1. Acknowledgements: We thank Steve Whitham, Jianzhong Liu, Chris Chunquan Zhang, Sehiza Grosic, Jaime Dittman, and Adah Leshem-Ackerman for various help. Walking into post-genomics era --Virus-induced gene silencing for soybean gene function-- Walking into post-genomics era --Virus-induced gene silencing for soybean gene function-- Jon Humston, Alan Petersen RET Summer Program 2009, Iowa State University, Ames, IA, 50010 Waterhouse, 2003 -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 (~300- 1,000 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. Background Figure 1. Virus-induced gene silencing Introduction 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 broad- spectrum 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. 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. Procedure Results DEPARTMENT OF PLANT PATHOLOGY I OWA S TATE UNIVERSITY 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 Figure 3. Insertion of soybean cDNA fragment into digested BPMV RNA2 vector Figure 8. Progress with MPK and MKK primers ligation Figure 2. Flowchart of the VIGS procedure BPMV:0 BPMV:GmMPK4a Figure 7. Example of plant phenotype caused by silencing MAPK4a 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 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 1. 2. 3. Figure 4. Example of PCR amplification gel analysis BPMV:PDS BPMV:0 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 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. Waterhouse, (2003) Figure 5. DNA sequence of cloned BPMV2 vector carrying soybean insert. Green= BPMV2 vector sequence Red= primer GmMPK14a Blue=GmMPK14a cDNA