Detection of killer-independent dsRNA plasmids in Ustilago maydis by a simple and rapid method of extraction of dsRNA

PLASMID

21,216-225 (1989)

Detection of Killer-independent dsRNA Plasmids in Ustilago may&s by a Simple and Rapid Method of Extraction of dsRNA EYAL SEROUSSI, TSAFRIRA PEERY, IDIT GINZBERG, AND YIGAL KOLTIN’ Department of Microbiology,

Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel

Received November 7, 1988; revised February 20, 1989 A novel method for efficient and rapid isolation of dsRNA molecules was developed. The dsRNA content of Ustilago maydis was reexamined; two distinct dsRNA cIasseswere identified. Class I includes the d.sRNA segmentsreported earlier for U. maydis virus systems and class II includes unencapsidateddsRNA moleculesthat were barely detectedby the conventional extraction methods despite their high titer. Segmentsof the classII, some of which are reported for the first time, were further characterized; all the segmentsare independent of the killer system and other encapsidated dsRNA molecules. These segments are cytoplasmically transmitted and, in sharp contrast with classIencapsidated dsRNA segments,their relative copy number decreasesrapidly while entering the stationary phase. 0 1989 Academic Press, 1~.

The dsRNA viruses and plasmids of fungi have been under investigation for more than three decades (reviewed by Buck, 1986). As more fungal species are studied and new dsRNA viruses and plasmids are detected, it appearsthat the dsRNA viruses and plasmids are ubiquitous in the fungi. The conventional method of extraction of dsRNA requires high density of cells and a long and tedious procedure to enable reliable characterization of the dsRNA content. The titer of the dsRNA molecules is low in some speciesand in some casesis associated with cell components that preclude efficient extraction of these molecules. Hence, general surveys of the dsRNA viruses and plasmids in fungal populations and the screening of many colonies, as performed routinely for DNA plasmids in bacterial cultures, are virtually impractical. A simple and rapid method of extraction of dsRNA from whole cells in the corn smut Ustilago maydis was examined. Three virus subtypes UmVPl,’ UmVP4, and UmVP6 were identified in U. maydis (re’ To whom correspondenceshould be addressedat Department of Antiinfectives, Research and Development, Smith Kline & French Laboratories, 709 SwedelandRoad, Ring of Prussia, PA 19046. ’ Abbreviations used:UmV, Ustilago maydis virus; ScV, Saccharomyces cerevisiae virus; PAGE, polyacrylamide 0147-619X3/89$3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

viewed in Koltin, 1988). The host cells carrying each type are designated Pl , P4, and P6, respectively. Each virus type consists of a typical dsRNA-segmented genome (described in Table 1) and the segmentsbased on their size have been referred to as heavy (H), medium (M), and light (L) (Koltin, 1986). The H segments of the UmV are associated with the maintenance functions of the entire segmented genome. M and L segmentsare encapsidated in virions consisting of the coat protein encoded by the H segment (Bozarth et al., 1981; Koltin et al., 1978) and strains that contain only the M or L segments without H were never found. In each viral subtype there is an homology between the major L segment and one of the M segments (Field et al., 1983; Chang et al., 1988). In each of the three types of viruses one M segment is correlated with the secretion of proteinaceous toxin called a killer toxin. A host cell that harbors one of the three viruses and secretesa specific toxin is called a killer cell (Koltin, 1988). Most strains of Ustilago contain at least part of the dsRNA complex found in killer strains (Day, 1981). We report the use of a simple method for dsRNA extraction that led to the detection for gel electrophoresis; EtBr, ethidium bromide; RsV, Rhizoctonia solani virus. 216

217

dsRNA PLASMIDS OF U. maydis

Growth conditions. Cells were grown in liquid medium containing 1%peptone, 1%yeast extract, and 2% glucose at 29°C in a gyratory shaker incubator. Minipreparations were grown either in glass vials (30 ml) or in glass test tubes, each containing 6 ml of medium. Care was taken to secure constant gas exchange since Ustilago is sensitive to microaerophyllic conditions. Unless described difMATERIALS AND METHODS ferently the extractions were performed from Strains. The strains of U. maydis used in cells that reachedthe stationary phase(3 X lo8 the study are from the collection at Tel Aviv cell/ml). University and are listed in Table 1. The sup Genetic procedures. Heterokaryon formapressive strain 18 (Koltin and Day, 1976) is tion and cytoplasmic transmission experialso the universal sensitive strain for identifi- ments (cytoduction) were performed accordcation of killer activity. The yeast strain 1293 ing to Stevens ( 1974). was obtained from R. B. Wickner, National Extraction of dsRNA. The standard proceInstitutes of Health (Bethesda, MD) and it dure used was as described by Vodkin et al. contains Saccharomyces cerevisiae virus (ScV) (1974) for extraction of the nucleic acids and of type Kl. the purification of dsRNA according to

the first time of new unencapsidated dsRNA speciesindependent of the UmV killer-related system. The method detects fully all the dsRNA molecules that have been described earlier for the killer systems.The distribution of these segments and the new segments in killer and nonkiller cells were reexamined.

