Toward Deregulation of Genetically Modified American Chestnut for Surface Mine Reclamation: Genomic Mechanisms of Transgene-Mediated Blight Resistance

We have been testing numerous candidate genetically modified (GM) lines of American chestnut (Castanea dentata) at the Powell River Project since 2014, with several showing promising levels of resistance and growth. Former surface mines represent an obvious context in which to begin restoration efforts using these GM trees, but extensive data on the environmental and ecological impacts of the transgenes is required before the US Department of Agriculture will consider their deployment outside of our current heavily monitored field trials. One of the first such pieces of data will be to show at the cellular level how a given transgene leads to resistance. As genes and their protein products function in regulatory networks that may involve thousands of other genes/proteins, assessing the risk (or lack thereof) when a transgene escapes requires an understanding of how that transgene functions in the tree, and whether it has undesirable off-target effects. One way to address this question is to leverage next generation sequencing to assess how gene regulatory networks are perturbed in the GM vs. wild-type lines. In this study, we have inoculated a number of trees in our GM field trial with Cryphonectria parasitica (chestnut blight) and sampled the resulting cankers for extraction of RNA. RNA-sequencing work is on-going, but we will achieve a detailed view of how the transgenes affect the rest of the gene regulatory network in these trees (that is, which genes are turned on/off and to what degree). The data arising from this project will supplement ongoing studies by collaborators toward the ultimate goal of de-regulation for one or more of these GM lines, and more extensive deployment of the trees for surface mine reclamation. Here we present some preliminary results from this study.