An endornavirus from a hypovirulent strain of the violet root rot fungus, Helicobasidium mompa

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<ul><li><p>Virus Research 118 (2006) 143149</p><p>An endornavirus from a hypoviruviolet root rot fungus, Helicoba</p><p>a a,ji Yo, Tsu-non</p><p>ber 22006</p><p>Abstract</p><p>We determ ble-identified as us, Hcontained a long open reading frame (ORF) potentially encoding a protein of 5373 amino acids (molecular mass 603,080 Da) with conservedmotifs characteristic of RNA-dependent RNA polymerase (RdRp) and helicase. The ORF is the longest so far reported in the fungal kingdom.The putative RdRp and helicase were shown to be related to putative RdRps and helicases of members of the genus Endornavirus. As is the casewith endornaviruses, the coding (sense) strand of L1 dsRNA contained a discontinuity (nick) at nt position 2552. A region between the RdRp andhelicase domains of the polyprotein also had an amino acid sequence, resembling UDP glycosyltransferases (UGTs) in Oryza sativa endornavirusand Phytophpresent at coand here, wenucleotide s 2005 Else</p><p>Keywords: H</p><p>1. Introdu</p><p>The violbasidiomycbelonging tage on agrieffective codrench of csoil microosoil microflintroducedproposed th</p><p> The nuclenucleotide seq</p><p> CorresponRegion, 1-3-1</p><p>E-mail ad</p><p>0168-1702/$doi:10.1016/jthora endornavirus 1. Regions in the L1 dsRNA-encoded protein presumed to contain putative helicase, UGT and RdRp motifs weremparable positions to those in other endornaviruses. L1 dsRNA of H. mompa strain V670 was assigned to the genus Endornavirus,designate it as H. mompa endornavirus 1-670 (HmEV1-670). This represents the first report of a fungal endornavirus whose complete</p><p>equence has been determined.vier B.V. All rights reserved.</p><p>elicobasidium mompa; Endornavirus; Helicase; RNA-dependent RNA polymerase; dsRNA; Hypovirulence; UDP glycosyltransferase</p><p>ction</p><p>et root rot fungus, Helicobasidium mompa Tanaka, aete, occurs on various plants, including 104 specieso 44 families (Ito, 1949). In spite of its serious dam-cultural crops, especially on fruit trees, there are fewntrol methods so far developed except biennial soilhemicals. Biological control of violet root rot, usingrganisms, is also inefficient, because the complexora is considered to prevent the establishment ofantagonists in the rhizosphere. Matsumoto (1998)e use of double-stranded (ds) RNA or mycoviruses</p><p>otide sequence data will appear in the DDBJ, EMBL and NCBIuence databases under the accession number AB218287.ding author at: National Agricultural Research Center for WesternSen-yu, Zentsuji, Kagawa 765-8508, Japan. Fax: +81 877 63 1683.dress: (H. Osaki).</p><p>to control root diseases of fruit trees because the viruses existwithin the cytoplasm of the host fungus and is free from micro-bial interactions in soil.</p><p>dsRNA mycoviruses or dsRNA elements exist in all majorclasses of fungi (Buck, 1986; Nuss and Koltin, 1990) andare classified into four families Partitiviridae, Chrysoviri-dae, Totiviridae, and Hypoviridae. In addition, a novel genus,Mycoreovirus has recently been added to the family Reoviridaeby the ICTV (Mertens et al., 2005). Although a large number ofthe viruses that infect plant pathogenic fungi have been reportedto be avirulent, it is becoming increasingly clear that pheno-typic consequences of harboring specific mycoviruses or cer-tain dsRNA molecules can range from symptomless to severelydebilitating, or from hypovirulence to hypervirulence (Ghabrial,1994; Nuss and Koltin, 1990).