TABLE 1 STRAINS OF U. maydis

Phenotype’

Designation

Segmentsb

1371 75-1

P6 killer P6 killer

H 1-6, M2-6, M3-6, L-6 Hl-6, M2-6, L-6

3162

P6 killer

Hl-6, M2-6, L-6

75lNK13

NK, P6 complementing mutant c

H 1-6, M2-6, L-6

75-lU1

NK

Hl-6

3038

NKP6 complementing mutant’

H l-6, M2-6, L-6

77

P4 killer

3126

Pl killer

HI-4, H2-4, H3-4, H4-4, M2-4, M3-4, L-4 Hl-1, H2-1, Ml-l, M21, M3-1, L-l

27

PI killer

18

NK, suppressive

HI-I, H2-1, Ml-l, M21, M3-1, L-l H4a

Source and remarks Contains wild-type P6 viruses Spontaneous viral variant of 75 (Ben-Zvi et al., 1984) Cytoduction from 3047 into a NK strain Cytoduction of viruses from 3038 to 75U1 (Peery et al., 1987) A spontaneous viral variant containing H 1 dsRNA only (Peery et al., 1987) NTG mutagenesisof a P6 killer strain 3047 (Koltin and Kandel, 1978) Wild type Cytoduction of Pl viruses into the nonkiller strain 3063 (Peery et al., 1982) Wild type Wild type

’ NK, nonkiller. b The dsRNA was extracted by the standard method (seeMaterials and Methods). The tirst number designatesthe segment number and the second number designatesthe virus type. ’ P6 complementing mutant secretesone of the two polypeptides of the P6 toxin (Peery et al., 1987).

218

SEROUSSI ET AL.

Franklin (1966) with the modifications described in Wigderson and Koltin (1982). The new method is described under Results. Electrophoresis. The dsRNA was examined in 1.5%agarosehorizontal slab gelscontaining 1 pg/ml ethidium bromide (EtBr) and in 5% PAGE. The buffer system was 40 IIIM Tris acetate with 2 mM EDTA, pH 7.4 (Maniatis et al., 1982). The gels were stained with EtBr (1 pg/ml) and viewed with a Chromato-Vue transilluminator and photographed with a Polaroid MP3 system. Estimation of copy number of dsRNA segments. The dsRNA that was extracted from 1 X 1O*cells was examined in agarosegels. Negatives of pictures of the stained gels were scanned with an LKB Ultrascan XL spectrophotometer. Reovirus type 3 dsRNA (obtained from W. Joklik, Duke University) was used as a standard for quantitation of each of the dsRNA segments. The copy number per cell of each dsRNA segment was determined by dividing its quantity (per cell) by its molecular mass estimated by PAGE (Bozarth et al., 198I), agarosegels, and electron microscopy (Field et al., 1983). Isolation of virions and RNA-dependent RNA polymerase activity. Virions were isolated from cells of U. maydis grown for 48 h in complete medium. The isolation of the viruses was according to Finkler et al. (1985). A volume of 1 liter of cell suspension was used and the protease inhibitor phenylmethylsulfonyl fluoride (0.05 mg/ml) was added to the lysis buffer. RNA polymerase activity in the fractionated sucrose gradients was examined as described by Ben-Zvi et al. (1984). Purification, 3’ end labeling, and blot hybridization of dsRNA were performed as described by Finkler et al. (1988).

