Enhanced Resistance to Phytophthora in Non-Transgenic Cacao Progeny Through CRISPR-Cas9 Mediated Mutagenesis of the TcNPR3 Gene

Abstract:
Black pod disease, caused by a complex of Phytophthora species poses a significant threat to the cacao industry. To explore the possibility of developing disease resistant cacao lines through genome engineering, we used CRISPR-Cas9 to mutate the TcNPR3 gene, a repressor of plant defense. Transgenic plant lines (T0) with npr3 mutations showed increased resistance in foliar infection assays in vitro. These plants were grown to maturity and then out-crossed with non-transgenic cacao plants in order to eliminate T-DNA carrying the transgenes. Whole genome and transcriptome sequencing of the T0 parents and 22 progeny seedlings revealed that the T0 plants contained one T-DNA insertion site each and we identified seven progeny that lacked a T-DNA insertion but retained npr3 mutations. Transcriptome analysis revealed that the mutations led to up-regulation of genes related to generation of reactive oxygen species (ROS) and transcription factors involved in the regulation of defense and other components such as pathogenesis related proteins. A set of genes were also downregulated in the T0 mutant plants, suggesting that in addition to its role as a repressor, NPR3 also plays an activating role in the basal transcriptional state of the defense response. The genome-edited plants appeared phenotypically indistinguishable from the control plants and exhibited a 42% reduction of lesion size in an in vitro Phytophthora infection assay. Future work will focus on field evaluations to confirm the long-term benefits and applicability of these modifications.

About the Speaker:
Mark Guiltinan is a Professor of Plant Molecular Biology and the J. Franklin Styer Professor of Horticultural Botany in the Department of Plant Science at Penn State University. He is also the Director of the Penn State Program in the Molecular Biology of Cacao and Director of The Penn State Plant Institute. He holds a B.S in Botany from California State University-Humboldt, and a Ph.D. from the University of California-Irvine. He pursued postdoctoral training at Texas A&M University, the University of North Carolina and inside industry, after which he joined the faculty at Penn State University in 1991 as a member of the Horticulture Department. His research has explored the molecular basis of plant development and applications of plant science to crop improvement with funding from the US Dept. of Energy, National Science Foundation, USAID, Gates Foundation, USDA and industry sponsors. He teaches a graduate course for the Plant Sciences Dept. entitled Ag Innovation. Visit http://guiltinanlab.cas.psu.edu for more details.

Contact

  Charles Anderson
  cta3@psu.edu