Studies on Five Viruses Infecting Mentha

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					       Studies on Five Viruses Infecting Mentha
                          Ioannis E.                      Tzanetakis 1,                         Joseph D.                    Postman 2                   and Robert R.                              Martin 1,3
                    1   Dept. of Botany and Plant Pathology and Center for Gene Research and Biotechnology, Oregon State University, Corvallis, 97331
                                                   2 National Clonal Germplasm Repository, USDA-ARS, Corvallis, OR 97333
                                                  3Horticultural Crops Research Laboratory, USDA-ARS, Corvallis, OR 97330

Mentha x gentilis L. 'Variegata' or ‘Golden Ginger Mint’ is an                                                                                                               A genomic region of more than 8 kilobases of MVBaV 3’ of the helicase
ornamental mint clone with vein banding and leaf yellowing. Symptoms                                                                                                         domain of the replicase has been sequenced. BLAST searches and
have been attributed to virus infection since they are graft                                                                                                                 phylogenetic analysis of the helicase and polymerase conserved motifs
transmissible and can be eliminated by heat therapy. The putative                                                                                                            (Candresse et al., 1990; Koonin, 1991 respectively) as well as the heat
causal agents of the symptoms are being studied. Two newly                                                                                                                   shock protein 70 homolog gene of the virus (data shown for the
described viruses as well as Strawberry latent ringspot virus have                                                                                                           polymerase motifs in Fig.3) indicate that MVBaV shares homology with
been associated with the symptoms. The first new virus is a                                                                                                                  all three genera of the family.
Potexvirus, related to Lily virus X and Strawberry mild yellow edge
virus. Sequence analysis of a second virus revealed a homology with                                                                                                          The characteristics of MVBaV indicate that it may be an ancestral
all three genera of the family Closteroviridae, making this virus a                                                                                                          member of the Closteroviridae family. Its unique characteristics suggest
possible “missing link” of the family. It may be an ancestral member,                                                                                                        it is either a precursor of the three genera identified today, or a product
or it may be a product of recombination between members of all three                                                                                                         of recombination between the three genera. The 3’ terminus of the
genera. A ‘Variegated Ginger Mint’ obtained from one nursery                                                                                                                 sequence encodes a coat protein gene. The uniqueness of the CP gene
exhibited yellowing symptoms different from the clone already studied.                                                                                                       sequence is such that it can not be identified as either the major or
This clone was also examined for the presence of viruses. A second                                                                                                           minor coat protein of the virus. The closest related genes of other
closterovirus with similarity to Beet yellows virus, and a Vitivirus                                                                                                         closteroviruses belong to both classes of proteins.
closely related to Grapevine virus B were identified in this plant, but
the three viruses found in the other ‘Golden Ginger Mint’ clones were                                                                                                        MVA is a new Vitivirus with homology with Grapevine virus B. It was
not found. Detection tools have been developed for all 5 viruses and                                                                                                         found in ‘Variegated Ginger Mint’ obtained from an Oregon nursery.
transmission studies are underway to identify vectors.                                                                                                                       Preliminary results show that the mint aphid is also able to transmit this
                                                                                           Figure.2. Symptoms on Mentha canadensis (clone MEN 617) infected with Mint vein
                                                                                           banding associated virus                                                          virus.
                                                                                                                                                                             RT-PCR detection tests have been developed for all five viruses (Figure
                                                                                           The sequence of SLRV revealed several unexpected characteristics. RNA 1           4) and are currently being used to estimate the presence of the viruses
                                                                                           is 6400 nt long and encodes for a polyprotein of over 1800aa with all the         in major mint producing areas of the United States.
                                                                                           motifs found in the members of the family Comoviridae. The virus bridges
                                                                                           the families Sequiviridae and Comoviridae, since the RNA1 polyprotein has
                                                                                           close homology to members of both families. Unlike what was previously
