A Novel Alcohol Oxidase/RNA-binding Protein with Affinity for Mycovirus Double-stranded RNA from the Filamentous FungusHelminthosporium (Cochliobolus)victoriae

Abstract

We have cloned and sequenced a novel alcohol oxidase (Hv-p68) from the filamentous fungusHelminthosporium (Cochliobolus)victoriae that copurifies with mycoviral double-stranded RNAs. Sequence analysis revealed that Hv-p68 belongs to the large family of FAD-dependent glucose methanol choline oxidoreductases and that it shares significant sequence identity (>67%) with the alcohol oxidases of the methylotrophic yeasts. Unlike the intronless alcohol oxidases from methylotrophic yeasts, a genomic fragment of the Hv-p68 gene was found to contain four introns. Hv-p68, purified from fungal extracts, showed only limited methanol oxidizing activity, and its expression was not induced in cultures supplemented with methanol as the sole carbon source. Northern hybridization analysis indicated that overexpression of Hv-p68 is associated with virus infection, because significantly higher Hv-p68 mRNA levels (10- to 20-fold) were detected in virus-infected isolates compared with virus-free ones. We confirmed by Northwestern blot analysis that Hv-p68 exhibits RNA binding activity and demonstrated that the RNA-binding domain is localized within the N-terminal region that contains a typical ADP-binding β-α-β fold motif. The Hv-p68 gene, or closely similar genes, was present in all species of the genusCochliobolus but absent in the filamentous fungus,Penicillium chrysogenum, as well as in two nonmethylotrophic yeasts examined. This study represents the first reported case that a member of the FAD-dependent glucose methanol choline oxidoreductase family, Hv-p68, may function as an RNA-binding protein. We have cloned and sequenced a novel alcohol oxidase (Hv-p68) from the filamentous fungusHelminthosporium (Cochliobolus)victoriae that copurifies with mycoviral double-stranded RNAs. Sequence analysis revealed that Hv-p68 belongs to the large family of FAD-dependent glucose methanol choline oxidoreductases and that it shares significant sequence identity (>67%) with the alcohol oxidases of the methylotrophic yeasts. Unlike the intronless alcohol oxidases from methylotrophic yeasts, a genomic fragment of the Hv-p68 gene was found to contain four introns. Hv-p68, purified from fungal extracts, showed only limited methanol oxidizing activity, and its expression was not induced in cultures supplemented with methanol as the sole carbon source. Northern hybridization analysis indicated that overexpression of Hv-p68 is associated with virus infection, because significantly higher Hv-p68 mRNA levels (10- to 20-fold) were detected in virus-infected isolates compared with virus-free ones. We confirmed by Northwestern blot analysis that Hv-p68 exhibits RNA binding activity and demonstrated that the RNA-binding domain is localized within the N-terminal region that contains a typical ADP-binding β-α-β fold motif. The Hv-p68 gene, or closely similar genes, was present in all species of the genusCochliobolus but absent in the filamentous fungus,Penicillium chrysogenum, as well as in two nonmethylotrophic yeasts examined. This study represents the first reported case that a member of the FAD-dependent glucose methanol choline oxidoreductase family, Hv-p68, may function as an RNA-binding protein. double-stranded RNA polyacrylamide gel electrophoresis flavin adenine dinucleotide glucose methanol choline touchdown-polymerase chain reaction degenerate oligonucleotide primer-polymerase chain reaction alcohol oxidase methanol oxidase base pair(s) kilobase pair(s) Helminthosporium (Cochliobolus)victoriae open reading frame glyceraldehyde-3-phosphate dehydrogenase peroxisomal targeting signal(s) nicotinamide adenine dinucleotide nickel-nitrilo-triacetic acid resin capsid protein RNA-dependent RNA polymerase nucleotide(s) It has recently become increasingly clear that cellular factors play important roles in the transcription and replication of RNA viruses (1Lai M.M.C. Virology. 