Proc. Natl. Acad. Sci. USA 84 (1987)

Correction. In the article "Structural properties of double-stranded RNAs associated with biological control of chestnutblight fungus" by James Tartaglia, Cynthia P. Paul, DennisW. Fulbright, and Donald L. Nuss, which appeared innumber 23, December 1986, of Proc. Natl. Acad. Sci. USA(83, 9109-9113), the data in the last line of Table 1 (p. 9111)are missing because of a printer's error. The table and itslegend and footnotes are presented below.

Table 1. Characterization of the 3'- and 5'-end-labeledheterogeneous RNase Ti-resistant oligonucleotidesfrom L-, M-, and S-RNA

Sensitivity

Treatment Nondenatured Denatured

3-End-labeledPancreatic RNase* - -RNase U2t - +(dT)1218 plus RNase Ht - +

S-End-labeledRNase U2RNase B. cereust + +

Nondenatured or denatured RNAs were digested with RNase asindicated. Digestions were terminated by incubation at 370C for 30min with proteinase K (Boehringer Mannheim) at 100 ,ug/ml. RNAwas recovered by precipitation following phenol extraction. RNaseT1 digestion and gel analysis (see Materials and Methods) were thenperformed to reveal the relative sensitivity of the heterogeneousRNase Ti-resistant material to the various RNases.*Pancreatic RNase (Sigma) was used under conditions in whichpoly(adenylic acid) tracts are resistant (22).tPurchased as part of an RNA sequencing kit (Pharmacia) and usedaccording to supplier's specifications.tRNase H digestion following hybridization to (dT)12 18 was amodification of that described previously (23). Approximately 1.0,ug of (dT)1218 was added to nondenatured or denatured ds RNA("10,000 cpm) in 80 mM Tris (pH 7.5)/40 mM MgCl2/400 mMKC1/0.4 mM dithiothreitol. RNase H (0.3 unit) was added and thereaction mixture was incubated at 37'C for 20 min. RNase H and(dT)1218 were kindly provided by A. K. Banerjee (Roche Instituteof Molecular Biology).

1350 Genetics: Correction

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Proc. Nati. Acad. Sci. USAVol. 83, pp. 9109-9113, December 1986Genetics

Structural properties of double-stranded RNAs associated withbiological control of chestnut blight fungus

(Endothia parasitca/transmissible hypovirulence/molecular hybridization/terminal-nucleotide analysis/internal deletion)

JAMES TARTAGLIA*, CYNTHIA P. PAULt, DENNIS W. FULBRIGHTt, AND DONALD L. NUSS*t*Department of Cell Biology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110; and tDepartment of Botany and PlantPathology, Michigan State University, East Lansing, MI 48824-1312

Communicated by Aaron J. Shatkin, August 18, 1986

ABSTRACT Double-stranded RNAs (ds RNAs) are thoughtto be the cytoplasmic determinants responsible for the phe-nomenon of transmissible hypovirulence in the chestnut blightfungus Endothiaparasitica [Murr.] Anderson. The three majords RNA components associated with the North Americanhypovirulent strain, Grand Haven 2, were characterized withrespect to molecular-hybridization specificity and RNase T1-digestion patterns. The large (L-RNA; %9 kilobase pairs) andmiddle-sized (M-RNA; -3,5 kilobase pairs) ds RNA compo-nents cross-hybridized under stringent conditions and exhib-ited indistinguishable partial and complete RNase T1 digestionpatterns relative to their 5' and 3' termini. These resultssuggest that M-RNA was derived from L-RNA by an internaldeletion event. The small (S-RNA; =1 kilobase pair) RNA wasunrelated to L- and M-RNA by these criteria. However, allthree ds RNA components contained RNase Ti-resistant oli-gonucleotides at one 5' terminus and at the corresponding 3'terminus of the complementary strand. These RNase T1-resistant species exhibited properties consistent with stretchesof poly(uridylic acid) and poly(adenylic acid), respectively. Thecombined results are discussed in terms of the structuralorganization of hypovirulence-associated ds RNA moleculesand their similarities to "double-stranded" RNA moleculesobserved in plant and animal cells infected with single-strandedRNA viruses.

