Proc. Nati. Acad. Sci. USAVol. 91, pp. 8680-8684, August 1994Genetics

A small mitochondrial double-stranded (ds) RNA element associatedwith a hypovirulent strain of the chestnut blight fungus andancestrally related to yeast cytoplasmic T and W dsRNAsJAMES J. POLASHOCK AND BRADLEY I. HILLMANDepartment of Plant Pathology, Rutgers University, New Brunswick, NJ 08903

Communicated by Myron K. Brakke, June 3, 1994

ABSTRACT A small double-stranded (ds) RNA elementwas isolated from a moderately hypovirulent strain of thechestnut blight fungus Cryphonectria parasitica (Murr.) Barr.from eastern New Jersey. Virulence was somewhat lower in thedsRNA-containing strain than in a virulent dsRNA-free controlstrain, but colony morphology and sporulation levels werecomparable. A library of cDNA clones was constructed, andoverlapping dones representing the entire genome were se-quenced. The 2728-bp dsRNA was considerably smaller thanpreviously characterized C. paraitica dsRNAs, which are12-13 kb and ancestrally related to the Potyvirldae family ofplant viruses. Sequence analysis revealed one large open read-ing frame, but only if mitochondrial codon usage (UGA = Trp)was invoked. Nuclease assays of purified mitochondria con-firmed that the dsRNA was localized within mitochondria.Assuming mitochondrial translation, the deduced amino acidsequence had landmarks typical of RNA-dependent RNA poly-merases. Alinents of the conserved regions indicate that thisdsRNA is more closely related to yeast T and W dsRNAs andsingle-stranded RNA bacteriophages such as Q(3 than to otherhvnovirulence-associated dsRNAs.

Cryphonectria parasitica, the filamentous ascomycete thatcauses chestnut blight, was responsible for the decline of thechestnut [Castanea dentata (Marsh.) Borkh.] in North Amer-ica. Virulence of C. parasitica can be reduced to variousdegrees by viral infection, a phenomenon referred to ashypovirulence (1). Considerable progress on the character-ization ofdouble-stranded (ds) RNA elements has been madein recent years (for reviews, see refs. 2-4). The most commonof these have been assigned to a newly described family ofviruses, the Hypoviridae (5). Members of this family havedsRNA genomes of 12-13 kb (6, 7) that are encapsulated inhost-derived lipid vesicles with a viral-encoded RNA-dependent RNA polymerase (8, 9). A full-length cDNA cloneof the type member of the Hypoviridae, CHV1-713 [previ-ously called HAV (6)] has been shown to be infectiousthrough stable transformation of C. parasitica spheroplasts(10).

Besides reducing virulence, the presence of viral dsRNAsmay have other effects on C. parasitica. These includereductions in growth rate, pigmentation, and sporulation (11);reduced laccase and cutinase production (12-14); and generaldown-regulation of fungal proteins and mRNAs (15, 16).Changes in culture morphology are often associated withdsRNA-containing strains such as EP713 or NB58, and suchstrains can easily be identified as infected.The isolation from a C. parasitica culture of a very small

dsRNA molecule not associated with any other dsRNAssuggested that it would be fundamentally different frommembers of the Hypoviridae. Results of this preliminary

characterization indicate that it is different not only in itsancestry but also in its localization within mitochondria ofinfected fungi.

MATERIALS AND METHODSFungal Culture and dsRNA Isolation. C. parasitica strain

NB631 was isolated from a recovering American chestnuttree in Howell Township, New Jersey, by P. J. Bedker.Other dsRNA-containing, hypovirulent strains described areNB58 (ATCC no. 76220), EP713 (ATCC no. 52571), and9B-2-1 from West Virginia (17). dsRNA-free, virulent strainsdescribed are EP155 (ATCC no. 38755) and NB58-19 (ATCCno. 76221). Maintenance of strains, dsRNA extraction, andanalysis were as described (18).

Virulence and Sporulation. Virulence studies were per-formed on Granny Smith apples (19, 20). Each apple wasinoculated with the test strains as well as EP155 as a virulentstandard. After inoculation, apples were incubated at 250C inplastic boxes with moist towels. Necrotic lesions were mea-sured after 3 weeks.

Sporulation under high light intensity was determinedessentially as described (11). Conidia were washed from eachplate after 5 days with 10 ml of distilled water, dilutedappropriately, and counted with a hemacytometer.