RESULTS

Simple and Rapid Method of Extraction of dsRNA from Cells The method is based on the stability of dsRNA to boiling in the presence of a high salt concentration. Cells were grown to the

stationary phase (2-5 X 10’ cells/ml) and divided to Eppendorf tubes (1.5 ml). The cells were pelletted in an Eppendorf centrifuge at room temperature (20 s, 14K rpm) and suspended in 1.5 ml of 2 X SSC (0.3 M Sodium citrate, 0.2 M NaCl). The tubes were placed in boiling water for 8 min. At this stage the nucleic acids that are releasedfrom the cells were examined and identified as single-stranded RNA; the dsRNA and DNA remained in the cells. After being boiled the cells were centrifuged (20 s) and the supernatant was completely removed. Acid-washed glass beads (0.45 mm) were added (equivalent to the volume of the pellet) along with 60 ~1of phenolchloroform ( 1:1). The mixture was mixed well with a toothpick. Double-distilled water ( 10 to 50 ~1) was added and the mixture was vortexed for 1 min. The tubes were then centrifuged (4 min) to obtain phase separation and the aqueous phase was transferred to a new tube. The aqueous phase from this stagecontaining the nucleic acids can be used directly to load on an agarose gel to examine the dsRNA content of the cells. A sample of 10 ~1 is sufficient to seeall the dsRNA segments typical to the Ustilago viruses. The phenolchloroform extraction is not essential since it appearsthat after the boiling step, in high salt, the dsRNA is not heavily proteinized and it can be readily extracted at this stageby breaking the cells in water. However, omission of the phenol-chloroform extraction resulted in release of DNA from the cells along with the dsRNA. The dsRNA samples extracted by this method contain salts and oligonucleotides. In order to further purify the dsRNA, ethanol precipitation in the presence of 1.25 M ammonium sulfate was performed. In 50% ethanol, the ammonium sulfate solution forms two visible phases.The heavy phasegradually disappearsby lowering the ethanol concentration to less than 40%. Centrifugation at this point precipitates the nucleic acids that are larger than ca. 100 bp to the bottom of the tube and the oligonucleotides remain in the supematant. One volume of 2.5 M (NH&S04 was added to the dsRNA sample followed by addition of about 1 vol of 100%ethanol. When

dsRNA PLASMIDS OF U. muydis

219

the two phases appear double-distilled water was gradually added with constant stirring until no heavy phase was observed. The tubes were centrifuged 10 min at 14K rpm, and the pellet was washed with 70% ethanol. At this stagethe dsRNA is sufficiently pure from oligonucleotides and can be used for enzymatic reactions. Detection of New Species of dsRNA The simple method was used for extraction of dsRNA from cells containing viruses of subtype P6. The dsRNA extracted from strain 75 lNK13 included in addition to the known P6 segmentstwo additional segments designated H3-6 and Mlb-6 (Fig. 1, lane a). These segmentswere not present in the killer strain 75-1 that contains only UmVP6 (Fig. 1, lane b). To verify that the new species are dsRNA segments,the dsRNA from strain 75- lNK13 was purified in CF- 11 (Franklin, 1966) that is designed to separatedsRNA from ssRNA and DNA. The new segmentswere purified along with the typical UmVP6 dsRNA that is present in this strain (Fig. 2, left). In addition, segments ab

-6

c

-

-6-6-

L-

FIG. I. Agarose gel electrophoresis of nucleic acids extracted by the simple method from strains containing P6 subtype viruses and a strain cured of the segmentsassociated with UmVP6: (a) strain 75-INK 13 containing segments HI-6, H3-6, Mlb-6, M2-6, and La-6; (b) strain 75 1 containing Hl-6, M2-6, and L-6; (c) the cured strain (3 179) containing only H3-6 and M 1b-6.

FIG. 2. The new segmentsare dsRNA. Agarosegel (1.2%) electrophoresisof dsRNA extracted from strain 75-1NK 13 and purified by cellulose chromatography displaying segments Hl-6, H3-6, Mlb-6, M2-6, and L (left). Lane anucleic acids extracted from strain 3038. Lane b-a mixture of the nucleic acids in (a) and a Hind111digest of X. Lane c--Hind111 digest of X. The mixture in lane b was tested for its sensitivity to DNase and RNase. Lane l30 min of digestion with DNase; lane 2-30 s of digestion with DNase. Lane 3-30 min of digestion with RNase in low ionic strength; lane 4-30 s of digestion with RNase in low ionic strength.

