Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Beyond Boundaries: Foxtail Mosaic Virus Drives Heterologous Protein Expression and Precision Gene Editing in Sorghum

Authors:

Can Baysal1,2,3* (cbaysal@umn.edu), Jon P. Cody2,3, Albert P. Kausch4, Daniel F. Voytas1,2,3, Andrew Leakey1

Institutions:

1DOE Center for Advanced Bioenergy and Bioproducts Innovation; 2Department of Genetics, Cell Biology, and Development, University of Minnesota–St. Paul; 3Center for Precision Plant Genomics, University of Minnesota–St. Paul; 4Department of Cell and Molecular Biology, The University of Rhode Island–Kingston

URLs:

Goals

The overall goal of this project is to develop viral vectors for delivering gene editing reagents and creating somatic and heritable mutations through infection in sorghum. Specifically, this work will:

  • Engineer and optimize novel plant RNA viruses to express heterologous proteins and CRISPR-Cas gene editing reagents.
  • Test systemic infections by tracking fluorescent protein “AmCyan” and determine the most effective plant RNA virus.
  • Develop viral vectors with a large genome cargo size and the ability to infect meristem or germline cells. Determine the viral cargo capacities and the stability of viral cargo at mRNA and protein level.
  • Develop a nontransgenic genome editing platform based on the engineered RNA virus vectors. Optimize novel viral vectors to express Cas9 endonucleases and gRNAs simultaneously from the viral genome.
  • Test different transformation techniques (particle bombardment, agro-injection, viral sap rub inoculation) to co-inoculate several viruses to determine their synergistic interaction to increase meristematic viral infection and targeted mutagenesis frequencies.

Abstract

Transformation is an important step in genome editing. The requirement for in vitro tissue culture and regeneration limits the technology’s application to commercially relevant varieties of many crop species. To overcome this issue, plant viruses have recently been used as vectors for foreign gene expression, endogenous gene silencing, and delivering gene editing reagents to induce mutations mostly in Cas9-expressing transgenic plants, especially in dicots. However, a limited number of viruses have been developed into viral vectors for the purposes of gene editing in monocotyledonous plants. The team engineered a set of (monopartite) Foxtail Mosaic Virus (FoMV) and (tripartite) Barley Stripe Mosaic Virus (BSMV) vectors to deliver the fluorescent protein “AmCyan” to track viral infection and movement in Sorghum bicolor. Researchers further used these viruses to express and deliver single guide RNAs (sgRNAs) to Cas9-expressing transgenic sorghum lines, targeting S. bicolor Phytoene Desaturase (PDS), Magnesium-Chelatase (MgCh) and Lemon White (Lw) genes. BSMV was unable to infect sorghum and express “AmCyan” or deliver sgRNAs. In contrast, FoMV systemically infected the sorghum lines and induced somatic mutations at frequencies up to 60%, which produced phenotypes that were visibly distinguishable from the wild type, indicating the potential applications of this virus for in planta gene editing and functional genomics studies in sorghum. Initial research indicates that FoMV vectors can be further engineered to gain the ability to induce mutations in the germline as well.

Funding Information

This work was funded by the DOE Center for Advanced Bioenergy and Bioproducts Innovation, U.S. DOE, Office of Science, BER program under Award Number DE-SC0018420.