The North American chestnut [Castanea dentata (March)Borkh.] has been eliminated as an important economic treespecies by the fungal pathogen Endothia parasitica (Murr.)Anderson (reviewed in refs. 1-3). Chestnut blight was alsointroduced into Europe, where it caused considerable dam-age to commercial chestnut (Castanea sativa Mill.) planta-tions (1-3). However, both in Europe and in North America,some chestnut trees survived and remained productive inspite of infection. Fungal isolates recovered from thesesurviving trees were shown to be reduced in virulence(hypovirulent) (4-7). Subsequent studies showed that thehypovirulence phenotype was transmissible by hyphal fusionto virulent strains (1, 2, 5, 6). Since normally susceptiblechestnut trees can control infection by hypovirulent strains,and the hypovirulence phenotype is transmissible, applica-tion of appropriate hypovirulent strains to trees infected withvirulent strains can have a curative effect (1-3, 6, 8, 9). Theuse of hypovirulent strains in Europe and, to a more limitedextent, in North America to control chestnut blight repre-sents one of the few examples of successful biological controlof a plant disease. By determining the molecular basis ofhypovirulence it may be possible to optimize the biologicalcontrol of chestnut blight and, perhaps, extend this strategyas a general mechanism for the control of fungal pathogens.

Correlative evidence strongly suggests that double-stranded RNA (ds RNA) is the cytoplasmic transmissibledeterminant responsible for hypovirulence in E. parasitica(9-11). ds RNAs associated with different hypovirulentstrains vary with respect to size, concentration, and sequencehomology (3, 9-12). Information concerning the relationshipof these ds RNAs to each other, their structural organization,and their role in hypovirulence is limited or nonexistent. Aprerequisite to understanding the molecular basis of hypo-virulence is an understanding of the structural and functionalproperties of the associated ds RNAs. We report an analysisof the relatedness and surprising structural properties of thethree major ds RNAs associated with a North Americanhypovirulent strain of E. parasitica.

MATERIALS AND METHODSFungal Strains and Growth Conditions. Grand Haven 2

(GH2) is a bark isolate of E. parasitica from Grand Haven,MI (7). Cultures of the isolate were maintained on potatodextrose agar (Difco) at 20°C under fluorescent lights. Cul-tures for ds RNA isolation were grown for 10-14 days in theliquid medium of Puhalla and Anagnostakis (13), which hadbeen modified by the omission of glucose.

Purification of ds RNA. The ds RNA was isolated asdescribed (7), with the following modification. Buffer (0.05MTris/0.1 M NaCl/0.001 M EDTA, pH 6.8) in the initial stagesof isolation contained 5 mM ethylene glycol bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), 5 mM N-ethyl-maleimide, 100 ,g of heparin per ml, 0.5% (vol/vol) 2-mercaptoethanol, and 50 ,g of spermine per ml (14). Afterphenol extraction, nucleic acids were precipitated with eth-anol and resuspended in buffer without additives prior toCF-11 chromatography.

5'- and 3'-End-Labeling of ds RNA. Total ds RNA waslabeled at the 3' termini with 3'-phosphocytidine 5'-[32P]phos-phate ([5'-32P]pCp, Amersham) and RNA ligase (Pharmacia)as described (15). After treatment with calf intestinal alkalinephosphatase (Boehringer Mannheim), total ds RNA waslabeled at the 5' termini using the 5' DNA terminus labelingsystem (Bethesda Research Laboratories).