1998; 244: 1-12Crossref PubMed Scopus (238) Google Scholar). The double-stranded RNA (dsRNA)1 genomes of the two mycoviruses (2Sanderlin R.S. Ghabrial S.A. Virology. 1978; 87: 142-151Crossref PubMed Scopus (43) Google Scholar, 3Huang S. Ghabrial S.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12541-12546Crossref PubMed Scopus (97) Google Scholar) known to infect the plant pathogenic fungusHelminthosporium victoriae (teleomorph: Cochliobolus victoriae; synonym: Bipolaris victoriae) consists of two genes that encode a capsid protein (CP) and an RNA-dependent RNA polymerase (RDRP). The CP and RDRP genes are either present on the same dsRNA segment, as in the case of the totivirus H. victoriae 190S virus, or on two separate dsRNA segments, in the case of the putative chrysovirus H. victoriae 145S virus (Hv145SV). It is thus not surprising that these viruses subvert host proteins for their own use. We have previously reported that host enzymes (a protein kinase and a protease) are involved in post-translational modification of the Hv190SV CP (4Ghabrial S.A. Havens W.M. Virology. 1992; 188: 657-665Crossref PubMed Scopus (25) Google Scholar, 5Huang S. Soldevila A.I. Webb B.A. Ghabrial S.A. Virology. 1997; 234: 130-137Crossref PubMed Scopus (24) Google Scholar, 6Soldevila A.I. Huang S. Ghabrial S.A. Virology. 1998; 251: 327-333Crossref PubMed Scopus (11) Google Scholar). H. victoriae 190S virus (Hv190SV) has been extensively studied (2Sanderlin R.S. Ghabrial S.A. Virology. 1978; 87: 142-151Crossref PubMed Scopus (43) Google Scholar, 3Huang S. Ghabrial S.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12541-12546Crossref PubMed Scopus (97) Google Scholar, 4Ghabrial S.A. Havens W.M. Virology. 1992; 188: 657-665Crossref PubMed Scopus (25) Google Scholar, 5Huang S. Soldevila A.I. Webb B.A. Ghabrial S.A. Virology. 1997; 234: 130-137Crossref PubMed Scopus (24) Google Scholar, 6Soldevila A.I. Huang S. Ghabrial S.A. Virology. 1998; 251: 327-333Crossref PubMed Scopus (11) Google Scholar, 7Ghabrial S.A. Bibb J.A. Price K.H. Havens W.M. Lesnaw J.A. J. Gen. Virol. 1987; 68: 1791-1800Crossref PubMed Scopus (23) Google Scholar, 8Ghabrial S.A. Havens W.M. J. Gen. Virol. 1989; 70: 1025-1035Crossref Scopus (18) Google Scholar, 9Soldevila A.I. Ghabrial S.A. J. Virol. 2000; 74: 997-1003Crossref PubMed Scopus (34) Google Scholar), and is classified as a definitive member of the genusTotivirus in the family Totiviridae (10Ghabrial S.A. Bruenn J.A. Buck K.W. Wickner R.B. Patterson J.L. Stuart K.D. Wang A.L. Wang C.C. Murphy F.A. Fauquet C.M. Bishop D.H.L. Ghabrial S.A. Martelli G.P. Mayo M.A. Summers M.D. Virus Taxonomy: Sixth Report of the International Committee on Taxonomy of Viruses. Springer-Verlag, New York1995: 245-252Google Scholar,11Ghabrial S.A. Patterson J.L. Granoff A. Webster R.G. Encyclopedia of Virology. Academic Press, London, New York1999: 1808-1817Crossref Google Scholar). The Hv145SV, on the other hand, has only been subjected to limited biochemical characterization (2Sanderlin R.S. Ghabrial S.A. Virology. 1978; 87: 142-151Crossref PubMed Scopus (43) Google Scholar). Because of similarity in size and number of dsRNA segments between the Hv145SV and viruses in the genusChrysovirus in the family Partitiviridae, the Hv145SV was tentatively classified as a member of the genusChrysovirus (12Ghabrial S.A. Bozarth R.F. Buck K.W. Yamashita S. Martelli G.P. Milne R.G. Murphy F.A. Fauquet C.M. Bishop D.H.L. Ghabrial S.A. Martelli G.P. Mayo M.A. Summers M.D. Virus Taxonmy: Sixth Report of the International Committee on Taxonomy of Viruses. Springer-Verlag, New York1995: 253-260Google Scholar). We have recently (13Soldevila A. Havens W.M. Ghabrial S.A. Virology. 