                                                                                           reported for RNA 2, 3900nt long, it probably encodes a movement protein
                                                                                           since the ORF is about 100aa longer (a frameshift found in the sequenced
                                                                                           strawberry isolate is absent in mint) and contains the LPL motif found in
                                                                                           other movement proteins.
                                                                                           MVX has a genome of under 6kb with all the characteristic potexvirus genes.
                                                                                           A polyprotein of 1300aa with methyltransferase and helicase motifs and
                                                                                           RdRp motifs at the 5’ and 3’ end termini respectively, followed by a triple
                                                                                           gene block involved in movement and the coat protein at the 3’ terminus of
                                                                                           the genome. Phylogenetic analysis utilizing the conserved motifs of the
                                                                                           polyprotein and the CP revealed the close relationship of MVX with lily virus
Figure 1.   A. ‘Golden Ginger Mint’ showing typical vein banding symptoms                  X and strawberry mild yellow edge virus.
            B. Another plant also marketed as ‘Variegated Ginger Mint’                     MLV is 15.5kb long and has 9 ORFs, a genome organization very similar to                  MiLV MVA MiLV- MVA-                 VB SLRV MVX MVX- SLRV- VB-
                                                                                           that of Beet yellows virus, the type member of aphid-borne genus
Introduction                                                                               Closterovirus. The virus has two ORFs at the 3’ terminus of the genome with       Figure 4. RT-PCR based detection of the five viruses.
Mentha x gentilis L. ‘Golden Ginger Mint’ has been used as an ornamental for more          no homology to proteins in the database, something not unusual for
than two centuries (Fig. 1A). The nature of the symptoms (graft transmissible,
eliminated after heat therapy) indicated that one or more viruses may be involved in       members of the family. The clustering of the virus in the genus Closterovirus
symptomatology. Three viruses were identified after cloning dsRNA extracted from           can be observed in the phylogram of figure 3. Preliminary transmission            Experiments are underway to determine the aphid acquisition and
‘Golden Ginger Mint’:                                                                      results using the mint aphid, Ovatus crataegarius, indicate that the virus is     transmission time of MLV and MVA. SLRV is known to be nematode-
   1. Strawberry latent ringspot virus (SLRV) which was already known to infect mint.      indeed aphid transmitted although no symptoms were observed (thus Mint            transmitted. The close relation of the virus with members of the
   2. A new virus belonging to the family Flexiviridae, genus Potexvirus we will call      “latent” virus). The presence of the virus has been verified in field material    Comoviridae, as well as it’s high incidence in California strawberry
      “Mint Virus X” (MVX)                                                                                                                                                   where methyl bromide is used extensively, suggest that beetle or aphid
   3. A new virus belonging to the family Closteroviridae we will call “Mint Veinbanding
                                                                                           from Oregon and Colorado.                                                         transmission may be more likely than nematode transmission.
      associated Virus” (MVBaV)                                                            The name Mint vein banding associated virus was given to the closterovirus
 A clone from an Oregon nursery also marketed as “Ginger Mint” did not show                found in NCGR clone MEN 454 after confirmation of it’s presence in another        Using a combination of virus purification, aphid transmission and
typical vein-banding symptoms (Fig. 1B). Two new viruses were identified in this                                                                                             mechanical inoculations to indicator plants, we have established single
                                                                                           mint clone (MEN 617, M. canadensis) with vein-banding symptoms (Fig. 2)           virus infections in mint for four of the viruses (MLV, SLRV, MVX and
   1. Another closterovirus we will call “Mint Latent Virus” (MLV)                                                                                                           MVA). Mechanical and/or graft transmissions are being used in an
   2. A Vitivirus we will call “Mint Virus A” (MVA)                                                                                                                          attempt to reconstruct symptoms observed in the two mint clones
                                                                                                                                                                             shown in Figure 1.
Materials and Methods                                                                                                                                                        Our next goal is to develop antibodies against the 4 new viruses. This
    Mechanical transmissions                                                                                                                                                 will permit immunological detection (i.e. ELISA) and make a broader
Tissue from several “Golden Ginger Mint” clones was ground in PBS + 2% nicotine                                                                                              survey for the presence of the viruses feasible.
and rubbed onto Mentha gentilis, Nicotiana benthamiana, N. occidentalis, N.
clevelandii, N. rustica, Chenopodium quinoa, Ch. amaranticolor, Cucumus sativus
and Tetragonia sp.                                                                                                                                                           Acknowledgments

