icvg 2009 part I pp 1-131.pdf - Cornell University

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NEW DEVELOPMENTS IN UNDERSTANDING GENE FUNCTIONS AND EVOLUTION
OF THE GRAPEVINE CLOSTEROVIRUSES
Valerian V. DOLJA
Department of Botany and Plant Pathology and Center for Genome Research and Biocomputing, Oregon State University,
Cordley Hall 2082, CORVALLIS, OR 97331, USA
Telephone: +1-541-737-5472; Fax: +1-541-737-3573; E-mail address: doljav@science.oregonstate.edu
Summary
There are ~10 viruses from the family Closteroviridae
associated with the grapevine leafroll disease (GLRD). Among
these, Grapevine leafroll-associated virus-2 (GLRaV-2) is a
member of the Closterovirus genus, whereas the remaining viruses
belong to the genus Ampelovirus. Recent work has resulted in
characterization of a novel GLRD-related ampelovirus with
unusually short genome. On the other hand, bioinformatics
analysis demonstrated that the limited nucleotide sequence that
reportedly belonged to GLRaV-8 was actually derived from the
grapevine genome. Availability of the biologically active cDNA
clone of GLRaV-2 allowed functional characterization of the
genes encoding two leader proteases. In addition, it was shown
that the AlkB domains found in many diverse plant viruses
including grapevine ampeloviruses, function in repairing RNA
damage by oxidative demethylation. Future progress in
understanding GLRD-associated viruses and using them as the
tools of grapevine biotechnology will depend on broader
application of the high-throughput sequencing, generation of the
full-length cDNA clones for ampeloviruses and, most pressingly,
on the development of the efficient technique for grapevine
inoculation with the genetically modified viruses.
NOVEL LEAFROLL-ASSOCIATED
AMPELOVIRUSES FROM GREECE
Recent analysis of the Greek grape varieties
Prevezaniko and Debina revealed two GLRD-associated
virus isolates, GLRaV-Pr and –De, of which at least the
former presents a novel ampelovirus (Maliogka et al.,
2009). Given that, among other things, ancient Greece was
known for large production and avid consumption of wine,
the chances are that these are the antique viruses, and
comparative genomic and phylogenetic analyses seem to
concur with this notion (Maliogka et al., 2008; Maliogka et
al., 2009). By Closteroviridae family standards, GLRaV-Pr
has the unusually small genome stripped to the bare
essentials, that is, the conserved replication and quintuple
virion assembly/transport gene blocks. It is not even clear if
the latter actually includes a minor capsid protein or CPm;
due to marginal similarity to the major CP, the gene
occupying CPm position could encode either an extremely
divergent or even unrelated protein. An additional
experimental analysis is required to solve this intriguing
issue.
Uncannily, the GLRaV-Pr genome looks exactly as
has been proposed for a common ancestor of the
ampelovirus genus that is just one step apart from such
ancestor of the entire family Closteroviridae (Dolja et al.,
2006). From an evolutionary prospective, however, smallest
and simplest does not necessarily imply the most ancient
origin. The secondary process of genome reduction typical
of parasitic organisms is also a viable possibility
compatible with the ‘retired’ life style of many grapevine
viruses that survive for up to a century in the same vine and
are transmitted predominantly via grafting.
THE UNTIMELY DECREASE OF GLRaV-8
Ironically, addition of GLRaV-Pr to the list of
currently recognized viruses related to GLRD did not
change the grand total because it became exceedingly clear
that the rumors of the existence for one of these viruses,
GLRaV-8, have been greatly exaggerated. A
straightforward BLAST analysis using the only available,
allegedly GLRaV-8-derived, sequence yielded no hits to
any viral sequences, leave alone closteroviruses (V. Dolja
& S. Bertsch, unpublished data). Furthermore, two
completely sequenced Vitis vinifera genomes each
contained the exact replica of the sequence in question. The
latter fact demonstrated unequivocally that the reported
‘GLRaV-8 sequence’ was nothing more than a cloning
artifact. Therefore, GLRaV-8 must be taken off the list,
moreover, utilization of the number 8 for another, even if
more real, GLRaV, would hardly be advisable.
FUNCTIONS OF THE GLRaV-2 TANDEM LEADER
PROTEASES
Generation of the infectious cDNA clones of GLRaV-
2 tagged via insertion of GFP, enzymatic, and epitope
reporters provided critical tools for addressing GLRaV-2
gene functions (Liu et al., 2009). Because most of GLRaV-
2 genes have orthologs in well-characterized Beet yellows
virus (BYV), the work was focused on a tandem of papainlike
leader proteases (L1 and L2) present in GLRaV-2 and
some other closteroviruses, but not in BYV that has only
one such protease. Although gene swapping experiments
suggested synergistic mode of action for tandem proteases
(Peng et al., 2001), their functional profiles remained
largely uncharacterized. The roles of L1 and L2 in RNA
accumulation were addressed using tagged GLRaV-2
minireplicons and agro-infiltration of an experimental host
plant Nicotiana benthamiana. It was found that the deletion
of genome region encoding the entire L1-L2 tandem
resulted in a ~100-fold reduction in minireplicon RNA
accumulation. Five-fold reduction in RNA level was
observed upon deletion of L1 coding region. In contrast,
deletion of L2 coding region did not affect RNA
accumulation. It was also found that the autocatalytic
cleavage by L2 but not by L1 is essential for genome
replication. Analysis of the corresponding mutants in the
Progrès Agricole et Viticole, 2009, Hors Série – Extended abstracts 16 th Meeting of ICVG, Dijon, France, 31 Aug – 4 Sept 2009