2000; 272: 183-190Crossref PubMed Scopus (27) Google Scholar) isolated a cellular protein, Hv-p68, that copurifies with viral dsRNA from H. victoriae isolates infected with both Hv190SV and Hv145SV. Additionally, Hv-p68 was demonstrated to be present as a minor component in the viral capsids (13Soldevila A. Havens W.M. Ghabrial S.A. Virology. 2000; 272: 183-190Crossref PubMed Scopus (27) Google Scholar). Our initial biochemical characterization studies of Hv-p68 indicated that it occurs in vivo as an octamer and that it is consistently present in higher levels in virus-infected H. victoriae isolates than in virus-free ones. We have also demonstrated by a gel retardation assay that Hv-p68 has RNA binding activity (13Soldevila A. Havens W.M. Ghabrial S.A. Virology. 2000; 272: 183-190Crossref PubMed Scopus (27) Google Scholar). In the present study, we report the isolation and complete nucleotide sequence of a cDNA clone of Hv-p68. We present evidence that Hv-p68 belongs to the FAD-dependent GMC family of oxidoreductases and that it has high sequence similarity to the alcohol oxidases of methylotrophic yeasts. Furthermore, we show that overexpression of Hv-p68 is associated with virus infection and that the RNA-binding domain of Hv-p68 is localized in the N-terminal region that contains a canonical ADP-binding β-α-β fold motif. In addition, we demonstrate by Southern hybridization analysis that theHv-p68 gene, or closely similar genes, is present in all Cochliobolus species examined but not in the filamentous fungus Penicillium chrysogenum nor in two nonmethylotrophic yeast species. Three H.(Cochliobolus) victoriae isolates that differ in their virus content were used. a known to contain both Hv190SV and Hv145SV (1Lai M.M.C. Virology. 1998; 244: 1-12Crossref PubMed Scopus (238) Google Scholar), was as a for and Hv-p68. victoriae has recently been demonstrated to be of virus and was as a of a fungal (13Soldevila A. Havens W.M. Ghabrial S.A. Virology. 2000; 272: 183-190Crossref PubMed Scopus (27) Google Scholar). The virus-free H. victoriae in virus studies S.A. Bishop D.H.L. RNA Viruses. New Scholar), was as an of a virus-free of and by were in Southern for theHv-p68 from the filamentous fungus chrysogenum by and were also in the Southern RNA was isolated from cultures of H. victoriae by the 1987; PubMed Scopus Google Scholar). RNA was purified from RNA by and cDNA were from RNA the cDNA and degenerate to the N-terminal sequence of the Hv-p68 protein and to acid of an were with the double-stranded cDNA to fungal mRNA to the of an Hv-p68 cDNA were polymerase and for for for for for for for for for The was and were subjected to and the was to a fragment to the of the Hv-p68 mRNA was from double-stranded cDNA by an and a on Hv-p68 cDNA nucleotide were polymerase and the for were as The was by an and subjected to RNA isolated from a of of H. victoriae was to a cDNA in the the for the The was and to a high Hv-p68 were isolated by with Hv-p68 by of the from were for in and of high were by two of with the cDNA were for in vivo the as by the and subjected to was by the and an was on both with and Sequence of and were the and J. Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). and sequence were with the and J. PubMed Scopus Google and were the S. PubMed Scopus Google Scholar). Sequence and analysis were the PubMed Scopus Google Scholar). by were 1996; Google Scholar). for protein and on the acid sequence were 1992; PubMed Scopus Google Scholar). cDNA to the region of Hv-p68 was by and of the isolated Hv-p68 cDNA as a to the and of the Hv-p68 and for and were and polymerase The was by gel electrophoresis by with with and an The was to to the of the Hv-p68, cultures of were in to a of was by with and for a of were by for in and with for by with in for were and for and the the was in in binding The protein was by on a for The Hv-p68 was purified by as by the The purified protein was by with of or or The protein was a were in the expression large