Isolation of the Individual ds RNA Components. To isolateindividual end-labeled ds RNA segments, the reaction mix-tures were fractionated in a 30-cm, 7.5% polyacrylamide gelfor 5000 V-hr at room temperature. Individual ds RNAcomponents were visualized by ethidium bromide staining,excised, and electroeluted according to the method of Sahaet al. (16). Nonlabeled ds RNA components used in thehybridization analysis were isolated from low-melting-pointagarose and run over a NACS column (Bethesda ResearchLaboratories) according to the manufacturer's specifica-tions.

Abbreviations: ds RNA, double-stranded RNA; bp, base pair(s).tTo whom reprint requests should be addressed.

9109

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 83 (1986)

Analysis of 3'- and 5'-End-Labeled RNAs. Individual 3'-end-labeled RNAs (15,000-20,000 cpm) were denatured andanalyzed by partial and complete RNase T1 (Sankyo) diges-tion as described (17). After elution of individual RNaseTi-resistant oligonucleotides with 0.5 M ammonium acetate,the 3'-terminal nucleotides were determined by PEI-cellulosethin-layer chromatography following RNase T2 (Sankyo)digestion (17).

Individual 5'-end-labeled RNAs (15,000-20,000 cpm) weredenatured (17), recovered by ethanol precipitation, andresuspended in 8.0 ,1 of 33 mM sodium citrate, pH 5.0/1.7mM EDTA/0.04% xylene cylanol FF/0.08% bromophenolblue/7 M urea. One microliter of this mixture was removedfor complete RNase T1 digestion (10 units) at 550C for 75 min.Partial digestions were performed by adding RNase T1 (0.005unit) to the remaining 7.0-1.l mixture with subsequent incu-bation at 559C for 20 min, followed by gel analysis andautoradiography as described (17). The 5'-terminal nucleo-tides were determined by PEI cellulose thin-layer chroma-tography after nuclease P1 digestion (18).

RESULTS

Hybridization Analysis of the Isolated GH2 ds RNAs. Anal-ysis of ds RNAs purified from the hypovirulent GH2 strain ofE. parasitica revealed three major ds RNA species (Fig. 1) ofapproximately 9.0 (L-RNA), 3.5 (M-RNA), and 1.0 (S-RNA)kilobase pairs (kbp), respectively. In order to determinewhether these ds RNA segments share sequence homology,hybridization analyses were performed using individuallyisolated species. Unlabeled L-RNA, M-RNA, and S-RNAwere isolated as described in Materials and Methods andimmobilized on Hybond N membrane (Amersham). Thepurity of these ds RNAs was confirmed by labeling a smallaliquot of each species with [5'-32P]pCp and visualizing themby autoradiography after NaDodSO4/polyacrylamide gelelectrophoresis (Fig. 2A). Similar gel profiles were obtainedwith both 5'- and 3'-end-labeled ds RNA fragments electro-eluted from polyacrylamide gels and employed as hybridiza-tion probes or as substrates for specific RNase analyses.The immobilized, unlabeled ds RNA species were probed

with 3'-end-labeled M-RNA or S-RNA. M-RNA hybridizedto both L-RNA and M-RNA but not to S-RNA (Fig. 2B, I),

WTV GH2

- L-RNA

3.5 kbp- - M-RNA

1.7 kbp -

0.9 kbp -Rg - S-RNA

FIG. 1. Electrophoretic pattern of GH2 ds RNA components.Total ds RNA extracted from the GH2 strain of E. parasitica wasfractionated in a 7.5% polyacrylamide gel according to the method ofLaemmli (19). The ds RNA species were visualized by silver stainingwith a kit purchased from Bio-Rad and used according to themanufacturer's specifications. These ds RNAs are shown in laneGH2. Wound tumor virus (lane WTV) ds RNA was used as a marker.The estimated sizes of WTV segments 1, 6, and 9, previouslyreported (20), are indicated. The asterisks indicate WTV segmentsfor which lengths were determined by sequence analysis of cDNAclones. Segment S5 (upper asterisk) is 2613 bp long (J. Anzola, T.Asamizu, and D.L.N., unpublished results), and segment S12 (lowerasterisk) is 851 bp long (21).