     RNA extraction, RT-PCR detection, cloning and genome amplification                                                                                                      This project was funded by the Mint Industry Research Council. We
                                                                                                                                                                             would also like to thank Dr. R. Gergerich (University of Arkansas) for
DsRNA was extracted from mint clone MEN 454. Following cloning, ssRNA was
utilized for reverse transcription polymerase chain reaction (RT-PCR) detection,
                                                                                                                                                                             performing the beetle transmissions of SLRV.
first- and second-strand cDNA synthesis (see Tzanetakis et al., 2004a, Tzanetakis
et al. 2004b). The original clones acquired by shotgun cloning were utilized for
development of oligonucleotide primers and amplification was achieved from either
ss or dsRNA as template for RT-PCR using Platinum Taq® (Invitrogen) or LA
Polymerase ® (Takara).                                                                                                                                                       References

                                                                                                                                                                             1. Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W. and
    Sequence and phylogenetic analysis                                                                                                                                       Lipman, D.J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein
The acquired sequences were compared using BLAST (Altschul et al., 1997) to                                                                                                  database search programs. Nucleic Acids Res. 25:3389-3402.
identify the regions of the genome that were cloned. Alignments were done using                                                                                              2. Candresse, T., Morch, M.D. and Dunez, J. 1990. Multiple alignment and hierarchical
the CAP3 software. Maximum parsimony was performed utilizing the PAUP* 4.0b                                                                                                  clustering of conserved amino acid sequences in the replication associated proteins of
10 (Swofford, 2001) utilizing heuristic search with ten random addition replications                                                                                         plant RNA viruses. Res. Virol. 141, 315-329.
and the tree bisection reconnection swapping algorithm. The bootstrap analysis                                                                                               3. Huang, X. 1996. An improved sequence assembly program. Genomics 33: 21-31
consisted of 1000 replications utilizing the same parameters as above.                                                                                                       4. Koonin, E.V. 1991. The phylogeny of RNA-dependent RNA polymerases of positive-
                                                                                                                                                                             strand RNA viruses. J. Gen. Virol. 72:2197-2206.
Results and Discussion                                                                                                                                                       5. Swofford D.L. 2001. PAUP*: Phylogenetic Analysis Using Parsimony (*and Other
                                                                                                                                                                             Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts.
The complete nucleotide sequence of Strawberry latent ringspot virus (SLRV), the
                                                                                                                                                                             6. Tzanetakis, I.E., Halgren, A. B., Keller, K. E., Hokanson, S. C., Maas, J. L., McCarthy,
potexvirus designated as Mint virus X (MVX) and the closterovirus identified in the
                                                                                                                                                                             P. L. and Martin R. R. 2004a. Identification and detection of a virus associated with
mint clone from Oregon, designated as Mint latent virus (MiLV) has been                                                                                                      strawberry pallidosis disease. Plant Dis. 88: 383-390.
determined, while for the other closterovirus, Mint vein banding associated virus          Figure 3. Phylogram of the Closteroviridae, utilizing the polymerase conserved
                                                                                                                                                                             7. Tzanetakis I.E., Keller K.E. and Martin R.R. 2004b. The use of reverse transcriptase
(MVBaV) and the vitivirus, Mint virus A (MVA) attemps are underway to obtain the           motifs. The oval shapes highlight Mint latent virus (MLV) and Mint vein banding
                                                                                                                                                                             for efficient first and second strand cDNA synthesis from dsRNA templates.
complete nucleotide sequence.                                                              associated virus (MVBaV).
                                                                                                                                                                             Phytopathology 94: S104.

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