</p><p>A large dsRNA (referred to as L1 dsRNA) in H. mompastrain V670 was identified as a hypovirulence factor (Ikeda etal., 2003). We subsequently analyzed L1 dsRNA to determine</p><p> see front matter 2005 Elsevier B.V. All rights reserved..virusres.2005.12.004Hideki Osaki a,, Hitoshi NakamurNaoyuki Matsumoto b, Ko</p><p>a National Institute of Fruit Tree Science, Fujimotb National Institute for Agro-Environmental Sciences, Kan</p><p>Received 25 October 2005; received in revised form 8 DecemAvailable online 18 January</p><p>ined the complete nucleotide (nt) sequence (16,614 nt) of a large douthe hypovirulence factor from strain V670 of the violet root rot funglent strain of thesidium mompa</p><p>Atsuko Sasaki a,oshida akuba 305-8605, Japandai, Tsukuba 305-8604, Japan005; accepted 8 December 2005</p><p>stranded (ds) RNA (referred to as L1 dsRNA), previouslyelicobasidium mompa. The positive-strand of L1 dsRNA</p></li><li><p>144 H. Osaki et al. / Virus Research 118 (2006) 143149</p><p>its complete RNA sequence and elucidated that L1 dsRNAbelonged to the genus Endornavirus which was proposed byGibbs et al. (2000) and newly accepted by the ICTV (Gibbset al., 2005). Fukuhara et al. (2003) also determined thepartial sequence of a dsRNA which was closely related toendornaviruses, from another strain of H. mompa. This is thefirst report to describe the complete nucleotide sequence of anendornavirus from a fungus.</p><p>2. Materials and methods</p><p>2.1. Extraction of dsRNA</p><p>A hypovirulent strain V670 of H. mompa from Corylusheterophylla in Japan (Ikeda et al., 2003) was maintained onpotato dextrose agar. For purification of dsRNAs, cultures weregrown on a cellulose membrane overlaid on potato dextroseagar plates at 25 C. The cellulose membrane with mycelia wasthen stripped from the agar and ground to a fine powder in liq-uid nitrogen. dsRNA was extracted according to the method ofArakawa eagarose gelNa2EDTA,For cloninggel and elu</p><p>2.2. Clonin</p><p>cDNAMoriyamaprimers forwere obtaiFinally, thecDNA endclones thatThe disco3-RACE.DNA sequCity). Ded</p><p>program DNASIS Version. 2.1 (Hitachi Software Engineering,Tokyo). Sequence comparison with those available fromnucleic acid and protein databases was performed using theBLAST program (Altschul et al., 1990). Multiple sequencealignments, construction of phylogenetic tree and bootstrappinganalysis were accomplished with the CLUSTAL W programs(Thompson et al., 1994). Neighbour-joining phylogenetictree was displayed using the program TREEVIEW (Page,1996).</p><p>3. Results</p><p>3.1. Nucleotide sequence of V670 L1 dsRNA and deducedamino acid sequence</p><p>The entire sequence of 16,614 nucleotides of L1 dsRNA wasassembled from overlapping clones (Fig. 1). One long openreading frame (ORF) was found in one strand. It has the poten-tial to encode a protein of 5373 amino acids (molecular mass603,080 Da). The first methionine codon at nt 1113 was in a</p><p>ableositiNA987kedgestusin</p><p>L1 dritiesers o</p><p>ICTendoOry</p><p>95),Oth</p><p>s. BLad n</p><p>mescleo</p><p>Fig. 1. Schem e orV670. cDNA tremitextremities by tentiapositive-strant al. (2002) and separated by electrophoresis on 1.0%in TAE buffer (0.04 M Tris, 0.02 M acetic acid, 1 mMpH 7.4). Gels were stained with ethidium bromide., L1 dsRNA was purified by excising bands from theting dsRNA.</p><p>g and sequencing of dsRNA</p><p>clones were obtained following the method ofet al. (1995). Briefly, random hexamers were used asthe synthesis of cDNA. Overlapping cDNA clones</p><p>ned using RT-PCR with sequence-specific primers.5- and 3-RACE system for rapid amplification of</p><p>s (Invitrogen, Carlsbad) was used to obtain cDNAcorresponded to the terminal regions of the dsRNA.