Penulis Utama : Supyani
Penulis Tambahan : -
NIM / NIP :
Tahun : 2007
Judul : Characterization of a Novel Reovirus Isolated From a Hypovirulent Strain (9B21) of The Chestnut Blight Fungus That is Infectious as Particles
Edisi :
Imprint : Surakarta - FP - 2007
Kolasi :
Sumber : UNS- FP Prog.Studi Devision of Resources management and Environmental Science -NIP 132046020
Subyek : FP
Jenis Dokumen :
ISSN :
ISBN :
Abstrak : A filamentous fungus Cryphonectria parasitica is the causal agent of the chestnut blight disease, o ne of the most devastating tree diseases in the world. The fungus penetrates from wound sites of trees, induces cankers and eventually destroys trees. This fungus is known to be a host of members of at least 5 virus families including Reoviridae, Hypoviridae, Narnaviridae partitiviridea and Chrysoviridae that often confer hypovirulence (attenuated virulence) to the fungal host, thus being potential or practical biological control agents. These viruses are classifled into two groups:”true” dsRNA viruses and “pseudo” dsRNA viruses. Fungal viruses (mycoviruses) are not infectious in the classic sense, as infection cannot be initiated by exposing uninfected hyphae to a cell extract that has been prepared from an infected strain. The cause/effect relationship is established o nly for a few pseudo dsRNA viruses by cDNA-based transfection or transformation protocols, but not for true o nes because of unavailability of inoculation methods. The former examples are represented by the prototype hypvirus, CHVI and other hypoviral strains, while the second o nes include members of the families Reoviridae, Partitiviridea and Chrysoviridae. Here an inoculation protocol was developed for a novel “true” dsRNA mycovirus, Mycoreovirus I (MyRV-I) isolated from a hypovirulent strain 9B21 of the chestnut blight fungus isolated from a superficial canker o n a chestnut tree in West Virginia. The virus was subsequently characterized biologically and molecularly. Virus particles purified by differential and sucrose gradient centrifugation had a double-shelled structure of approximately 80 nm in diameter. Furthemore, cryo-electron microspcopy showed turrets o n MyRV-I core particles. Spheroplasts of a virus-free isogenic strain were transfected with purified MyRV-I particles. Regenerated colonies harbored virus particles with the same morphology and possessed identical phenotypic attributes to 9B21, like reduced growth of aerial mycelia and enhanced production of brown pigments, representing the fulfillment of Koch’s postulates. This is the first example of a mycovirus that is infectious as particles. Virulence tests with apples revealed that MyRV-I caused higher levels of host hypovirulence than CHVI as a reverence, the established biocontrol agent. While host pigmentation was enhanced by MyRV-1 infection, sporulation was not significan- tly affected. This is in stark contrast to CHVI infection which reduced both host pigmentation and conidiation. These properties may contribute to enhance environmental fitness of MyRV-1-infected strains as a biocontrol agent against the chestnut blight disease. Molecular analysis showed that MyRV-1 had 11 dsRNA genome segments that were termed S1 to S11 with an increasing order of mobility in acrylamide gel. The sizes of genome segments ranged from 4127 bp for the longest segment (S1) to 732 bp for the shortest segment (S11). Each segment had a single open reading frame (ORF) o n its plus-sense strand. All the 11 genome segments of the virus had common terminal sequences o n both 5’- and 3’- termini (5’GAUCA----GCAGUCA) and 5’ –cap structures o nly o n their plus-sense strands. Together with particle morphology, these molecular characteristics clearly indicate that the virus is a member of the family Reoviridae that belongs to a new genus. In 2004 the International Committee o n Taxonomy of Viruses (ICTV) approved a proposal that a new genus Mycoreovirus within the family Reoviridae contains MyRV-1 as a type member and two other member MyRV-2 (CpMyRV2/C18) and MyRV-3 (RnMyRV-3/W370), as suggested in this study. It is known from previous studies o n other reoviruses that their particles play important roles in intracellular replication that is similar irrespective of members. A motif search showed that S1 –coded VP1 and S6-coded VP6 are RNA-dependent RNA polymerase and NTP binding protein. VP1 and VP6 are assumed to be constituents of core particles based o n their predicted roles. For further molecular dissection of core particles, the 11 dsRNA genome segments of MyRV-1 were expressed via a baculovirus vector system. This system was utilizer to identify the capping enzyme and determine gene-product assignment for some viral Proteins. The observation that S2-coded VP2 expressed in insect cells comigrated with a major protein in purified virus preparations showed that VP2 is a structural protein. Autoguanylylation assay revealed that VP3, a protein encoded by S3 of the virus, is a guanylytransferase (capping enzyme). VP3 migrated in SDS-polyacrylamide gel electrophoresis to the same position as a GTP-labeled protein in purified virus particles, suggesting VP3 is a structural protein. Delection and site-directed mutational analysis revealed that its enzymatic activity domain residing at the N-terminal region that contains amino acid residues H233, H242, Y243, F244, and F246. An extensive search of available reoviral sequences with the sequence of the activity domain lead to the identification of as-yet-unraveled motif Hx8H of guanylytransferase of the turrected group and the genus Coltivirus of the family Reoviridae. This raise a possibility that the two histidines strictly conserved, rather than lysine residues suggested in earlier reports, may bind GMP via their imido-amino group. Phylogenetic analysis supported that MyRV-1 is placed in a group that contains other mycoreoviruses and closely related to mammal pathogenic members of the genus Coltivirus. It is interesting to note that both mycoreoviruses and coltiviruses are associated with the Acari. Coltiviruses are vectored by ticks, while some species ot mites and C.parasitica inhabit a common ecological niche. These reoviruses might have originated from an ancestor infecting members of the Acari. These findings will contribute to progress o n the study of the mycoreovirus as a potential biocontrol agent and to exploration into reovirus intracellular replication.
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Fakultas : Fak. Pertanian