H3-6 and M 1b-6 were identified as RNA by their insensitivity to DNase and as dsRNA by their sensitivity to digestion with RNase A at low ionic strength (Fig. 2). These segmentsare resistant to RNase A at high ionic strength (not shown). The dependence of H3-6 and M 1b-6 on the other dsRNA segments was tested by curing experiments. Strain 3 162 was treated with 3% EMS (Peery et al., 1987) and a nonkiller strain (3 179) was obtained. This nonkiller lost the UmV segments H 1-6, M2-6, and L-6 and it contains only the dsRNA segmentsH3-6 and Mlb-6 (Fig. 1, lane c). These results indicate that these segmentsbelong to a different class of segmentsnot related to UmV since in UmV the maintenance of M2-6 is dependent on the HI segment. It was also observed, that cells were always cured from segments H3-6 and M 1b-6 simultaneously, suggesting an interdependence between these two segments. Characterization of the New dsRiXA Species The cytoplasmic nature of the new dsRNA segments was demonstrated in cytoduction experiments. When cytoplasm was exchanged between strain 3038 and strain 75 lU1, the

220

SEROUSSI ET AL

segments H3-6 and M lb-6 were transmitted along with UmVP6 (Fig. la). Also, the new dsRNA segmentswere transmitted in crosses and were found to segregateas a non-Mendelian trait. To determine if the new dsRNA segments are encapsidatedin distinct vii-ions asUmVP6, strains 3 179 and 75-l (as a control) were examined for the presence of virions after purification in a sucrose gradient. The presence of virions can be detected by a highly sensitive assay that tests for the coat-associated RNA polymerase activity (Ben-Zvi et al., 1984). Such activity was clearly detected in the preparation from 75-l but not detected in the preparation from strain 3 179. The distribution of RNA polymerase activity in a sucrosegradient from strain 3 179 and from strain 75- 1 was compared (Fig. 3A), and the dsRNA was extracted from the fractionated gradients (Fig. 3B). RNA polymerase activity was detected in tubes 6 to 10 (strain 75-l) and was associated with viral particles detected in the same tubes by electron microscopy (not shown). dsRNA that was extracted from tubes 6 through 10 showed the typical UmVP6 segments: Hl-6, M2-6, and L-6 (Fig. 3B). No polymerase activity was detected in the gradient fractions from strain 3 179 (Fig. 3A). The activity detected at the bottom of the gradient from strain 75-1 was associatedwith aggregatesof viruses.Also, very low activity was detected in tubes 16 to 2 1 in the gradient from strain 3 179, but no dsRNA was noted in these tubes. In addition, strain 75-INK13 that contains both UmV and new dsRNAs (Fig. la) was tested for the presence of H3-6 and M lb-6 in the fractions eluted from a sucrose gradient. Only segments H l6, M2-6, and L-6 were detected in the gradient fractions. Therefore, it appears that H3-6 and M 1b-6 are not encapsidated as other segments of UmV. Unencapsidated dsRNA segments were detectedearlier by Koltin and Day ( 1976) in U. maydis in two strains. One of these strains ( 18) contains one dsRNA segmentdesignated H4a. The relatednessof M lb-6 and L-6 wastested in hybridization studies. L-6 was usedto probe the M size dsRNAs since it is known to be

81 0 b x E a ”

82

6

10 Tube

2

68lO141822M

14

18

22 T

No. M2

6

8 10141422

FIG. 3. (A) RNA polymerase activity in fractions of the sucrosedensity gradients.Symbols:(0) gradient from strain 75-1, (0) gradient from strain 3 179. (B) Electrophoresis of virion dsRNA from the fractionated sucrose density gradients. Left: dsRNA marker(M) and the extraction of the gradient fractions for the detection of dsRNA from a P6 killer strain (75-l). Right: the same as in left but from the NK strain (3 179).

homologous to the 3’ end of M2-6 which is one of the UmVP6 segments (Chang et al., 1988). The probe hybridized to M2-6 as expected, but did not hybridize to Mlb-6 (Fig. 4). Also, the same probe did not hybridize to H3-6 (not shown). Hence, the new dsRNA segments, H3-6 and Mlb-6, have no homology to L-6, indicating that they are distinct from the UmV dsRNA. The Distribution of dsRNA Segments in Strains Containing Urn V PI, P4, and P6 The new method of extraction was used to reexamine the dsRNA segments in strains carrying UmVPl and P4. The dsRNA was characterized in agarosegels and in polyacryl-

221

dsRNA PLASMIDS OF U. may&s A

B

C

MI b6 -

-Ma-a

dsRNA and the new segmentsdescribed, the minimal copy number of the dsRNA segments per cell was estimated. Samplesof dsRNA extracted from strains containing UmVPl , P4, or P6 and dsRNA from strain 18 and from a strain of S. cewisiae containing ScVKl were

