1. Gene silencing for crop improvement and pathogen resistance: Transgenic lines for nematode resistance using gene targets previously validated in Arabidopsis and produced during the previous funding cycle were shipped to Melissa Mitchum at the University of Missouri for nematode feeding assays. The results have been mixed and could not be validated. Levels of small RNAs do not correlate with the cyst count or zygosity of the plant. That being said, there were cases in which there was resistance, the gene was present and there were high levels of the small RNA.

Work continues to advance the tasiRNA nematode target lines. These employ an alternative silencing vector that contains an endogenous microRNA (miR1514) target. Putative homozygous lines have been identified for seven events and seed produced for nematode feeding assays.

Work has progressed to engineer susceptible soybean with 2 different SHMT (Rhg4) alleles, these may confer different levels of resistance to SCN. Two stable T1 events have been generated in the susceptible background, other events are in progress.

2. Soybean Promoter Analysis: Work progressed with urease and GmScream promoter families. Urease promoter analysis confirmed strong expression of two promotes in embryos and seeds, with low level expression in vegetative tissues. Studies evaluating components of the GMScream promoters continue and have uncovered some novel G-box like elements. Modification of the core promoter and some of the elements result in altered expression. Experiments continue to try to understand how these elements work. The first promoter patent, “Highly active soybean promoter from the SUBI-3 polyubiquitin gene and uses thereof, John J. Finer and Robert A. Bouchard. US Patent# 8,395,021” was issued. A second patent has been published.

3. The CRISPR/Cas9 vector system has been developed: The system has been adapted to soybean and 95% of the events generated were transgenic and every target had some modifications. New gene targeting vectors can be produced for as little as $20 per target and requires only three days of work with minimal hands-on time. Stable transformation experiments for gene deletions (targeting MET1 and DDM) are underway and plants are currently being regenerated. Work continues to recover stable plants via Agrobacterium that then can simply be engineered with guideRNA/Hyg constructs using the gene gun, or conversely, CRISPR/Hyg lines will be made with the gun, that then can be simply engineered with guideRNA/Bar using Agrobacterium. In addition, efforts to replace the pathogen-derived promoter, terminator and nuclear localization signal (NLS) of the CRISPR vectors are underway. The well-characterized GmUbi promoter and pea rubisco terminator were chosen.

Two manuscripts (one accepted for publication) have been prepared describing tasi-RNA silencing and CRISPR in soybean.

Not achieving objectives:
The RNAi studies continue to be inconsistent. The researchers are finishing the last experiments and will soon be deciding whether to abandon this work if it does not pan out.