Cloning and Sequencing of NB631 dsRNA. A randomlyprimed cDNA library of purified, denatured dsRNA wasconstructed in the plasmid vector pUC9 as described byHillman et al. (18). Clones representing the ends of thedsRNA were obtained by using the 5' RACE (rapid amplifi-cation ofcDNA ends) procedure (21, 22) on 2.5-pg templatedsRNA. Primers and reagents from BRL were used essen-tially according to the manufacturer's recommendations.Representative clones were mapped and sequenced using theSequenase kit (United States Biochemical) and specific prim-ers where required. The entire genome was sequenced fromoverlapping clones on both strands.* Ninety-five percent ofthe genome was sequenced on multiple clones. To verify thesequence at the ends, three independent 5' RACE cloneswere analyzed. Sequences of all oligonucleotides used in thisstudy are available on request.

Isolation and Analysis of Mitochondria. Strain NB631 wasgrown for 5 days in liquid medium (250 ml/i-liter flask). Thiscomplete medium was made essentially as described byPuhalla and Anagnostakis (23), except that the salt solutionwas replaced with Murashige and Skoog basal salt mixture(24). Flasks were inoculated with -5 x 105 conidia. Tissuewas prepared and mitochondria were purified by the methodof Cramer et al. (25) as described by Taylor and Natvig (26).Nuclease assays were performed on aliquots of purified

mitochondria that were lysed and extracted once with an

Abbreviations: ds, double stranded; ss, single stranded; RACE,rapid amplification of cDNA ends.*The sequence reported in this paper has been deposited in theGenBank data base (accession no. L31849).

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A Silver-stainedpolyacrylamide1 2 3

A 12.5

2.6

( 1.7

B Northern blots

ss ds

2.4>.

1 .35o.2.6

1 .7

NB58 NB631

FIG. 1. Colony morphologies of virulent and hypovirulent C.parasitica isolates. EP155 is a standard, orange, dsRNA-free virulentculture. EP713 and NB58 are, respectively, white and brown hypo-virulent cultures with large (>12 kb) cytoplasmic dsRNAs. NB631 isorange and hypovirulent, and it contains a 2.7-kb mitochondrialdsRNA.

equal volume of phenol/chloroform/isoamyl alcohol(25:24:1). Restriction digestions were performed with bufferssupplied by the manufacturer. Nuclease digestions were as

described by Hillman et al. (11), except that high-salt RNasedigestions were performed in 0.6 M rather than 0.3 M NaCl.Northern Blot Analysis. dsRNA was separated on a 1%

agarose gel and alkaline blotted to Zeta-Probe nylon mem-brane (Bio-Rad) according to the manufacturer's protocol.Single-stranded (ss) RNA was isolated and separated on a1.2% formaldehyde gel as described (27) and blotted asdescribed above. Blots were probed with cDNA clones thatwere 32P-labeled by random hexamer priming as recom-mended by the manufacturer (BRL).

RESULTS AND DISCUSSIONStrain Characteristics. Many hypovirulent strains of C.

parasitica exhibit grossly altered morphology compared tovirulent strains (3, 4). NB631 did not appear abnormal inculture but looked similar to virulent, dsRNA-free strainEP155 (Fig. 1). One of the screens we use to identify

1 09

Virulence 0a

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EPISS EP713 NB58 NB631

Isolate

FIG. 2. Sporulation and virulence of dsRNA-containing and

dsRNA-free C. parasitica isolates. Sporulation was determined byconidial counts from colonies grown on potato dextrose agar. Esti-mation of virulence was determined by growth on apple fruits asdescribed in the text and is presented as a percentage of virulentstrain EP155.

FIG. 3. Gel electrophoresis and Northern blot analysis of ds- andssRNAs from NB631 tissue. (A) Silver-stained 8% polyacrylamidegel of dsRNAs from three hypovirulent strains of C. parasitica.Lanes: 1, strain 9B-2-1 [a hypovirulent strain from West Virginia(17)]; 2, strain NB631; 3, strain NB58. Size standards are NB58dsRNA (12,507 bp; GenBank accession no. L29010) and woundtumor reovirus segments 4 (2565 bp) and 7 (1726 bp) (29). (B)Northern blots of 2 M LiCl-precipitated RNA (lane ss) and purifieddsRNA (lane ds) fractions from NB631 tissue separated on 1.2%agarose, formaldehyde-permeated denaturing gel or 1% nondenatur-ing gel, respectively. Both gels were probed with 32P-labeled clone631-9 (see Fig. 4). Size standards are from BRL ssRNA ladder (left)or wound tumor virus dsRNA segments 4 and 7 (right).