FIG. 4. Hybridization of PmVP6 segmentL as the probe with the M size dsRNAs extracted from strains (A) 3038, (B) 75lNK13, and (C) 1371. Upper: ethidium bromide stain of the gel used for the hybridization. Lower: autoadiograph of the blot. -ii3 -Iha amide gels (Fig. 5). All the typical dsRNA segments of U. maydis viruses PI and P4 appear -Mtb in the gel (Field et al., 1983; Shelbourn et al., 1988) (Fig. 5A). However, the Pl cells examined contain two additional segmentsdesignated H3-1 and Ml b- 1 that have the same mobility in the gels as H3-6 and Mlb-6. Hybridization experiments have shown that M 1b6 and M lb-l are homologous (not shown). size C Strains harboring UmVP4 that were examined Pl P4 P6 (bp) did not contain these segments. A very proHl-4 Hl-6 6700 - HZ-1 nounced additional new dsRNA segment was HZ-1 H2-6 4500 H2-4 noted in P4 and Pl cells. This segment was H3-6 3625 designated H4a-4 and H4a- 1. Previously, this - H3-1 -3200 H3a-4 I3100 =H3b-4 segment was occasionally recovered with a -H4b-4 2600 - H4a-1 -H4a-4 very low titer in P 1 cells (Wigderson and Kol=2300 -2210 tin, 1982).This segmentis not present in those strains tested containing UmVP6. PAGE was usually used in most studies to 1416 Mla-1 Mlb-6 z132g =Mlb-1 examine the patterns of dsRNA in the H seg-M2-6 1197 ments. However, in the present study it was - M3-6 - MZ-1 980 M2-4 noted that agarosegels offer a good separation _ 920 --M3-4 - M3-1 of the H dsRNA segments.Using PAGE, the segments H3a-4 and H3b-4 appear as one band, and segment H4b-4 runs faster than H4a-4 (a very thick band). In agarosegelsH3a4 and H3b-4 separate and the order of H4b-4 and H4a-4 is reversed. An agarosegel pattern -L-6 _ 360 L-4 L-l of the dsRNA segmentsfound in strains containing UmVPl , P4, or P6 is described in Fig. 5C. This pattern should serve as a guide in FIG. 5. Gel electrophoresis of dsRNA extracted by the future characterization of additional dsRNA simple method from strains containing UmVPl, P4, or P6. (A) Agarose gel electrophoresis. (B) As in (A), but the segmentsfound in strains of U. maydis.

Copy Number of the dsRNA Segments To determine the stochiometric relations between the two dsRNA systems, the UmV

sampleswere run in 5% PAGE. (R) Reovirus (type 3) was used as molecular mass marker. (C) A schematic description of the dsRNA patterns in agarose gel from strains containing UmVPl, P4, or P6. Reovirus dsRNAs were used as additional size markers.

222

SEROUSSI ET AL.

run in agarosegel and scanned in a densitometer (Fig. 6). Reovirus type 3 dsRNA was used as a standard for calibration. The segmentsof reovirus have been well characterized based on electrophoretic separation and sequence data (Joklik, 1985).The dsRNA were extracted in each casefrom 2 X 1O*cells/ml. The results indicate that the copy number of the different dsRNA segmentsis related to the class of the dsRNA segment and the growth phase. The copy number of the extracted dsRNA from late log phase cultures is presented in Table 2. The typical segmentsof UmV are all within a range of 50-500 copies per cell, except for the L segment that is present in at least 1000 copies per cell. The copy number of the new H segment (H3) is only 50 and is the lowest among the H segments.The copy number of the H4a segments is in the range of a few thousands while the other H segmentsare one order of magnitude lower. The high copy number segment H4a from strain 18 migrates in the gels to the same position as H4a-1 and H4a-4 (Fig. 6). Within the M size segments

/--RR

PI

P4

P6

18

y

L-A

-M

TABLE 2 COPYNUMBEROFdsRNA

SEGMENTS PER CELL

Segment

Size (kbp)

Copy number

Hl-1 HI-4 HI-6 H2-1 H2-4 H2-6 H3-1 H3-6 H3a-4 H3b-4 H4-4 H4a-1 H4a-4 H4a(18)

6.1 6.1 6.7 4.5 4.5 4.5 3.8 3.8 3.2 3.1 2.9 2.5 2.5 2.5

120 170 130 250 4301 260 50 60 280 460 340 2870 6990 1730

Mla-1 Mlb-1 Mlb-6 M2-6 M2-1 M2-4 M3-6 M3-1 M3-4

1.5 1.3 1.3 1.2 1.0 0.96 1.0 0.92 0.92

250 390 430 380 300 580” 220 240 460”

L-6 b L-l L-4 L-6 L-lb

0.38 0.36 0.36 0.36 0.34

200 430 1100 1030 200

a M2-4 and M3-4 appear in the gel as one band. The estimation of the copy number of these segmentswas according to their relative amounts in PAGE. b L size minor species.