A 1 2 3 M

MMa.

_p _

B L MSI **

II 0

FIG. 2. Dot blot hybridization of 3'-end-labeled M-RNA andS-RNA against isolated L-, M-, and S-RNA. (A) Isolated ds RNAswere 3'-end-labeled and run in a 7.5% polyacrylamide gel to test theirpurity. The ds RNA species were visualized by autoradiography onKodak XAR-2 film. Lanes 1-3: L-, M-, and S-RNA, respectively.Lane M: end-labeled WTV ds RNA used as markers (see Fig. 1). (B)Unlabeled, individually isolated ds RNA components (-50 ng) weredenatured by incubation at 65°C for 30 min in the presence ofN2-flushed dimethyl sulfoxide and then were precipitated. Theprecipitates were resuspended in 2.0 ,ul 5x SSC (ix is 0.15 MNaCl/0.015 M sodium citrate, pH 7) and applied immediately ontoHybond N membrane (Amersham) and immobilized by UV irradi-ation. Prehybridization and hybridization buffers were similar tothose described previously (17). Prehybridization was at 42°C for 3hr, and hybridization was at 42°C for 18 hr with 100,000 cpm ofheat-denatured, 3'-end-labeled M-RNA or S-RNA in a final volumeof 2.0 ml. After hybridization, the dot blots were washed four times(30 min each) at room temperature with lx SSC/0.1% NaDodSO4/0.1% sodium pyrophosphate. Three additional washes (30 min each)were performed at 72°C with 0.2x SSC/0.1% NaDodSO4/0.1%sodium pyrophosphate. The blots were analyzed by autoradiographyon Kodak XAR-2 film. The letters L, M, and S correspond to thepositions of the immobilized L-, M-, and S-RNAs, respectively. RowI: L-, M-, and S-RNA probed with radiolabeled M-RNA. Row II: theL-, M-, and S-RNA probed with radiolabeled S-RNA.

whereas S-RNA hybridized exclusively to itself (Fig. 2B, II).These results suggest that L-RNA and M-RNA share se-quence homology but lack extensive sequence homologywith S-RNA.RNase T1 Analysis of 3-End-Labeled RNAs. Since L-RNA

and M-RNA are related by sequence homology, the possi-bility existed thatM-RNA was derived fromL-RNA by eitheran internal deletion or an early termination event. To distin-guish between these possibilities, partial and completeRNase T1 digestion patterns of 3'-end-labeled moleculeswere compared. The partial digestion patterns for L-RNAand M-RNA were indistinguishable (Fig. 3, lanes 1 and 2),indicating that guanosine positions relative to the 3' terminifor each strand of L- and M-RNA are identical for at least 40nucleotides from each end. The partial digestion pattern ofS-RNA, however, differed from that of L-RNA and M-RNA(Fig. 3, lane 3). The complete RNase T1 digestion patterns forL-RNA and M-RNA were also indistinguishable but differedfrom the digestion pattern of S-RNA (Fig. 3, lanes 4-6).Oligonucleotides a and a' (Fig. 3) both contained 3'-terminalcytidine, while oligonucleotides b and b' both contained3'-terminal adenosine (data not shown). These results con-firm the relationship between L-RNA and M-RNA demon-strated by the hybridization analyses and are consistent withthe possibility that M-RNA was derived by an internaldeletion of L-RNA.

