Developing tolerance to Huanglongbing disease in citrus by using gene editing

Research by: Zhonglin Mou, Ph.D., Jude Grosser, William Dawson, Vladimir Orbovic, Amit levy, Manjul Dutt, Choaa El Mohtar, Ozgur Batuman, and Michael Irey

Article written by: Dr. Zhonglin Mou
Article edited by:  Ed Stover, Lukasz Stelinski, Peggy G. Lemaux

What is the research?

The overall goal of our research is to produce citrus varieties that are either resistant or tolerant to Huanglongbing (HLB). Our strategies are based on understanding the mechanisms that regulate the plant immune system. It is well-known that the plant immune system has both positive and negative regulators and the balance between the two can be shifted towards resistance or tolerance by adding positive or removing negative regulators. In this project we have engineered overexpression of more than 20 positive immune regulators in sweet orange and/or grapefruit. We found several immune regulators that conferred robust tolerance to HLB. Although these HLB-tolerant lines are valuable for the citrus industry, they are considered genetically modified organisms (GMOs). In this project, we attempt to generate non-GMO plants by creating HLB tolerance in commercial citrus varieties by silencing certain genes, using an editing technology termed CRISPR/Cas9 (see “New Genome Editing Technology” snapshot). These are not considered GMO under USDA regulatory laws. We first employ a delivery vehicle, based on the Citrus tristeza virus (CTV), that inserts the edits into citrus. By studying how tolerance happens in these GMO plants, we identify targets for our non-GMO editing approach to achieve the same tolerance outcome.

What are some of the major successes to date?
We developed CTV delivery vehicles for different editing strategies, that can shut down 21 different genes in citrus that control negative regulators in the citrus immune system. We evaluated how shutting down these negative regulators affect citrus plants, using a study system in the laboratory.
These plants are used to provide budwood for inoculation of commercial varieties with the editing CTV. All successful editing approaches have been graft-transmitted into sweet orange. The resulting trees were screened in the greenhouse for HLB tolerance. We have identified several gene targets that, when silenced, provide strong tolerance to HLB.
Meanwhile, a series of genome editing systems have been tested and vectors with high editing efficiency have been found. A number of different target genes have been edited and these edited plants are being screened for HLB tolerance currently.

Zhonglin Mou, Jude Grosser, William Dawson, Vladimir Orbovic, Amit levy, Manjul Dutt, Choaa El Mohtar, Ozgur Batuman from the University of Florida; Michael Irey from United States Sugar Corporation Technical Operations.

What are the challenges and opportunities?

We have successfully identified citrus genes encoding negative immune regulators that, when silenced, lead to strong HLB tolerance. We used various gene editing strategies to silence these genes to produce HLB-tolerant plants that are not considered GMO. While we have improved the editing efficiency, obtaining non-GMO edited plants is still a major challenge because current methods still leave DNA in the plants that make it a GMO. We are using new editing strategies to increase the chance of producing non-GMO edited plants.

Funding Source: NIFA ECDRE 2018-70016-27392