M 1b is the most pronounced in cells that harbor this segment. The extraction method was effective for extraction of dsRNA from a killer strain of S. cerevisiae. This strain contains ScV that encodes the killer toxin (Wickner, 1986). The extraction yielded the L and M segmentstypFIG. 6. Agarose gel electrophoresis of dsRNA extracted ical to the Kl killer of ScV and their minimal by the simple method from Ustilago strains and a yeast copy numbers per cell (shown in Fig. 6) were killer strain. Such a gel was used for the estimation of the estimated as 600 for L-A (the large segment) copy number of daRNA segments.From left to right: reo- and 60 for M (the toxin encoding segment). virus (type 3) dsRNA (1.8, 0.3, and 0.15 jog).dsRNA ex- The same method of extraction was used with tracted from strains containing UmVPl, P4, and P6, dsRNA extracted from strain 18, and dsRNA extracted a filamentous fungus Rhizoctonia solani but was unsuccessful for recovery of the dsRNA from S. cerevisiae containing ScV and expressing the Kl of RsV described by Finkler et al. ( 1985) and toxin.

dsRNA PLASMIDS OF U. maydis

223

pattern (Fig. 7). The behavior of the new segments H3- 1, H4a- 1,and M 1b- 1during the different phases of cell growth is quite distinct from that of the other UmV-related segments. Variation of the Titer of the dsRNA The samephenomenon wasobservedwith H3Segments during the Growth of the Host 6 and M lb-6 (not shown). It is conceivable The simple extraction method was used in that these segmentsthat are not encapsidated a time course study to determine the variation as UmV-related dsRNA are more susceptible in the titer of the dsRNA segments in cells to degradation at a phase in which cells cease containing UmVPl . The advantage of the to divide and some cell death is initiated. method is that such a study does not require a major effort and can be performed using DISCUSSION small volumes of the same cell culture. The results shown in Fig. 7 indicate that contrary New cytoplasmic dsRNA specieswere deto the continuous accumulation of H 1-1, H2tected in U. maydis strains by using a novel 1, Ml-l, M(2 + 3)-l, and L-l toward the stamethod of dsRNA extraction from whole cells. tionary phase, the titer of H3- 1, M 1b- 1, and The method is based on the insensitivity of H4a-1 follows an entirely different pattern. the dsRNA molecules to boiling in the presThe titer of these segmentsincreasesuntil the ence of high salt concentration. Its simplicity early stationary phase(46 h) and declines durenabled us to examine many killer and noning the stationary phase (between 46 and 77 killer strains of Ustifago for the presence of h). Their titer in cultures after 96 h was below additional dsRNA segments. the level of detection in agarosegels. The titer The simple and rapid method has some per cell of the viral encapsidated dsRNA segmajor advantages over the standard method ments kept increasing even in cultures after that has been used commonly, and in com168 h. When cells from a 168-h culture were parison to the rapid method developed by inoculated into fresh media and grown for 46 Fried and Fink (1978) for yeast cells the folh, all the dsRNA segmentswere recovered and lowing are observed: (a) The method is very their pattern was identical to the zero time efficient; a high yield of dsRNA segments is obtained from a relatively small number of cells. (b) The procedure is short and results are Time (h) o 4 6.5 24 46 77 96 168 obtained within 1 h; hence, many extractions can be performed on the same day: (c) DNA and ribosomal RNA, which are the main contaminants in the dsRNA samplesextracted by the conventional procedures, do not contamHI inate the dsRNA extracted by the simple #‘J H4 method: (d) After ethanol precipitation of the dsRNA samplesin the presenceof ammonium Ml a MI b sulfate, they are sufficiently pure even from oligonucleotides and can be used for enzyM2+3 matic reactions (such as 3’ end labeling) and hybridization experiments. The method of Fried and Fink ( 1978) was tested in U. maydis but managed to resolve only the heavy segment of UmV (Koltin, unpublished). The newly detected dsRNA speciesappear FIG. 7. Agarose gel of dsRNA extracted from Pl cells to be independent and distinct from the UmVat different times in a growing culture. Note the stability related dsRNA segments. Curing of P6- and of the UmV related dsRNA and the variation in H4 and Mlb. P 1-related segmentsis not accompanied by the the unencapsidated dsRNA segment (Finkler et al., 1988) found in this strain.

