Florida researchers have taken an important step toward combating a deadly disease devastating crops around the U.S. by developing a viral vector that generates a response without changing a citrus plant's genome.

The inventors hope their tool can eventually be used by scientists and growers to prevent the infection and spread of huanglongbing, an incurable citrus greening disease also known as HLB. The researchers have applied to patent their vector and the technology to get it inside citrus plants, and the World Intellectual Property Organization published the application April 15.

Vectors have been used in plants and recently humans, including in the Russian-developed Sputnik V COVID-19 vaccine, to deliver RNA into cells. But the new system, which carries a modified version of the citrus tristeza virus, or CTV, does not involve a transfer of foreign genes.

"We wanted to create a process or a strategy to help scientists solve the problem of the HLB in a non-transgenic way," said inventor Yosvanis Acanda Artiga, a scientist at Idaho-based agribusiness company J.R. Simplot Company who worked on this pending patent as part of his research as a postdoctoral associate of plant biotechnology at the University of Florida. "This is to open the door to other scientists for newer strategies to combat HLB in citrus."

Although the technology has not yet been shown to guard against HLB, the researchers have demonstrated that the vector can express foreign proteins — meaning that once scientists can identify the proteins associated with HLB, the vector could be used to combat the disease. Researchers have been searching for these proteins for more than a decade.

Over the last decade, the Floridian citrus crop has declined by about one-third, according to Acanda Artiga, and HLB is responsible for much of the destruction. It is a bacteria that spreads between trees rapidly through their phloem, essentially their vascular system. Aphids, fly-like insects, can infect trees with HLB through their leaves, and it spreads through their phloem and eventually to the entire tree. 

The fruit borne by a tree infected by HLB is small, splotchy and bitter-tasting with a green bottom half. It is unusable, unsellable and practically inedible. From 2015-20, citrus greening caused by HLB wrought economic damages of about $1 billion and caused 5,000 jobs to be lost per year.

Where a transgenic solution to this crisis would involve changing a part of a plant's genome to enact some protective change, a non-transgenic approach can do the same thing transiently, without altering any DNA. This is important for growers who might be leery about having to put genetically modified orange juice on the market, as changing any genes or DNA would turn the growers' oranges into GMOs. 

The vector works non-transgenically because it does not introduce new genetic material to the plant. 

"It carries something to the plant cell, and makes it express something that is going to be necessary to combat disease and protect the plant," Acanda Artiga said in an interview with The Academic Times. "The vector is just RNA based on CTV that replicates inside the cell, and disappears eventually. But we can put something in that vector that, when it is replicating in the cell, can protect the plant from the attack of the other pathogen. That's what makes it a vector."

The invention allows scientists to alter certain proteins within the virus to evoke a particular response inside a plant as the viral vector replicates within the plant. The vector can either silence certain genes, or express new proteins that provide resistance to a bacterium such as HLB.

Those scientists, however, will not include Acanda Artiga, who states that he is a bioengineer and not a plant pathologist. Creating a vector that protects from HLB will require better understanding of the molecular relationship between the disease and its hosts. Without knowing this, scientists cannot change the proper proteins or peptides in the vector to make the relevant changes inside the plant.  

But the pending patent is a start. Acanda Artiga's vector is an updated version of one previously invented in a lab at the University of Florida. CTV ruined millions of citrus trees across South America during the 20th century. Spanish-speaking farmers from that region gave the disease its name for the despair it caused over the massive crop loss it inflicted; in English, "tristeza" means sadness. 

The new vector will also work better than prior CTV-based vectors, the researchers say. For example, it is less likely to cause CTV and spread it to other plants, a possible side effect of using viral vectors. 

Once scientists discover the proper target proteins to protect from HLB, they will be able to implant the vector using a gene gun, or particle bombardment, to create new varieties of HLB-resistant citrus in vitro. This process involves encoding viral RNA into small particles of metal and blasting them into the cells of a plant. In experiments, Acanda Artiga and his team used gold, which would be too expensive for regular use. In the field, growers or scientists will be able to implant the vector using laser technology being developed at the University of Florida, rather than metal particles. 

The application for the patent, "CTV-based RNA replicon transferable to multiple cells and tissues in citrus," was filed on Oct. 12, 2020 with the World Intellectual Property Organization. It was published April 15 with the application number PCT/US2020/055281. The earliest priority date was Oct. 10, 2019. The inventors of the pending patent are Amit Levy, Yosvanis Acanda Artiga and Choaa Amine A. El Mohtar. The assignee is the University of Florida Research Foundation.

Parola Analytics provided technical research for this story.