1a.Objectives (from AD-416):
1. Sequence the Rps2, Rps3 and Rps8 loci from the resistant parents and use expression data to identify expressed candidate resistance genes.
2. Use virus induced gene silencing (VIGS) to assay the function of candidate resistance genes in response to Phytopthora sojae infection.
1b.Approach (from AD-416):
Our approach will identify candidate Phytophthora sojae resistance genes for Rps2, Rps3 and Rps8. We will leverage the Williams82 and PI 96983 genome sequences and use it to identify candidate genes from the resistant parents. Our approach will be to compare sequence from the Williams82 soybean genome and sequence from PI96983, which does not have an Rps gene in these regions, to sequence from lines with Rps2, Rps3 and Rps8. Real-time PCR will be used to monitor expression of candidate genes in resistant and susceptible parents following P. sojae infection. The function of expressed candidate resistance genes will be assayed using virus induced gene silencing to "turn off" resistance.
We have sequenced the Rps2 Phytophthora sojae (Phytophthora root and stem rot) resistance locus in the resistant parent L76-1988. This region, which spans ~370 kb, contains 25 candidate resistance genes. The corresponding region from the publicly available genome sequence (from a susceptible genotype) was also analyzed. While the gene content and gene order were conserved across the two lines, the susceptible line had only 22 candidate resistance genes. In order to narrow the number of candidate genes for Rps2 we used multiple sequence alignments to group related genes. The 25 candidate genes fell into four major groups. Working with collaborators we have developed Virus-Induced Gene Silencing (VIGS) constructs that should distinguish between groups. We are currently awaiting the delivery of the constructs.
In order to identify particular genes that could be conferring resistance, we have developed primers for use with quantitative Polymerase Chain Reaction (PCR). We would like to compare gene expression in the resistant line infected and mock infected with P. sojae. When used on genomic DNA, these primers amplified from all full-length resistance genes in the Rps2 region. When used for qPCR, sequencing of the PCR product should distinguish between differentially expressed genes. We are preparing to isolate RNA from infected plants to use in this experiment.
In addition to identifying candidate resistance genes, we have also been focused on modifying the VIGS protocol for use with P. sojae. Ideally, we would like to infect resistant plants with the VIGS vectors two weeks prior to infection with P. sojae. Using the existing VIGS protocol, plants would be infected too late with P. sojae and could out grow the disease. Therefore, we are exploring a vascular puncture inoculation (VPI) protocol for infecting seeds with the VIGS vectors. Using a vector overexpressing green fluorescent protein (GFP), we were able to confirm virus infection as early as 14 days, which would be an ideal time to infect with P. sojae. However, results have been inconsistent. We are trying a number of methods to improve the VPI protocol for use with soybean and VIGS.