KOLTIN and DAY 1976a). Recently, studies were initiated to determine the organization of the viral genome in terms of the distribution of * The research wa5 supported in part by a grant from the Commission for Basic Research, Israel National Academy of + On leave from California State University a t Fullerton. Science Genetics 88: P67-XG February, 1978. 268 Y. KOLTIN A N D J. S. KANDEL the information in the various segments, using mutants with partial genomes. The study of the genetic behavior and the dsRNA pattern of these nonkiller mutants (KOLTIN 1977) suggested that the information in the different dsRNA molecules is not redundant and that a specific molecule contains the information for viral replication and coat formation, whereas other molecules contain the information for the killer substance and immunity to the killer toxin. An additional series of nonkiller mutants is described in the present paper. The results obtained with this series, together with the earlier reported findings (KOLTIN 1977), allow further assignments of viral functions to the different components of the viral genome. In uitro complementation data among nonkiller mutants also shed some light on the nature of the killer toxin. MATERIALS A N D METHODS Strains: Nonkiller mutants were induced in an auxotrophic P6 killer strain (C-28-12), An adenine-requiring auxotrophic strain was obtained from a cross between a PG killer and an auxotrophic sensitive strain. The induced nonkillers are designated NK-3, NK-5, NK-9, NK-12, NK-13, NK-15, NK-16 and NK-27. The nonkillers designated T-4, T-7, T-9 and T-11 were recovered as nonkiller progeny from a cross of the P6 killer No. 75 with the sensitive strain No. 7. Other nonkiller strains used in the study (NK-10 and NK-11) were those described by KOLTIN (1977). Strains used in the genetic analysis were sensitive strain No. 12, P1 killer No. 1268, P4 killer No. 1407 and P6 killer No. 75. All the strains are from the collection of DR. P. R. DAY, The Connecticut Agricultural Experiment Station, New Haven, Conn. Mutagenesis: The mutagenic treatment with N-methyl-N-nitro-N-nitrosoguanidine (NTG) was as described by KOLTIN (1977). Among a control sample of 534 untreated cells, no nonkillers were detected. Among 400 treated cells, eight independent nonkiller mutants were recovered. The mutant NK-16 was induced by UV irradiation. Genetic analysis: Two types of genetic procedures were used; (a) formal crosses and (b) heterokaryon transfer experiments. The former is designed to test the effect of nuclear genes on the phenotype; the latter is designed to test the inheritance of certain phenotypes by cytoplasmic exchange The crosses, dissection of tetrads, and the isolation of spores were performed as described by STEVENS (1974) and PUHALLA (1968). Heterokaryons were formed between compatible strains on synthetic media, as described by DAY and ANAGNOSTAKIS (1973). The nonkiller parent always carried a nutritional requirement, whereas the strain that was to be tested for the acquisition of the cytoplasmic function was prototrophic. The unstable heterokaryon was dissociated on minimal medium, thus eliminating the auxotrophic parent. The prototrophic cells were then tested for specific phenotypic changes resulting from the cytoplasmic mixing in the absence of the exchange of nuclear information. Identification of phenotypes as killers, nonkillers and sensitives was described by KOLTIN and D ~ Y (1976a,b). Analytical procedures: dsRNA was extracted following the procedure of DAY et al. (1977). The cells were disrupted in a Braun Homogenizer, as described by KOLTIN and DAY (1976a). Final purification of the dsRNA was performed on a cellulose CFll column according to FRANKLIN (1966). Electrophoresis was performed in 5% polyacrylamide gels at 2 mA/gel for 2.5 to 3 hr. All other conditions of the electrophoresis as well as the isolation of the VLPs were as described by KOLTIN and DAY (197Ga) and KOLTIN (1977). The killer protein was recovered from the supernatant of cultures grown for 40 hr in liquid complete medium (STEVENS 1974) on a rotary shaker at 25". The protein was concentrated 10-fold by lyophilization. followed by dialysis against 0.01 M phosphate buffer, pH 7.1. Samples KILLER PHENOMENON I N USTILAGO 269 (100 pl) were electrophoresed on 10% SDS polyacrylamide gels according to the procedure of WEBER and OSBORN (1969). The gels were stained overnight with 0.2% Coomassie Blue in 10% trichloroacetic acid and destained in methanol: acetic acid: water (2: 3:35 v/v) . Polyacrylamide disc gel electrophoresis at pH 8.3 was performed according to the procedure of DAVIS (1964). Activity was eluted from sliced duplicate unstained gels.