Characterization of the Heterogeneous RNase Tl-ResistantSpecies. In addition to the presumptive 3Y-terminal oligonu-

9110 Genetics: Tartaglia et A

Proc. Natl. Acad. Sci. USA 83 (1986) 9111

M 1 2 3 4 5 6

..,_, A

Cii,0'--,xcslipXC--StV

BPB- a ..,a

FIG. 3. Partial and complete RNase T1 analysis of 3'-end-labeledL-, M-, and S-RNA. Partial and complete RNase T1 reactions ofend-labeled RNAs were done as described (17). The reactionproducts were fractionated in a 20% polyacrylamide gel containing 7M urea and were visualized by autoradiography on Kodak XAR-2film. Lanes 1-3: partial RNase T1 digests of L-, M-, and S-RNA,respectively. Lanes 4-6: complete digests of L-, M-, and S-RNA,respectively. The complete RNase T1 digestion products of L-RNAare designated a, b, and c, and the complete products ofM-RNA aredesignated a', b', and c'. The complete digestion products of theS-RNA are designated d and e. Lane M (marker): partial hydrolysisproducts of end-labeled RNA in half-volume reaction mixturescontaining 50 mM NaHCO3 plus 1 mM EDTA (pH 9.0) at 90°C for15 min and 30 min, respectively. Migration positions ofbromophenolblue (BPB) and xylene cylanol FF (XC) are indicated at left.

cleotides a, a', b, b', and d, complete RNase T1 digestion ofthe 3'-labeled ds RNAs generated material that migrated in aheterogenous manner at a position expected for oligonucleo-tides composed of30-60 nucleotides (Fig. 3, oligonucleotidesc, c', and e). Exposure of the RNase Ti-resistant heteroge-neous material to various nucleases (Table 1) revealedresistance to pancreatic RNase in high salt (330mM KCl) andsensitivity to RNase U2 [an adenosine-cleavage-specificnuclease (24)] or to RNase H after hybridization of thematerial with oligo(dT). However, sensitivity to RNase U2and RNase H required prior denaturation. These propertiessuggest that the RNase Tl-resistant heterogeneous materialrepresents a collection of 3'-terminal oligonucleotides com-posed of 30-60 adenosine residues [oligo(A)] that exist in abase-paired form.The generation of presumptive 3'-terminal oligonucleo-

tides a, a', b, and b' as well as oligo(A) (oligonucleotides c andc') from both L- and M-RNAs indicates a degree of 3'heterogeneity. Hybrid-selection studies with bacteriophageM13 recombinants harboring cDNA sequences specific for L-and M-RNAs revealed that the oligo(A) and oligonucleotidesb and b' were located at the 3' end of the same-polarity strand(data not shown). As mentioned previously, oligonucleotidesa and a' have a 3'-terminal cytidine, whereas oligonucleotidesb and b' have a 3'-terminal adenosine. It is concluded that L-and M-RNAs each exist as a population of molecules thatcontains oligonucleotide a/a' with a terminal cytidine at the3' end of one strand. The 3' terminus of the other strandcontains either oligonucleotide b/b' with a terminal adeno-sine, or oligonucleotide b/b' extended with oligo(A) (i.e.,oligonucleotide c/c'). Consistent with this interpretation is

Table 1. Characterization of the 3'- and 5'-end-labeledheterogeneous RNase Ti-resistant oligonucleotidesfrom L-, M-, and S-RNA

Sensitivity

Treatment Nondenatured Denatured

3'-End-labeledPancreatic RNase*RNase U2t +(dT)1218 plus RNase Ht - +

S'-End-labeledRNase U2ANase B.. cereust ............................................

Nondenatured or denatured RNAs were digested with RNase asindicated. Digestions were terminated by incubation at 370C for 30min with proteinase K (Boehringer Mannheim) at 100 ,ug/ml. RNAwas recovered by precipitation following phenol extraction. RNaseT1 digestion and gel analysis (see Materials and Methods) were thenperformed to reveal the relative sensitivity of the heterogeneousRNase Ti-resistant material to the various RNases.*Pancreatic RNase (Sigma) was used under conditions in whichpoly(adenylic acid) tracts are resistant (22).tPurchased as part of an RNA sequencing kit (Pharmacia) and usedaccording to supplier's specifications.tRNase H digestion following hybridization to (dT)12_18 was amodification of that described previously (23). Approximately 1.0,ug of (dT)1218 was added to nondenatured or denatured ds RNA(-10,000 cpm) in 80 mM Tris (pH 7.5)/40 mM MgCl2/400 mMKC1/0.4 mM dithiothreitol. RNase H (0.3 unit) was added and thereaction mixture was incubated at 37°C for 20 min. RNase H and(dT)1218 were kindly provided by A. K. Banerjee (Roche Instituteof Molecular Biology).