</p><p>ntinuity of coding strand was also analyzed byDNA sequence was determined using an automatedencer (PRISM 310NT, Applied Biosystems, Fosteruced amino acid sequence was predicted using the</p><p>favourG) in pL1 dsRet al., 1and lacthe lonsearchdictedsimilamembby thethora2005),al., 191998).strandacids hing fraThe nu</p><p>atic representation of the strategy used in cDNA cloning and predicted genomand RT-PCR clones are represented by filled boxes, 3-RACE clones of the exopen boxes. The positive-strand features a long ORF encoding 5373 aa long po</p><p>d is interrupted by a discontinuity at nt 2552 (arrow).context for translation initiation, with a purine (A oron -3 and a G in position +4 (GCA G in cDNA of), according to Kozaks rules (Kozak, 1986; Lutcke). The 3-untranslated region (UTR) was 485 bp longa poly(A) tail. The ORF of L1 dsRNA is apparentlyso far reported in the fungal kingdom. A BLASTg the complete amino acid sequence of the pre-sRNA-encoded protein showed significant sequenceto the predicted polyprotein sequences encoded byf the novel genus Endornavirus, recently acceptedV (Gibbs et al., 2005). These included Phytoph-rnavirus 1 (PEV1, E value: 3e77; Hacker et al.,za sativa endornavirus (OsEV, 4e72; Moriyama etand Vicia faba endornavirus (VfEV, 8e69; Pfeiffer,er short reading frames were also found in bothAST searches revealed that their deduced aminoo clear similarity to any proteins. These short read-are considered unlikely to be translated into proteins.tide sequence is deposited into the DDBJ, EMBL and</p><p>ganization of the L1 dsRNA from Helicobasidium mompa strainies are indicated by hatched shading, and 5-RACE clones of thel protein with a 10 nt long 5-UTR and a 485 nt long 3-UTR. The</p></li><li><p>H. Osaki et al. / Virus Research 118 (2006) 143149 145</p><p>Fig. 2. (A) Multiple alignment of the amino acid sequences of the RdRp-like regions of the polyproteins encoded by L1 dsRNA and other endornaviruses. The positionsof motifs AE are indicated by lines above the sequences. Symbols below the sequence alignment: * indicates identical amino acids residues; indicates chemicallysimilar residues. (B) Neighbour-joining phylogenetic tree derived from the alignment of amino acid sequences of RdRps of L1 dsRNA, other endornaviruses, andpositive-strand RNA viruses. Bootstrap values (1000 replicates) of over 500 are indicated on the branches. L1 dsRNA, Helicobasidium mompa V670 L1 dsRNA(accession number AB218287). Endornaviruses: HvEV, Hordeum vulgare endornavirus (AB185251); LsEV, Lagenaria siceraria endornavirus (AB185247); OsEV,Oryza sativa endornavirus (D32136); PEV1, Phytophthora endornavirus 1 (AJ877914); and VfEV, Vicia faba endornavirus (AJ000929). Positive-strand RNAviruses: PMWaV1, Pineapple mealybug wilt-associated virus 1 (AF414119); LCV, Little cherry virus (AF416335); CrTV, Crucifer tobamovirus (AB003936); TMV,Tobacco mosaic virus (Z29370); and TBSV, Tomato bushy stunt virus (AY579432). The tree was rooted with TBSV. Bar, number of amino acid substitutionsper site.</p></li><li><p>146 H. Osaki et al. / Virus Research 118 (2006) 143149</p><p>Fig. 3. Multipmotifs IVI arresidues. L1 dendornavirus</p><p>NCBI nuclber AB218</p><p>Endornagenomes rsingle ORFal., 2005).gle, long Obelongs to tpresent in talso foundpositioned</p><p>3.2. Helica</p><p>BLASTcharacterishelicase inthe genus Eacid seque1989; Brueing L1 dsRregions of Rle alignment of amino acid sequences of the helicase-like region of the polyproteinse indicated by lines above the sequences. Symbols below the sequence alignment: * indsRNA, Helicobasidium mompa V670 L1 dsRNA (accession number AB218287); O1 (AJ877914); PvEV, Phaseolus vulgaris endornavirus (AB185245); VfEV, Vicia fa</p><p>eotide sequence databases under the accession num-287.viruses are endogenous dsRNA elements with</p><p>anging between 14 and 18 kbp in size and contains spanning most of the length of the RNA (Gibbs etThe size of V670 L1 dsRNA, the presence of a sin-RF, and sequence similarity indicate that L1 dsRNAhe genusEndornavirus. Furthermore, a discontinuityhe coding strand of all members of Endornavirus wasto exist in L1 dsRNA at nt 2552, which was similarlyto the discontinuity of VfEV (nt 2735) (Fig. 1).