Transcript The (Indirect) Costs of Conducting Research: A study of

Global BYDV/CYDV Genome Sequencing Project
1
Hansen ,
2
Siguenza ,
2
Beckett ,
2
Miller
Jill
Chris
Randy
W. Allen
1Bay High School, Panama City Florida, 2Iowa State University, Ames Iowa
RESEARCH QUESTION
ABSTRACT
Yellow Dwarf Viruses (YDVs) are plant diseases with a significant effect in
determining crop yield and viability. With a global impact, developed and
developing countries alike are looking for resources to combat this
economically important group of viruses. Members of YDV have a
remarkable diversity that is causing taxonomic confusion yet they possess
very similar replication genes. For all their diversity, there exist few fulllength genome sequences of the multitude of isolates. As a service to the
scientific community, plant breeders, and farmers worldwide, we attempted
to sequence samples taken from a variety of locations to determine
homology and recombination. A database of these and other sequences
could provide researchers with avenues to combat new types of infections,
better understand genetic resistance, and determine possible phylogenetic
relationship. Samples analyzed from California and Iowa showed
significant variation that may lead to the a better grasp of viral replication
and recombination. Future research may lead to increase in crop yields.
The goal was to determine and disseminate the complete genomic
sequences BYDV and CYDV as a free service to researchers and plant
breeders in developed and developing countries. A large database of
full-length YDV sequences will be invaluable to help facilitate breeding
for resistance, to undertake epidemiological studies, and to guide
research on molecular mechanisms that may lead to more sustainable
cropping practices and increased yields that benefit the farmers and
ultimately the consumer.
From L to R: 1) an Oat farmer with a BYDV-PAS infected crop, 2) the classic
symptoms of YDVs, and 3) aphids likely responsible for transmission
BACKGROUND
Yellow Dwarf viruses are the most economically important viruses of
wheat, barley, oats, corn, and rice. Transferred only by aphids, Barley
Yellow Dwarf Virus (BYDV) and Cereal Yellow Dwarf Virus (CYDV)
have caused more than $500 million dollars damage in one year and
have shown a 100% infection rate in some areas. Reductions in yield
considered insignificant in developed countries can be devastating to
subsistence farmers trying to provide for their families. In these
developed countries, risk of significant loss is high enough that vast
quantities or pesticides are sprayed to control the aphid vector. This
may cause environmental damage and harm non-target organisms.
The epidemiology and ecology of YDV has been referred to as “a study
in ecological complexity”. As members of the Luteoviridae family, they
share substantial homology to each
other in the virion proteins (see Fig.1)
The few full-length YDV sequences
known reveal amazing variation in
different parts of the genome. The
differences are so great that BYDV
and CYDV have recently been placed
Fig 1: Similarities in isolates
in different genera. The few isolates
of these viruses that have been sequenced suggest that CYDV and
possibly BYDV may be split again into more viruses by the International
Committee on the Taxonomy of Viruses.
YDV outbreaks are highly variable, being dependent on the time of
planting and aphid migrations. Natural host plant resistance has met
with limited success since it tends to be isolate/serotype specific, and
some viral isolates can break resistance. A great deal of YDV
molecular mechanistic biology and resistance breeding has been done
using a small number of virus isolates as “lab rats”. Yet, it is clear that
the viruses identified as BYDV or CYDV exist as a complex of variants
in the field and the predominant populations will change according to
host, vector, and environment.
METHODS
Isolates were provided by collaborators from every arable continent. As
common sequences appear in all isolates, we used the rapid procedure
of cloning and sequencing additional isolates with defined primers.
Total RNA was extracted from leaf tissue of oats (Avena sativa) by
grinding under liquid nitrogen and homogenized with TRIzolTM. The
creation of a full length first strand via a reverse transcriptase (RT)
reaction off the viral RNA messenger is often difficult on genomes of this
size because the secondary structure can cause RT to terminate
prematurely. Creation of a first strand and amplification was achieved
using ThermoscriptTM reverse transcriptase and Pfx DNA polymerase.
PCR products were visualized and extracted on GelRed stained
agarose gels, and purified using a QIAquick Gel Extraction Kit (from
QIAGEN). For rapid and efficient cloning of the
PCR products we used the proven TOPO®
blunt cloning method. This allows rapid,
efficient (95%) in vitro recombination of any
insert with no need for restriction enzyme
digestion and ligation. Any pUC derived vector
provides convenient restriction sites, high copy
Fig 2: the TOPO plasmid number, and Fwd/Rev primer sites. This
provided for easy screening of transformants and purification of high
quantities of recombinant plasmids to be used for sequencing.
Sequencing was performed at the ISU DNA Sequencing and Synthesis
Facility, using an ABS 3730xl high throughput capillary system.
RESULTS
Sequences obtained from the DNA facility were examined and
compared with previously sequenced genomes from common isolates.
Tissue sample 104 from California matched BYDV-PAV (IL), one of the
most common isolates. Sample 106, also from California, matched the
sequence of a previously known CYDV-RPV isolate originally from New
York. Subsequent testing failed to reveal the presence of an RMex
isolate in the California samples.
Samples taken from an oat field near Albia IA (see pictures 1,3) were
found to match the sequences of BYDV-PAS. Previously only
sequenced in wheat, BYDV-PAS (also known as PAV-129) did not
cause striking symptoms. However in oats it has been shown to be
lethal and breaks resistance that is effective against other PAV isolates.
BYDV-PAS is a PAV isolate based on serology and aphid transmission,
but the severe symptoms and nucleotide sequence, especially in the
replication genes, reveal a very different virus.
DISCUSSION
A goal of this project was to sequence resistance breaking isolates
towards an understanding of their mechanisms of resistance avoidance.
Further research in samples obtained from southern Iowa may yield such
data. YDVs offer great potential to understand
transmission of circulative viruses due to their
unique and extreme vector specificity. Knowing
the aphid transmission phenotype and the
corresponding virus protein sequence of numerous
YDV isolates within each serotype would provide
correlative evidence on which amino acid sequences
should be targeted for subsequent mutational analysis.
A comprehensive database created and supported by
Iowa
State University could facilitate this effort.
Discovery of a polerovirus (CA sample 104) and
a luteovirus (CA sample 106) in the same region Fig 3: A possible
phylogenetic tree of YDVs
may help to trace the phylogeny of YDVs (see
Fig 3). Luteoviruses undergo striking recombination on an evolutionary
time scale and sequencing large numbers of full-length, unbiased
genomes may shed light on the evolution of YDVs and similar plant
viruses. Future research in other fields may also help to explain the
replication and recombination of important human viruses. For example
with HIV there is a major variant that exists in just a handful of people in
very small geographic regions in Africa. Such a variant might be missed
without full-length, unbiased sequencing of large numbers of genomes.
REFERENCES
ACKNOWLEDGEMENTS
Thanks to Aurelie Rakotondrafara, Jelena Kraft, Krzysztof Treader, Zhaohui
Wang, Betty Chung, Jackie Jackson, Adah Leshem-Ackerman, Eric Hall, Sharon
Andrews, Allen Miller, and especially Randy Beckett.
•Much of this poster was taken from the 2003 United States Department of
Agriculture Microbial Genome Sequencing Program Grant Proposal.
Other sources:
•The Aphid: A Virus Vector CD-ROM, APS Press 2004
•Invitrogen and Qiagen Corporation Websites