the observation that RNase T2 digestion of 3'-labeled L- andM-RNAs yields pA and pC in a ratio of 60% to 40% (data notshown). The 3'-terminal heterogeneity observed with L- andM-RNA was not evident with the S-RNA. Complete RNaseT1 digestion of the S-RNA generated two RNase Ti-resistantproducts (Fig. 3, lane 6): oligonucleotide d, which containeda 3'-terminal cytidine, and oligonucleotide e, which wascomposed of poly(adenylic acid). Furthermore, RNase T2digestion of S-RNA yielded equal amounts of pA and pC(data not shown).RNase T1 Analysis of 5'-End-Labeled RNAs. Since the L-,

M-, and S-RNA molecules contain 3'-terminal stretches ofpoly(adenylic acid) and their removal required prior dena-turation (Table 1), the possibility existed that they contain acorresponding 5' stretch of poly(uridylic acid). Therefore,5'-end-labeled L-, M-, and S-RNA were isolated, treated withRNase T1, and analyzed (Fig. 4). Partial digestion patternsagain revealed the relatedness of L- and M-RNA (Fig. 4A;lanes 1 and 2). The partial digestion pattern of the 5'-end-labeled S-RNA (Fig. 4A, lane 3), as expected, differed fromthose of the L- and M-RNA, although all three RNAs appearto be rich in guanosine residues at one 5' terminus.A significant feature of the complete RNase T1 digests was

the presence of heterogeneous RNase Ti-resistant species(Fig. 4B; lanes 1-3) similar to those observed after completeRNase T1 digestion of 3'-end-labeled L-, M-, and S-RNA(Fig. 3; lanes 1-3). However, the 5'-terminal, RNase T1-resistant heterogeneous species were resistant to RNase U2but sensitive to RNase from Bacillus cereus [a cytidine- anduridine-cleavage-specific nuclease (23)] (Table 1). The sen-sitivity of this material to RNase B. cereus, its insensitivityto RNase U2, and the presence of a base-paired 3'-terminalstretch of poly(adenylic acid) strongly suggested that one 5'terminus of these ds RNA molecules contains a stretch ofpoly(uridylic acid). A portion (40%) of the poly(uridylic acid)moiety was sensitive to RNase B. cereus in the presence of330 mM NaCl without prior denaturation of the ds RNA,

Genetics: Tartaglia et al.

Proc. Natl. Acad. Sci. USA 83 (1986)

(-) C M-RNA (U)n3

(+) 5' GCrich (A)n(Gh(-) C S-RNA (U)n

3'

I I.

FIG. 4. Partial (A) and complete (B) RNase T1 analysis of5'-end-labeled L-, M-, and S-RNA. Partial and complete RNase T1digestions of 5'-end-labeled RNA species were described in Mate-rials and Methods. Digestion products were analyzed as describedfor Fig. 3. Lanes 1-3: L-, M-, and S-RNAs, respectively. Migrationpositions of BPB and XC are indicated. In B, the 5'-heterogeneousRNase Tl-resistant material for each RNA species is indicated by anasterisk.

suggesting that some of the molecules have a 5' poly(uridylicacid) extension.