A Scale, kb

NB631 dsRNA

5-2 5-1 Y-1 3-2-~ 5'-RACE =~~ =~~-I _ RACER.C- 64'''''4

9 12

21

Randomly-primedcDNA clones 13 1

15

81B- STRAND

AMito.Cvt

2 Mito. iLL 1 II LLCyt. -EIIT'1 ---r-----V--177 7t77ZI ] 1 a11-7

IMito.! I. LL. I1,; 1 iK.I..i.LACyL Ir] i IIE I''::I.- STRAND

1 Mito.LIl ::1;cyt. 11 !1 . :

2 Mito.;| I |ili l l

Cyt 1 1J'EYTIIF-[- l I-T -

3Mito. o i L 1 LI .ICy1.II' I 1! I V1

FIG. 4. Map of NB631 cDNA clones and potential deducedtranslation frames. (A) Randomly primed clones (solid boxes) weremapped by restriction digest and sequence analysis. cDNA reactionmixtures for 5' RACE clones were primed with oligonucleotides 5'-1(upper strand) or 3'-1 (lower strand) and amplified with primers 5'-2or 3'-2, in conjunction with external, dC-tail-specific primers (BRL).(B) Possible translation frames deduced from the sequence in Fig. 5.Mitochondrial translation (Mito) assumes UGA = Trp; cytoplasmiccodon usage (Cyt) assumes UGA = Stop. Each line represents atranslation terminator, with some appearing heavier than othersbecause two or more lines are immediately adjacent to each other.

EP155 EP713

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8682 Genetics: Polashock and Hilman Proc. Natl. Acad. Sci. USA 91 (1994)

potentially interesting C. parasitica isolates is laccase pro- extractions were performed on NB631 tissue. A singleduction, as determined by color reaction on malt extract/ dsRNA substantially smaller than CHV2-NB58 dsRNA (12.5tannic acid agar medium (MTAA) (28). Lighter color of kb) and slightly larger than wound tumor phytoreoviruscolonies on MTAA correlates quite well with dsRNA pres- segment 4 (2.6 kb) was identified (Fig. 3A). Even on greatlyence (12). NB631 was identified as significantly lighter than overloaded gels, no other dsRNA species were evident (datavirulent isolates during such a screen (data not shown), not shown).prompting further study. cDNA Coigand Nucleotide Sequence Analysis. To char-

Sporulation 'was similar in NB631 and in dsRNA-free strain acterize the dsRNA associated with NB631, a cDNA libraryEP155 (Fig. 2). Virulence of NB631'using Granny Smith encompassing the entire genome was generated. Clones wereapples as an indicator was intermediate between hypoviru- demonstrated to represent dsRNA sequences by hybridiza-lent strain EP713 and virulent strain EPi55. tion to Northern blots (Fig. 3B). A single dsRNA speciesTo examine whether reduced virulence and laccase pro- hybridized, confirming that the 2.-7-kb dsRNA was not a

duction were associated with dsRNA presence, dsRNA deletion of 'a' larger species as is the case with CHV1-713

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CAA OCT BOA AAA COC ABA ATC OTT OCA ATA ACO AAT TCQOnGAATC CAB ACC OCG TTT TAT TCC TTA CAT CTA CAT OTC TTT AAG TTA TTA 1111o A B K A K I V A T NS8 WjI 0 T A F Y S L H L H V F K L L 86

AAG AAT ATA SAC CAB OAT BOA ACG TTT OAT CAA BAA CBA CCT TTT AAA CTA CT C ATT AA GA TTA AAT BAA CCT ACA CAA AAG TTT TAT 1271K N D a 0 D a T F D a E K P F K L L I KLI Wj L N E P T a K F Y 315

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TTCTCTOACBOCATATCATAAATABTTTCAAACTACOCTTTBBTATTBCCACTBABOCTTTAOCCOTCAATBBB 2728

FIG. 5. Nucleotide sequence (presented as cDNA) and deduced translation of NB631 dsRNA. Translation is shown in the frame deducedfrom the first AUG. Single boxes surround UGA codons, assumed to encode Trp. Double and triple boxes denote UAG and UAA codons,respectively. Underlined sequence represents core RNA-dependent RNA polymerase motifs A-D in Fig. 6.

