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Walking into post-genomics era
IOWA STATE
--Virus-induced gene silencing for soybean gene function--
UNIVERSITY
DEPARTMENT OF PLANT PATHOLOGY
Alan Petersen, Jon Humston RET Summer Program 2009, Iowa State University, Ames, IA, 50010
Introduction
Procedure
Over the past decade, our understanding of genes and the size of
our genomic databases have been growing substantially. As the
complete set of genomes for more and more species are being
brought to light, we are passing from the era of genomics to the era
of functional genomics, answering the question: What do all of those
genes do?
Soybean is one of the world’s most valuable plant commodities; it
accounts for 70% of the world’s edible protein, is the leading U.S.
agricultural export, and is responsible for the vast majority of
feedstock for biodiesel production. The soybean crop is perennially
subjected to attack by a variety of pathogens. The average
estimated annual soybean loss due to disease in the United States
was over US$4 billion. Therefore, there is a critical need to
understand the soybean disease resistance pathways so that
soybeans may be improved to express novel, stable, and broadspectrum resistance.
At the end of 2008, the DOE and USDA announced a complete draft
of the soybean (Glycine max) genetic code and made it widely
available for research into various fronts. Virus-induced gene
silencing (VIGS) using Bean pod mottle virus (BPMV) as vector has
been established for functional genomics in soybean (Zhang et al.
2009). With the soybean genome information and the established
VIGS approach at hand, we determine to interrogate the functions
of soybean genes in disease resistance pathways.
ligation
-The Bean pod mottle virus (BPMV) has a
bipartite positive-strand RNA genome
consisting of RNA1 and RNA2.
-BPMV RNA1 and RNA2 have been cloned
into two binary vectors (Zhang et al. 2009).
-RNA2 vector can be modified to carry a
partial sequence of gene of interest (~3001,000 bp) from soybean.
-BPMV RNA1 and modified RNA2 are
simultaneously delivered into soybean
leaves by co-bombardment to cause
infection and subsequent silence of
soybean genes carried by modified RNA2
vector.
Figure 5. DNA sequence of
cloned BPMV2 vector
carrying soybean insert.
Figure 2. Flowchart of the VIGS procedure
Figure 1. Virus-induced gene
silencing
Background
Figure 3. Insertion of soybean
cDNA fragment into digested
BPMV RNA2 vector
Green= BPMV2 vector sequence
Red= primer GmMPK14a
Blue=GmMPK14a cDNA
Figure 8. Progress with MPK and MKK primers
Results
1.
Figure 4. Example of PCR
amplification gel analysis
BPMV:0
BPMV:PDS
BPMV:0
2.
Figure 7. Example of plant
phenotype caused by
silencing MAPK4a
Why MAPK genes?
Studies in Arabidopsis and other plant
species showed that the Migoten-activated
protein kinase (MAPK) cascade is a key
component in disease-resistance pathway
(Menke et al. 2004). We aim to silence
each member of MAPK pathway genes in
soybean genome and to investigate their
functions in disease resistance.
Waterhouse, 2003
3.
Waterhouse, (2003)
1. Double-strand RNA (dsRNA) is
recognized and cleaved into small
interference RNA (siRNA) by Dicer enzyme.
2. RNAi silencing complex (RISC) is guided
by the antisense strand of siRNA to cleave
homologous mRNAs
3. mRNA degradation is promoted, thus
silencing gene expression
Figure 6. Example of bleaching phenotype of
soybean with phytoene desaturase (PDS)
gene silenced by BPMV-induced gene
silencing (Zhang, 2009)
BPMV:0= vector only
BPMV:GmMPK4a
Summary: Sixteen out of twenty-four primer pairs have successfully
amplified gene fragments. These fragments have been cloned into the
BPMV RNA2 vector (BPMV2). Thirteen BPMV2 constructs showed
positive DNA insertions, ready for sequencing.
Three BPMV2
constructs were sequenced and of those, two were co-bombarded with
BPMV RNA1 into soybean plants and await phenotype analysis.
Acknowledgements: We thank Steve Whitham, Jianzhong Liu, Chris Chunquan Zhang, Sehiza Grosic, Jaime Dittman, and Adah Leshem-Ackerman for various help.
Funding: NSF Plant Genome Research Program grant awarded to Drs. Steven Whitham,
John Hill & Thomas Baum grant # DBI-0820642.
And NSF Plant Genome Research Program grant awarded to Drs. Jonathan Wendel & Adah
Leshem-Ackerman grant # DBI-0638418
Literature Cited:
Zhang C. et al. (2009) Development and use of an efficient DNA-based viral gene silencing vector for soybean. MPMI 22,123-131
Menke F. et al. (2004) Silencing of the Mitogen-Activated Protein Kinase MPK6 compromises disease resistance in Arabidopsis. The Plant Cell 16, 897-907
Waterhouse, P.M. et al. (2003) Exploring plant genomes by RNA-induced gene silencing. Nature Reviews Genetics 4 29-38