Identification of the presumptive terminal RNase T1 frag-ment of the non-poly(uridylic acid) 5' terminus of L-, M-, andS-RNAs was complicated by the inability to achieve com-plete digestion even under denaturing conditions (Fig. 4). Asindicated by the partial RNase T1 digestion pattern (Fig. 4A;lanes 1-3), the non-poly(uridylic acid) 5' termini of each dsRNA is rich (Q15 nucleotides) in guanosine residues. Thepotential for extensive G-C base-pairing in this region ispresumably responsible for the failure to achieve completeRNase T1 digestion. RNase P1 digestion of each ds RNAyielded 5'-terminal pU, pG, and pA in a 50%:20%o:30% ratiofor L- and M-RNA and a 50%:40%:10% ratio for S-RNA. Thecombined results suggest that one strand of each ds RNAcontains a 5'-terminal stretch of poly(uridylic acid) partiallybase-paired to the poly(adenylic acid) stretch found at the 3'terminus of the opposite strand (Fig. 5). The 5' terminus ofthe poly(adenylic acid)-containing strand is composed ofa 15-to 20-nucleotide-long, guanosine-rich region terminated witheither a guanosine or an adenosine residue.

DISCUSSIONThis study was undertaken to determine the relatedness andstructural properties of the double-stranded L-, M-, andS-RNA species associated with the North American hypo-virulent strain of E. parasitica, GH2. Results from hybrid-ization analyses have demonstrated that L- and M-RNAshare extensive sequence homology. However, neither theL-RNA nor the M-RNA cross-hybridized with the S-RNA.RNase T1 digestion patterns ofboth 3'- and 5'-end-labeled L-and M-RNA were found to be indistinguishable. These datastrongly suggest that the M-RNA was derived from theL-RNA by an internal deletion event.

Variant ds RNAs that are terminally conserved remnantsof genomic structures generated by internal deletion eventshave been observed in RNA preparations from wound tumorvirus (17), cytoplasmic polyhedrosis virus (M. Arella and Y.Furuichi, personal communication), and Saccharomycescerevisiae virus, ScV (25-28). Additionally, terminal se-

FIG. 5. Schematic representation of the L-, M-, and S-RNAcomponents associated with the GH2 hypovirulent strain of E.parasitica. (A) The 9.0-kbp L-RNA. (B) The 3.5-kbp M-RNA isillustrated as being derived from L-RNA by an internal deletionevent. (C) The 1.0-kbp S-RNA is depicted and, as indicated, does notshare any extensive sequence homology with L- and M-RNA.Physical characteristics common to all three ds RNA species areindicated. The RNA strand containing the 3'-terminal poly(adenylicacid) sequence is arbitrarily designated as the (+)-strand, while thecomplementary strand with the 5'-terminal poly(uridylic acid) se-quence is designated as the (-)-strand. Also noted is the 3'-terminalheterogeneity for L- and M-RNAs relative to polyadenylylation, the5'-heterogeneity noted for all three species, and the apparentG+C-richness of one of the 5'-terminal sequences of all three dsRNA molecules. The G+C-rich regions are not drawn to scale andrepresent 15-20 nucleotides of the 5' terminus.

quences are conserved in the formation of defective inter-fering RNA genomes of poliovirus, Sendai virus, Sindbisvirus, and influenza virus (29-32). In all of these systems, theinternal deletion events yielded genomic RNA remnants thatwere functional with respect to transcription and, in theappropriate context, to replication and packaging.Although M-RNA of GH2, obviously, contains the se-

quences essential for its replication, it may not be functionalwith respect to the hypovirulent phenotype. There existhypovirulent GH2 isolates that either contain a decreasedconcentration ofM-RNA or totally lack this component whileretaining the full complement ofL-RNA (8). Close inspectionof the ds RNAs associated with most hypovirulent strains(refs. 5 and 8-11; Fig. 1) reveals a minor heterogeneouspopulation of molecules that are smaller than the largest dsRNA present in the isolate. These molecules may representremnants of larger ds RNA segments that are maintained inhypovirulent strains as a consequence of the retention ofsequence domains essential for their replication. The gener-ation of remnant molecules by internal deletion events maybe a general feature of ds RNA replication in E. parasiticathat contributes to the diverse ds RNA banding patternsobserved among hypovirulent isolates. S-RNA, however, isclearly not derived from L- or M-RNA (Figs. 2 and 3). Sincethe hypovirulent phenotype persists in GH2 isolates that lackS-RNA (D.W.F., unpublished observation), it is tempting tospeculate that species such as S-RNA may play a role inmodulating the level of hypovirulence.

