2020-21 OWB Funded Research | Developing a methodology to generate transgene-free edited grapevine

In its 2020-21 fiscal year, the Oregon Wine Board of Directors granted $350,000 to researchers for eight projects with the potential to advance quality grape growing and winemaking in Oregon. The update below is part of a series to let industry members know about the status of these projects.

Dr. Laurent Deluc, associate professor of grape genomics at Oregon State University and the Oregon Wine Research Institute, has prepared the update below. Dr. Satyanarayana Gouthu, research associate at Oregon State University, is co-PI on this project.

In addition to the Oregon Wine Board grant, this project receives support from the Oregon Wine Research Institute and Erath Family Foundation.

Developing a methodology to generate transgene-free edited grapevine

Project objectives:
The overall objective of this project is to establish a gene editing methodology to produce transgene-free edited grapevine material. As proof of concept, we propose to develop a stepwise approach to 1) confer resistance to Grape Powdery Mildew (GPM) by disrupting a certain class of genes (MLO genes) via conventional genetic engineering; and 2) identify the resulting transgenic grapevines that are resistant to GPM and make them transgene free.

The main research objectives are to:

1. Generate GPM-resistant transgenic grapevine plants by disrupting the function of four MLO genes (VitviMLO3, 4, 13, and 17) likely involved in the grapevine susceptibility to powdery mildew.
2. Facilitate the delivery of a modified molecular scissor, CRISPR/Cas9, to remove all foreign DNA sequences from the edited grapevines that are resistant to GPM.

Importance to the Oregon wine community:
The current application of gene editing offers the opportunity for rapid and precise modifications of the grapevine genome for genetic improvement of essential traits such as disease resistance, tolerance to abiotic stress, and plant architecture. However, the current methodology results in the generation of stable transgenic material, which is not accepted by the public and falls under strict regulations by USDA. The development of a methodology for edited grapevine material that could be transgene-free has a significant potential for the wine industry to create elite cultivars with desirable characteristics that have better public acceptance and fewer restrictions from USDA regulations.

Progress so far:
Since the start of project in July 2019, we have reached several milestones in our project.

From Objective 1, we have successfully transformed microvine plants to create a series of mutants to determine which MLO or combination of MLO genes may confer the resistance to Grape Powdery Mildew. Transformed plants are currently under the regeneration phase before being assessed for their resistance to Grape Powdery Mildew.

We have also made progress against Objective 2 with regards to the delivery of the modified CRISPR/Cas9 protein complex—a small tag (Cell-Penetrating Peptide also known as CPP) to facilitate entry into intact cells like embryogenic cells. This tag facilitates the penetration through the cell wall and the plasma of large protein complex like CRISPR/Cas9.

For this specific purpose, we have been using genetically engineered embryogenic lines, developed in the lab, that express a fluorescent marker (Green Fluorescent Protein from Jelly Fish, [GFP]). We were able to prove that the molecular scissors CRISPR/Cas9 complexed to the CPP tag (CPP-CRISPR/Cas9) could penetrate the intact cells and could disrupt the expression of GFP in the resulting microvine embryos.

Unfortunately, few embryos could survive to the CPP-CRISPR/Cas9 complex treatment. This was likely caused by an extreme sensitivity of embryogenic cells’ embryos to the CPP solution that can cause toxicity in plant cells. We have made few adjustments in the methodology to maximize the delivery of the protein complex and the regeneration of the resulting individual plantlets.

Next steps:
Our next steps are to:

• characterize the MLO mutants plant to determine which MLO or combination of MLO genes confer the resistance to Powdery Mildew; and
• optimize the facilitated delivery of the molecular scissors CRISPR/Cas9 to make the microvine MLO mutants that are resistant to Powdery Mildew transgene-free.