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Proc. Nadl. Acad. Sci. USA 91 (1994) 8683

dsRNA (4). Northern blots of the 2 M LiCl-precipitatedssRNA fraction were used to examine whether a singletranscript or multiple transcripts were associated withNB631. Only one species of ssRNA hybridized, suggestingthat a single transcript is associated with NB631 expression.

Overlapping randomly primed cDNA clones were se-quenced, and dsRNA terminal sequences were determinedfrom 5' RACE clones (Fig. 4A). At each terminus, all threeclones sequenced were identical. Sequences and termini ofthe 5' RACE clones were confirmed by direct dideoxynucle-otide sequencing of dimethyl sulfoxide-denatured dsRNAtemplates (data not shown). Neither terminus of NB631dsRNA was polyadenylylated. In this respect, NB631dsRNA differs from dsRNA genomes of the Hypoviridae,which have poly(A) sequences at the 3' ends of the codingstrands and complementary poly(U) sequences on the minus-sense strands (6). The size of the sequence was 2728 residues(Fig. 5).

Analysis of the sequence revealed no long open readingframes on either strand when normal cytoplasmic codonusage was invoked (Fig. 4B). A reading frame on one strand,however, contained nine UGA codons, as well as a singleUAG codon shortly after the first AUG. The role ofthe UAGcodon is unclear; however, the UGA codons may be moreeasily explained. Mitochondria in other fungi use UGA toencode tryptophan rather than as a translation terminator(reviewed in ref. 30), although this has not been formallydemonstrated for C. parasitica. Allowing for mitochondrialtranslation opens this reading frame to a size that couldencode a functional RNA-dependent RNA polymerase(RDRP). Analysis of the deduced amino acid sequence re-vealed all four domains identified by Poch et al. (31) asconserved among RDRPs (Fig. 6A).Alignments of the conserved core polymerase region of

NB631 dsRNA with other viral RDRPs suggested an ances-tral relationship not with previously sequenced C. parasiticadsRNAs (6, 7) but with two small dsRNA elements identifiedin the yeast Saccharomyces cerevisiae (37, 38, 40). Theseelements, named T and W, are similar in size to NB631dsRNA (2.5-2.9 kb) and also appear to be autonomous. They

Aa betoA GAb tM82SpS.c. TS.C. WNB6 31

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S.c. T8.c. WNB631

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BDLR-ISPKGRL-PDO4RVVTYEKISSM4NNYTFELESLI--FA----SLAR$RIRTISFTMI---DORLHKWOLFSTMONGFTFELESMI--F ----AL8KSRIR-jSHYGIV--DOETIRWELFSTM3NGFTFELESMI--F ----AIVKADLR-jSDEGIL-PDORVVTYEKI8SMGNGYTFELESLI--FA----AIARS_.__ PYHLHYPDOSEVTVRQGILMOLPTTWPLLCLIHLF VELODWAPARACTjPQHLVYPD4OEITTRRGILMOLPTTWAILNLMHL- CWDSADRQYRIK- ----YK8PQOFL-TYAVOQPMGAY88FAMLALTHV IVCVAA----

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D- FTTNTKKTFSE----PFRE8CO-NFLPNEEKTFTT----YFRESCG- FKPNLRKTFVS--- GLFRESCG- FTPNRKKTFCD---GPFRESCGC QFSAGKHLESKTWGIFTEKVF- ILOPOKHFRONRRGVFLERLL- L8188GKSVI$SEFTEFAKKLK

are in the same lineage as plus-sense RNA bacteriophagessuch as Q( (38, 41). Our alignments indicate that NB631 andthe yeast elements form a separate phylogenetic cluster (Fig.6B). No evidence has been presented for a mitochondrialassociation of T or W dsRNAs, and neither has been dem-onstrated to cause disease in yeast (42).

Mitochondrial Assocation ofNB631 dsRNA. The possibilityofmitochondrial association was further pursued by nucleasedigestion analysis of purified mitochondria from strainNB631. Total mitochondrial nucleic acid preparations in-cluded a band that comigrated with column-purified NB631dsRNA (Fig. 7). The sharp banding pattern evident when themitochondrial nucleic acid was digested with EcoRI suggeststhat the preparation was virtually free of contaminatinggenomic DNA (43). Nuclease sensitivity assays indicate thatthe 2.7-kb element associated with the mitochondrial fractionis resistant to DNase and high-salt RNase digestion, con-firming its identity as dsRNA.

