Update:
Progresses: - Rag5: We reported in the last quarterly report that although we were able to express the putative Rag5 gene in soybean hairy roots, the high mortality rates of soybean aphids on hairy roots made it unreliable for assessing the functionality of Rag5. During the current quarter, we initiated the new approach of generating soybean transgenic plants that express Rag5. Specifically, we have cloned Rag5 into another vector compatible with the soybean transformation protocol in Dr. John Finer’s lab. This part of work was successful and the sequence of Rag5 was confirmed. We are currently propagating immature embryo tissue culture. Once these tissues are ready, we will transform the Rag5 gene into these cultured immature embryos using particle bombardment. The actual transformation will occur during the coming quarter. - Rps8: We reported in the last quarterly report about using three novel reporter genes to assess the effectiveness of VIGS to silence R genes in soybean roots. These three genes are: GmRAR1, GmSGT1-1, and GmSGT1-2, which have been shown to be needed for the function of many R genes. The VIGS constructs for all three of these genes have been successfully assembled and tested in soybean plants in collaboration with Dr. Dorrance lab. Unfortunately, the first round of P. sojae inoculation was not as successful as we had hoped. We are currently repeating this experiment for another round, and expect to report the results in the next report. - QTLs on Chromosome 19: We have been focusing on three genes, AS5, AS7a, and AS9, that are part of one of the QTLs. Recent results suggest that silencing of these three genes caused the QTL-containing Conrad variety to become more susceptible to P. sojae, suggesting that these genes play a role in partial resistance. We also found that at least AS9 is preferentially expressed in the roots, and is strongly induced by P. sojae inoculation. These experiments are being repeated to ensure the reproducibility of the results. In addition, we have also initiated experiments that silence AS5 and As9 in hairy roots. Briefly, transgenic hairy roots will be generated that express short double-stranded (ds) RNAs that target AS5 and AS9. Upon successful generation of the transgenic hairy roots, we will first ensure that both AS5 and AS9 are indeed silenced in these hairy roots. They will then be tested to determine whether they become more susceptible/resistant to P. sojae.