224

SEROUSSI ET AL.

loss of the segmentsH3, H4a, and M 1b. Segment Ml b does not hybridize to L-6 segment indicating that Mlb-6 belongs to an entirely different viral system. However, H3-6 and M 1b-6 may be interdependent since in curing experiments they are lost simultaneously. Strains that contain only M segments were never found in Ustilago and in S. cerevisiae. This is due to the fact that M cannot code for proteins needed for maintenance functions such as the coat protein, the RNA-dependent RNA polymerase, and the replicase. In view of this, H3 may be essential for M 1b maintenance and may play a role similar to that of HI that is required for maintenance of Ml and M2. In contrast to the dsRNA segments described earlier in Ustilago (Koltin, 1986) H36 and Ml b-6 do not have capsids that can be detected by fractionation in sucrosegradients. An unencapsidated dsRNA segment was described earlier in Ustilago (H4a in strain 18) (Koltin and Day, 1976) and it appearsthat the same high copy number segment is also present in the PI and P4 killer cells examined. Therefore, it appearsthat these segmentshave no relation to the different biotypes of UmV and can coinfect cells containing any one of the virus subtypes. H4a is the smallest dsRNA segment maintained independently in Ustilugo (ca. 2500 bp) and probably encodes its own polymerase. However, from its coding capacity it is predicted that the polymerase would be significantly smaller than the reovirus polymerase (135 kDa) (Joklik, 1985). The maintenance functions of this segment should be further explored. The pattern of behavior of the new dsRNA segments during the growth of the culture is different from that of UmV and is probably related to the fact that these segments are unencapsidated. Apparently, naked dsRNA molecules are more sensitive to RNase digestion than the UmV-related dsRNA. The question why were these segmentsnot noted earlier in studies using the conventional extraction procedure can be answered by two probable reasons: (a) the unencapsidated dsRNA may be more accessibleto RNases;(b) if this RNA is membrane associated,as has been described

for other fungi (Van Alfen, 1988), it may have evaded extraction and the boiling procedure in high salt releases such molecules from membranes. Unencapsidated dsRNA segments, W and T, were detected in the yeast S. cerevisiae (Weselowski and Wickner, 1984). These segments are independent of ScV and their location in the cell is unknown. The hlamentous fungus R. solani also harbors unencapsidated dsRNAs (Finkler et al., 1988). In the fungus Endothia parasitica in hypovirulent strains, the dsRNA is associated with cellular membrane vesicles.Thesevesiclescontain an RNAdependent RNA polymerasethat is not present in the vesicles of virulent strains lacking dsRNA (Van Alfen, 1988). The detection in Ustilago and in other fungi of two completely distinct classesof dsRNA segments, encapsidated and unencapsidated, may indicate that this phenomenon is not uncommon in these lower eukaryotes. ACKNOWLEDGMENT The study was supported by the US-Israel Binational ScienceFoundation Grant 0035/86.

REFERENCES BEN-ZVI, B. S., KOLTIN, Y., MEVARECH,M., AND TAMARKIN,A. (1984). RNA polymerase activity in virions from U&ago maydis. Mol. Cell Biol. 4, 188-194. B~ZARTH,R. F., KOLTIN, Y., WEISMAN,M. B., PARKER, R. L., DALTON, R. E., AND STEINLAUF,R. (198 1). The molecular weight and packaging of dsRNA in the mycovirus from Ustilago killer strains. Virology 113,492502. BUCK, K. W. (1986). Fungal virology-An overview. In “Fungal Virology” (K. W. Buck, Ed.), pp. 2-64. CRC Press,Boca Raton, FL. CHANG, T. H., BANEFUEE,N., BRUENN,J., HELD, W.. PEERY,T., AND KOLTIN, Y. (1988). A very small viral double-stranded RNA. Virus Genes, in press. DAY, P. R. (1981). Fungal viruses populations in corn smut from Connecticut. Mycologia 73, 379-391. FIELD, L. J., BRUENN,J. A., CHANG, T. H., PINCHASI, O., AND KOLTIN, Y. (1983). Two Ustilago maydis viral dsRNAs of different size code for the same product. Nucleic Acids Rex 11, 2765-2778.