If NB631 dsRNA is in fact intimately associated withmitochondria, we would predict maternal inheritancethrough ascospores, which is not normally a characteristic ofC. parasitica dsRNAs (4), and a high rate of transmissionthrough conidia. Both of these characteristics have beenobserved (J.J.P., P. J. Bedker, and B.I.H., unpublishedresults).

Conchudons. We have described an unusual dsRNA ele-ment that appears to be localized for at least part of its lifecycle in mitochondria of C. parasitica and is not related topreviously described hypovirulence-associated dsRNAs. Inaddition to the relationship between NB631 dsRNA and yeastT and W dsRNAs, several other intriguing connections canbe made. A small dsRNA was identified in C. parasiticaisolate RC-1 from Michigan by Fulbright (19), but this has notbeen characterized. Of particular interest, a mitochondrialassociation was demonstrated for one ofthe dsRNA elementsfrom Ophiostoma ulmi, the Dutch elm disease pathogen (44).The 0. ulmi system is somewhat more complicated and lesswell-characterized at the molecular level than C. parasitica.Finally, intriguing parallels may be drawn from similar-sizedautonomously replicating RNAs from mitochondria of plants

B

FIG. 6. Alignment and phenogram of putative RNA-dependent RNA polymerase domains [A-D from Poch et al. (31)] of NB631 dsRNA andseveral other ss- and dsRNA elements. Alignments in A were performed with the program CLUSTAL v (32). The phenogram in B was drawn withthe DRAWGRAM program of PHYLIP (33). Sequences came from the following sources: MS2 phage (34), GA phage (35), SP phage (36), QB(GenBank accession no. X14764), S. cerevisiae (S.C.) dsRNA W (37), S. cerevisiae dsRNA T (38), and yellow fever virus EYFV; used as anoutgroup in B (39)].

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TotalM it ochon d r ial

Z Nucleic Acid

< _A

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c

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FIG. 7. Nuclease assays of mitochondria isolated from strainNB631. Total mitochondrial nucleic acid extracts were treated withRNase A in water (RNase), RNase A in 0.6 M NaCl (HS RNase),RNase A in 0.6 M NaCl + DNase I in 20 mM MgCl2 (HS R + D),DNase I in 20mM MgCl2 (DNase), or EcoRI as described in the text,electrophoresed through a 0.8% agarose gel, and stained with ethid-ium bromide. Size standards (kb) are wound tumor virus dsRNA and1-kb dsDNA ladder (BRL). Column-purified NB631 dsRNA is shownfor reference.

with S-type male-sterile cytoplasm (45). Since there arecurrently no available sequence data for the RNAs describedabove, it is unclear whether there is a direct relationshipbetween these elements and NB631.

Properties of NB631 dsRNA and its relationships withother viruses and virus-like elements bring up interestingevolutionary questions. Koonin and Dolja (41) have specu-lated that RNA bacteriophages may have originated byhorizontal transfer of a eukaryotic progenitor virus commonalso to S. cerevisiae T and W dsRNAs. Since mitochondrialdsRNA elements such as NB631 are rare, it seems likely thatthey too evolved from such a cytoplasmic progenitor bytransfer into mitochondria and subsequent specialized adap-tation. The apparent absence of a coat protein encapsidatingNB631 dsRNA (and the S. cerevisiae dsRNAs) is a propertythese virus-like dsRNAs share with the more common Po-tyvirus-related dsRNAs that cause hypovirulence in C. par-asitica (46). This supports the idea that loss of coat proteinfunction may be a common adaptation ofviruses to fungi (46).

Since NB631 dsRNA is small, it is an attractive candidatefor manipulation and reintroduction into C. parasitica. In-corporation of full-length cDNA clones into mitochondria bybiolistic particle bombardment and mutagenesis of a full-length NB631 clone for possible cytoplasmic, nonmitochon-drial replication remain to be investigated.

We wish to thank Dr. P. J. Bedker for helpful discussions through-out the course of this research. This work was initiated duringresearch supported by U.S. Department of Agriculture/NationalResearch Initiative Competitive Grants Program Grant 91-37303-6651 and was further supported by New Jersey Agricultural Exper-iment Station/McIntyre-Stennis Project 11310.

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