either the or sequence of the Hv-p68 for expression of a acid N-terminal was by an fragment from to in the Hv-p68 a acid was by the fragment from to of cultures of with the Hv-p68 and the and were in to a of was by with and for a of were by for and in The were and for to separate the and the component The proteins were purified from the by of the by by of of the in of proteins from the Hv-p68 The were in of and an of and the proteins were by for Northwestern RNA was isolated by a modification of the of and 1987; PubMed Scopus Google Scholar). H. victoriae isolates and were in cultures in of supplemented with yeast for the victoriae was by and with supplemented with glucose Scopus Google Scholar). The was in a and the was to supplemented with and for on a The was and with and to supplemented with either glucose or methanol and on a for from and cultures were by two of and in and of a of The was for and for and the was by of of The was with alcohol and the RNA was by of of The RNA were in and subjected to two of high of and of by in Northern of the RNA was on and to a by and the blot was subjected to were with Hv-p68 cDNA for in hybridization and high for for The Hv-p68 of a fragment from a Hv-p68 was isolated from filamentous fungal species by a modification of the by Scopus Google Scholar). from cultures of the filamentous were in a complete glucose for H. isolates and were by two of and in The were in of of and for with by The was to with and the was for The was with The was by alcohol and the was and in of The was with for the to with and the was with alcohol by with alcohol The was in the of and the was and in The was by electrophoresis and the isolation of genomic from cultures of S. were in with to a of were by for and the was in of and of alcohol Three of were and the was for of was and the were by for The was with alcohol and The were in of and with of for and the was by of of and of The was in and by electrophoresis and of the genomic was on a gel for and the was to a by and the blot was subjected to were with an Hv-p68 cDNA for in hybridization and high for for The Hv-p68 of a fragment from a a cDNA of Hv-p68. oxidase activity in Hv-p68 from H. victoriae and the Hv-p68 was by a the of of methanol to 188: PubMed Scopus Google Scholar). of of methanol to the reaction was by the in a acid as and purified alcohol oxidase was in for content was a protein was as of protein. were by in transcription from a a cDNA of the dsRNA S. Ghabrial S.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12541-12546Crossref PubMed Scopus (97) Google Scholar), and a cDNA of the in the The were by of and previously with with RNA polymerase for in the of The transcription were by with for and the reaction were with and in The activity of the was by of the were on and to a a The were in to the proteins were by with the in the same for The were for the same and were in and for in were for the Hv-p68 (13Soldevila A. Havens W.M. Ghabrial S.A. Virology. 2000; 272: 183-190Crossref PubMed Scopus (27) Google Scholar) and as and were as for cDNA for the Hv-p68 gene were isolated by a of and The acid previously by analysis (13Soldevila A. Havens W.M. Ghabrial S.A. Virology. 2000; 272: 183-190Crossref PubMed Scopus (27) Google Scholar) were to two of degenerate for of to the N-terminal sequence of the protein and to an were to the of an Hv-p68 with as a for a in This was a and subjected to The for the region of the fragment was to a for of the region of the Hv-p68 to the region of Hv-p68 was as and the and an The in Southern with the not The was cloned the and The acid sequence of the the acid sequence from the N-terminal sequence of the purified Hv-p68 protein as well as that from the sequence acid in the by the and to a we a cDNA victoriae in of from the with the four were purified by two of and with the and the were The from all four were with that of the cDNA the Three of the four cDNA with of the The clone in was found to contain the region the and an from the The is present in a to J. 1989; PubMed Scopus Google Scholar) and the region the with the sequence for filamentous Scopus Google Scholar) or or the to the is by the of the of Hv-p68 The complete sequence and acid sequence for the Hv-p68 cDNA is in The region of Hv-p68 for a acid protein with a of This is in with of on analysis (13Soldevila A. Havens W.M. Ghabrial S.A. Virology. 