FINKLER,A., BEN-ZVI, B. S., KOLTIN, Y., AND BARASH, I. (1988). Transcription and in vitro translation of the dsRNA virus isolated from Rhizoctonia solani. Virus Genes 1, 205-2 19.

FINKLER, A., KOLTIN, Y., BARASH,I., SNEH, B., AND

dsRNA PLASMIDS OF U. maydis POZNIAK,D. (1985). Isolation of a virus from virulent strains of Rhizoctonia solani. J. Gen. Virol. 66, 12211232. FRANKLIN,L. J. (1966). Purification and properties of the mphcative intermediate of the RNA bacteriophageR 17. Proc. Nat/. Acad. Sci. USA 55, 1504- 1511. FRIED, H. M., AND FINK, G. R. (1978). Electron microscopic heteroduplex analysis of “killer” double-stranded RNA speciesfrom yeast. Proc. Natl. Acad. Ski. USA 75, 4224-4228.

JOKLIK, W. K. (1985). Recent progress of Reovirus research.Annu. Rev. Genet. 19, 537-575. KOLTIN, Y. (1986). Ustilago maydis virus encoded killer system. In “Extrachromosomal Elements in Lower Eukaryotes” (R. B. Wickner, A. Hinnebusch, A. M. Lambowitz, I. C. Gunsalus, and A. Hollaender, Ed%), pp. 239-26 1. Plenum, New York. KOLTIN, Y. (1986). The killer systems in Ustilago. In “Fungal Virology” (K. W. Buck, Ed.), pp. 109-143. CRC Press,Boca Raton, FL. KOLTIN, Y. (1988). Killer system of Ustilago maydis. In “Viruses of Fungi and Simple Eukaryotes” (Y. Koltin and M. Leibowitz, Eds.), pp. 209-242. Dekker, New York. KOLTIN, Y., AND DAY, P. R. (1976). Suppression of the killer phenotype in Ustilago maydis. Genetics 82,629637.

KOLTIN, Y., AND KANDEL, J. (1978). Killer phenomenon in Ustilago maydis: The organization of the viral genome. Genetics 88, 267-276. KOLTIN, Y., MAYER,I., AND STEINLAUF,R. (1978).Killer phenomenon in Ustilago maydis: Mapping viral functions. Mol. Gen. Genet. 166, 181-186. MANIATIS,T., FRITSCH,E. F., AND SAMBROOK,J. (1982).

225

Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. PEERY,T., KOLTIN, Y., AND TAMARKIN, A. (1982). Mapping the immunity function of the Ustilago maydis Pl virus. Plasmid 7,52-58. PEERY,T., SHABAT-BRAND,T., STEINLAUF,R., KOLTIN, Y., AND BRUENN,J. (1987). Virus-encoded toxin of Ustilago maydis: Two polypeptides are essential for activity. Mol. Cell. Biol. 7,470-477. SHELBOURN,S. L., DAY, P. R., AND BUCK, K. W. (1988). Relationships and functions of virus double-stranded RNA in P4 killer strain of Ustilago maydis. J. Gen. Virol., in press. STEVENS,R. B. (1974). Genetics studies with Ustilago maydis. In “Mycology Guidebook (R. B. Stevens,Ed.), pp. 506-524. Univ. Washington Press,Seattle/London. VAN ALFEN,N. K. ( 1988). Viruses of Endothia parasitica. In “Viruses of Fungi and Simple Eukaryotes” (Y. Koltin and M. Leibowitz, Eds.), pp. 371-386. Dekker, New York. VODKIN, M., UTTERMAN, F., AND FINK, G. A. (1974). Yeast killer mutants with altered double-stranded ribonucleic acid. J. Bacterial. 117, 68 l-686. WESELOWSKI, M., AND WICKNER,R. B. (1984). Two new double-stranded RNA molecules showing non-Mendelian inheritance and heat inducibility in Saccharomyces cerevisiae. Mol. Cell. Biol. 4, 181-187. WICKNER,R. B. (1986). Double-strandedRNA replication in yeast: The killer system. Annu. Rev. Biochem. 55, 373-395.

WIGDERSON,M., AND KOLTIN, Y. (1982). Dual toxin specificities and the exclusion relations among the Ustilago dsRNA viruses. Curr. Genet. 5, 127-l 36. Communicated

by P. J. Farabaugh