</p><p>se-like, UGT-like, and RdRp-like regions</p><p>searches implied the presence of conserved motifstic of RNA-dependent RNA polymerase (RdRp) andL1 dsRNA. They resembled those of members ofndornavirus. Fig. 2A shows an alignment of amino</p><p>nces within the conserved motifs AE (Poch et al.,nn, 1993) of RdRps in six endornaviruses, includ-NA. The next most similar alignments were withdRps of the alpha-like virus superfamily of positive-</p><p>strand RNAand tobamomight havecapsid-codderived froRdRps, is swith other eamino acidand Dolja,</p><p>The largassumed toproteinaseshave yet beet al. (2005enzymic roWe also fothat contain(aa 11981</p><p>A BLARdRp andsignificanttransferaseencoded by L1 dsRNA and other endornaviruses. The positions oficates identical amino acids residues; indicates chemically similarsEV, Oryza sativa endornavirus (D32136); PEV1, Phytophthoraba endornavirus (AJ000929).</p><p>viruses (Buck, 1996), particularly closterovirusesviruses. It supports a suggestion that endornavirusesevolved from an alpha-like virus that has lost its</p><p>ing region (Gibbs et al., 2000). A phylogenetic tree,m the alignment of amino acid sequences of thehown in Fig. 2B. L1 dsRNA RdRp obviously clustersndornaviruses in RdRp. Fig. 3 shows an alignment ofsequences within the conserved motifs IVI (Koonin1993) of helicases with known endornaviruses.e proteins encoded by the endornaviruses have beenbe polyproteins that are processed by virus-encoded. No proteinase motifs or proteolytic cleavage sitesen reported for the endornaviruses. However, Hacker) identified cysteine-rich regions, which might playles, in the polyproteins of VfEV, OsEV and PEV1.und no proteinase motifs, but a cysteine-rich regioned 16 cysteine residues in a sequence of 72 residues269) in the L1 dsRNA-encoded large protein.ST search also revealed that a region between thehelicase domains of the L1 dsRNA polyprotein hadamino acid sequence similarity to UDP glycosyl-s (UGTs), as was reported in the case of OsEV and</p></li><li><p>H. Osaki et al. / Virus Research 118 (2006) 143149 147</p><p>Fig. 4. Multip roteinSymbols belo ates cbetween moti V670endornavirus ectria(AY307099).</p><p>Fig. 5. Compendornavirus.UGT-like (doof the long re</p><p>PEV1. Fig.tive UGT mdsRNA, othypoviruseendornavirtria hypoviclosely pos</p><p>The regof the L1 dtinuity, wein other ensupport thaEndornavir</p><p>4. Discuss</p><p>Based onetic analymompa V6navirus an1-670 (Hm(Fukuharabean (Waknavirus, Phle alignment of amino acid sequences of the UGT motifs A and B of the polypw the sequence alignment: * indicates identical amino acids residues; indicfs A and B for each virus are indicated. L1 dsRNA, Helicobasidium mompa(D32136); PEV1, Phytophthora endornavirus 1 (AJ877914); CHV3, Cryphonarison of genome organizations of Helicobasidiummompa endornavirus 1-670, PhytoLong rectangular boxes represent the coding region for the polyproteins. Small boxe</p><p>tted) and RdRp-like (black) regions within the polyproteins. Arrowheads point to thectangular boxes indicate the 5- and 3-UTRs.</p><p>4 shows the amino acid sequence alignment of puta-otifs A and B (Hacker et al., 2005) encoded by L1</p><p>her endornaviruses and hypoviruses, Cryphonectrias 3 and 4 (CHV3 and 4; Linder-Basso, 2002). Theuses could not be distinguished from the Cryphonec-ruses in UGT motifs except that the motifs were moreitioned in the endornaviruses.ions presumed to encode helicase, UGT, and RdRpsRNA polyprotein, as well as the point of discon-</p><p>re all arranged in positions similar to those founddornaviruses (Fig. 5). These observations stronglyt L1 dsRNA represents the genome of a member ofus.</p><p>ion</p><p>n sequence analyses, homology searches, phyloge-sis, and genomic organization, we propose that H.70 L1 dsRNA be assigned to the genus Endor-</p><p>d designate a new species, H. mompa endornavirusEV1-670). Endornaviruses exist in plants, i.e., riceet al., 1993), broad bean (Pfeiffer, 1998), and kidneyarchuk and Hamilton, 1990). Recently an endor-ytophthora endornavirus 1 (PEV1), has also been</p><p>identified i2005), belo1997), whi(Hawkswowhich wasof...</p></li></ul>