2000; 272: 183-190Crossref PubMed Scopus (27) Google Scholar). of the acid sequence of Hv-p68 revealed the of for with sequence for cellular with the a the of the acid sequence in for to the peroxisomal The Hv-p68 all the RNA-binding the S. PubMed Scopus Google Scholar, PubMed Scopus Google we have not isolated genomic that contain the gene, we genomic sequence from a genomic clone by the degenerate and with victoriae genomic as a The was the and of the sequence of the genomic fragment with the sequence for the Hv-p68 cDNA indicated the region is by four in size from to the first the sequence of the region to the of Hv-p68 between acid and four are in with the filamentous sequence PubMed Scopus Google Scholar) for or or or or and as well as putative of protein the revealed that the acid sequence of the Hv-p68 cDNA has significant similarity with of the large family of FAD-dependent GMC oxidoreductases J. 1992; PubMed Scopus Google Scholar). The sequence identity were with the alcohol oxidases of the methylotrophic yeasts higher than of the acid for Hv-p68 and for reported for methanol oxidases are in Hv-p68 shares the S. PubMed Scopus Google of the in the GMC oxidoreductase of the ADP-binding region with its typical β-α-β fold also J. PubMed Scopus Google Scholar). The between the first and as well as the acid the of the are in these as well as in other oxidoreductases J. R.S. A. S. S. J. PubMed Google Scholar, 1992; PubMed Scopus Google Scholar). the of the other are not their putative have been in a study J. 1998; PubMed Scopus Google Scholar). the flavin J. 1998; PubMed Scopus Google Scholar). region the that is by GMC oxidoreductases 1992; PubMed Scopus Google J. PubMed Scopus Google Scholar) is to to the in of alcohol oxidases or are to in the of these enzymes J. PubMed Scopus Google Scholar). The FAD-dependent GMC oxidoreductases have been reported to be J. 1992; PubMed Scopus Google Scholar). The of the of Hv-p68 and of the family of FAD-dependent GMC four alcohol oxidases from methylotrophic yeasts are in A. Hv-p68 showed significantly high similarity and identity to the alcohol the were for the from with similarity and identity analysis showed that Hv-p68 a with the alcohol oxidases from methylotrophic yeasts that is by analysis The region of the Hv-p68 to a was in the expression The protein was purified on a and by and The protein with Hv-p68 thus for the Hv-p68 cDNA The to Hv-p68 also with alcohol oxidase (a from but the of the blot was than that with the The RNA binding activity of Hv-p68 has previously been demonstrated in Hv-p68 purified from fungal (13Soldevila A. Havens W.M. Ghabrial S.A. Virology. 2000; 272: 183-190Crossref PubMed Scopus (27) Google Scholar). the RNA-binding domain of Hv-p68, we compared the RNA binding activity of and of Hv-p68 Northwestern analysis Hv-p68 purified from the fungus and the Hv-p68 were subjected to and a and the were with from cloned to Hv190SV or Hv145SV in binding to binding of the RNA to Hv-p68 were were either the or The on the to the protein on of the and the Hv-p68, with to Hv-p68 The binding of the to the and 145S was only in an compared with that for either the or Hv-p68. base with the acid sequence of Hv-p68 indicated the of for the RNA-binding previously reported for RNA-binding proteins S. PubMed Scopus Google Scholar, PubMed Scopus Google studies with and oxidoreductases have revealed that these enzymes may also function as RNA-binding proteins Sci. PubMed