IMPROVING AND CHARACTERIZING RESISTANCE TO BROWN STEM ROT
Corn Insects and Crop Genetics Research
2012 Annual Report
1a.Objectives (from AD-416):
1. Use virus induced gene silencing (VIGS) to turn off candidate resistance genes in the Brown Stem Rot resistant genotypes. Test silenced plants for susceptibility to Brown Stem Rot.
2. Use Solexa transcript sequencing to determine which resistance genes in the Rbs3 locus are expressed preferentially in the resistant parent and correlate with Brown Stem Rot resistance.
3. Use the Williams 82 genome sequence to fine map Rbs3 in a population segregating for Brown Stem Rot resistance (BSR101 x PI437654, developed by Lewers et al. 1999). Develop SSR markers to be used for marker-assisted selection of BSR resistance.
1b.Approach (from AD-416):
Our approach will identify candidate Brown Stem Rot resistance genes for Rbs3. We will leverage the Williams 82 genome sequences and use it to identify candidate genes from the resistant parents. We have compared sequence from the Williams 82 soybean genome to markers that have been used to map Rbs3. The region corresponding to Rbs3 has been bioinformatically screened for the presence of genes with similiarity to known resistance genes. The sequences of these R-genes has been used to develop Virus Induced Gene Silencing Constructs. These constructs will be tested on Brown Stem Rot resistant genotypes to determine if we can turn off resistance to Brown Step Rot. This information will identify a cluster of genes responsible for resistance. To identify the actual genes, we will use solexa transcript sequences to compare Brown Stem Rot resistant and susceptible genotypes before and after infection with Brown Stem Rot. Significantly differentially expressed genes in the Rbs3 cluster will be candidates for Rbs3. In addition, we will use fine mapping to further define the candidate genes.
Plant mechanisms for controlling infection by Phiolophora gregata (the causal agent of Brown Stem Rot (BSR) in soybeans) are poorly understood. Unlike most soybean-pathogen systems, scoring resistance or susceptibility to BSR takes six to seven weeks. It is unclear if resistance is induced late or whether the human eye is unable to score resistance earlier. Understanding when resistance is initiated will help us design better experiments for testing candidate resistance genes. To that end, we have set up time course experiments to compare resistant and susceptible genotypes infected with P. gregata compared to mock-infected controls. We are using a stem inoculation method to infect BSR resistant and susceptible genotypes. Plant tissue was harvested at early (24, 48, 72 and 288 hours after infection) and late (6, 14 and 21 days post-inoculation) time points. In addition, control resistant and susceptible plants were grown out for six to seven weeks to confirm their resistance response. RNA from these plants is currently being isolated and will be used for microarray analyses. Bioinformatic approaches will be used to determine how and when resistance occurs.