This communication also presents a structural analysis ofhypovirulence-associated ds RNAs. The results have beeninterpreted as indicated in Fig. 5. RNA strand polarity hasbeen arbitrarily assigned. The 5' terminus ofthe (+)-strand ofL-, M-, and S-RNAs is rich in guanosine residues (15-20nucleotides) and is terminated at the 5' end with either aguanosine or an adenosine residue. The 3' termini of the(-)-strands are uniformly terminated with a cytidine residue.

B1 2 3

Om

XC

A1 23

.: a.4

.x

4'

;FBPB

WsAO

A 3'-IA~n

-BPB.. r:0* a

9

5'

5

3'

5'

9112 Genetics: Tartaglia et al.

(A) G:Crich

FA-l G 9=6-

Proc. Natl. Acad. Sci. USA 83 (1986) 9113

The 3' termini of the (+)-strands contain a stretch of 30-60adenosine residues, while the 5' termini of the (-)-strandsappear to be a complementary stretch of poly(uridylic acid).However, partial sensitivity of the poly(uridylic acid) moietyto RNase B. cereus without prior denaturation of the ds RNA(Table 1) suggests that some of the 5' uridine residues are inan unpaired form. The precise nature and possible functionalroles of the heterogeneity found at the 5' ends of the (+)- and(-)-strands are unknown. Heterogeneity is also found at the3' termini of the (+)-strands of L- and M-RNAs, in that someof the molecules appear not to be polyadenylylated (Fig. 5).This heterogeneity is not observed for S-RNA.The structural features determined for the GH2 ds RNAs

provide valuable landmarks for characterization and se-quence analysis of cDNA clones of the ds RNAs. Usinggene-transfer methods recently developed for fungal systems(33) and appropriate cDNA clones, it may be possible toidentify precisely the genetic information responsible for thehypovirulence phenotype. This information, in turn, could beexploited to engineer hypovirulent fungal strains.The presence of ds RNAs associated with hypovirulent

strains of E. parasitica suggests that virus-like pathogens ofthe fungus are involved, since this is the genetic material ofmost mycoviruses (34). However, structures resemblingmycovirus particles (34, 35) have not been observed in thesehypovirulent strains (36-38). It is conceivable that the dsRNAs are not genomic elements but are replicative interme-diates (RI) and/or replicative forms (RF) of a single-strandedRNA genome. Several structural and functional features ofthe E. parasitica ds RNA species are strikingly similar tothose exhibited by RI and RF molecules of single-strandedRNA genomes (39, 40). For example, RNA species ofvaryingsize classes, including full-length molecules, are observed byelectrophoretic analysis of the L-, M-, and S-RNAs understrand-separating conditions (data not shown), a situationsimilar to that reported for poliovirus RI molecules (41). Inaddition, both GH2 ds RNAs and poliovirus RI and RFmolecules contain poly(adenylic acid) at the 3' terminus ofone strand and a stretch of poly(uridylic acid) at the 5'terminus of the complementary strand (42-44). Finally, theGH2 ds RNAs are found associated with membrane struc-tures that contain RNA-dependent RNA polymerase activity(38), much like the replication complexes found withinpoliovirus- and tobacco mosaic virus-infected cells (45-49).Based on the structural properties of GH2 ds RNAs de-scribed in this report, we propose that hypovirulence-associated ds RNAs may represent replicative forms ofsingle-stranded genetic elements.

We are grateful to A. Hernandez for assistance in preparing themanuscript. This is article 12073 ofthe Journal Series ofthe MichiganAgriculture Experiment Station.

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Genetics: Tartaglia et al.