Scopus Google Scholar). The that these enzymes have in is a region a typical β-α-β dinucleotide binding fold It has been that the and the RNA-binding are Sci. PubMed Scopus Google Scholar). It was of to the ADP-binding domain in Hv-p68 also function as an RNA-binding the RNA-binding we and of the Hv-p68 protein in and compared their RNA-binding by Northwestern blot The proteins were purified from the as protein with of and for the and In Northwestern binding of either of the RNA was similar for both the and the Hv-p68. the other hand, of the N-terminal region of Hv-p68 all binding to the RNA that the RNA-binding domain is localized within the region of Hv-p68. The methanol oxidizing activity of Hv-p68 purified from fungal and the purified Hv-p68 was by of of alcohol oxidase was as a The of the assay are in was with activity of of protein, Hv-p68 activity was detected with the Hv-p68. We Hv-p68 purified from fungal extracts, purified as well as that been for the activity was that have been subjected to and showed the not assay for alcohol oxidase oxidase Hv-p68 Hv-p68 activity was by the of from of is as is to of methanol to and of protein. in a activity was by the of from of is as is to of methanol to and of protein. Northern hybridization analysis was with RNA isolated from cultures of the virus-infected and the virus-free isolates and (13Soldevila A. Havens W.M. Ghabrial S.A. Virology. 2000; 272: 183-190Crossref PubMed Scopus (27) Google Scholar). In Northern with a an mRNA in size was detected in all fungal The size of mRNA is with the size for the Hv-p68 cDNA the of the Hv-p68 mRNA in the virus-infected was to higher than that in the virus-free This that overexpression of Hv-p68 is associated with virus The levels of Hv-p68 transcription are with the higher of Hv-p68 that are associated with cultures of the virus-infected (13Soldevila A. Havens W.M. Ghabrial S.A. Virology. 2000; 272: 183-190Crossref PubMed Scopus (27) Google Scholar). of the alcohol oxidases of the methylotrophic yeasts is the by a of by glucose and by methanol 1992; PubMed Scopus Google Scholar). We examined the that the Hv-p68 gene may be similar by the virus-free in a for in a either glucose or The cultures on a supplemented with methanol as the sole carbon showed or in compared with the cultures supplemented with showed significant the Northern blot analysis with of RNA isolated from these cultures revealed that the of Hv-p68 was not significantly between the glucose and the cultures of methanol to the not to the transcription of the Hv-p68 We examined the of the Hv-p68 gene or closely similar genes by Southern hybridization analysis genomic isolated from fungal species and an Hv-p68 The with enzymes known to the within the Hv-p68 region or with fungal species. species the Hv-p68 gene or a closely similar gene The for victoriae were from for victoriae isolates and that victoriae is not genes closely similar to Hv-p68 were detected in the filamentous fungus chrysogenum nor in the nonmethylotrophic yeasts S. and S. We have previously isolated an RNA-binding cellular protein, Hv-p68, that copurifies with viral dsRNA from the filamentous and demonstrated that it to higher levels in virus-infected isolates than in virus-free isolates (13Soldevila A. Havens W.M. Ghabrial S.A. Virology. 2000; 272: 183-190Crossref PubMed Scopus (27) Google Scholar). In the present study, we isolated and sequenced a cDNA the of the Hv-p68 and indicated that Hv-p68 belongs to the family of FAD-dependent GMC oxidoreductases J. 1992; PubMed Scopus Google Scholar) and that it is closely to the alcohol oxidases of methylotrophic yeasts. Unlike the genes of the closely are the Hv-p68 gene contains four introns. This has that Hv-p68, by a gene from a filamentous has significantly high sequence identity with of from methylotrophic yeasts higher than Hv-p68 gene may have to the of yeasts and filamentous Furthermore, it is that methylotrophic yeasts may viruses that are to Cochliobolus species. number of and oxidoreductases have recently been reported to RNA binding J. PubMed Scopus Google Scholar, Sci. PubMed Scopus Google Scholar, A.L. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). The of these enzymes a typical β-α-β fold is to the RNA-binding The that RNA binding activity of Hv-p68 is localized the N-terminal region the putative ADP-binding domain the first reported of an FAD-dependent oxidoreductase that may function as an RNA-binding protein. The RNA binding activity of Hv-p68 has been demonstrated by gel (13Soldevila A. Havens W.M. Ghabrial S.A. Virology. 2000; 272: 183-190Crossref PubMed Scopus (27) Google Scholar) and Northwestern blot analysis proteins that to viral RNA may as of RDRP or may to of a viral RNA to transcription or replication (1Lai M.M.C. Virology. 1998; 244: 1-12Crossref PubMed Scopus (238) Google J. 1996; PubMed Scopus Google Scholar). viral not to viral RNA or all (1Lai M.M.C. Virology. 1998; 244: 1-12Crossref PubMed Scopus (238) Google Scholar). that the of the Hv145SV by a S. and S. A. in and of the Hv190SV A.I. Ghabrial S.A. J. Virol. 2000; 74: 997-1003Crossref PubMed Scopus (34) Google Scholar) are of the Hv-p68 may to the binding of RDRP to its RNA The of has also been to limited RNA binding activity for viral was significantly than that of Hv-p68. In to RNA binding activity, Hv-p68 also exhibits A. Ghabrial and in In the glyceraldehyde-3-phosphate dehydrogenase an for a cellular protein with a protein, is known to a number of RNA of gene and a M.A. PubMed Scopus Google Scholar). Furthermore, that is involved in and viral M.A. PubMed Scopus Google Scholar). The activity of the purified Hv-p68 that either methanol is not the or that the of the protein in purified is present in an It is well that binding is a in alcohol oxidase and that the octamer the of PubMed Scopus (18) Google Scholar). Our have that Hv-p68 occurs as an octamer in the and thus it is to be in its The of from Hv-p68 be It is that methanol is not the for Hv-p68 as by the that Hv-p68 expression was induced by methanol nor by glucose these two were as the sole carbon in cultures of virus-free H. It is the high of sequence identity in the region of the and show significant 1992; PubMed Scopus Google Scholar). It is thus that may their of Hv-p68 expression levels in virus-infected and virus-free isolates demonstrated that overexpression of Hv-p68 is associated with virus It may not be surprising that the Hv-p68 was in the alcohol oxidase on the Hv-p68 is to nor to and thus oxidase proteins are to be involved in binding and PubMed Scopus (18) Google Scholar, J. 1987; PubMed Scopus Google Scholar). This is by the that the from H. to and to in the J. 1987; PubMed Scopus Google Scholar). The of in the these The that Hv-p68 is in virus-infected fungal isolates that the S.A. Buck K.W. Virology. Press, Scholar) is of We have by Northern hybridization analysis that Hv-p68 mRNA levels were to higher in virus-infected isolates than in virus-free the identity of the for the oxidase activity of Hv-p68 is not the similar alcohol oxidases from methylotrophic yeasts or filamentous in a to are J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). of Hv-p68 is in virus-infected isolates may to the in virus-infected victoriae isolates S.A. Buck K.W. Virology. Press, Scholar). we have only examined a limited number of fungal it is that all species in the Cochliobolus contain genes closely similar to Hv-p68. In a study of the Cochliobolus it was that isolates victoriae are not S. Scopus Google Scholar). This is in with on Southern analysis of from isolates of victoriae also showed that be from other isolates of It be of to the of the Hv-p68 gene or in the fungal species to the family or the important plant of the Hv-p68 gene or to of and a for victoriae) be as a novel for of important plant