Potential treatments for citrus greening

In 40 years, biologist Sharon Long has become an expert in symbiotic bacteria that promotes the growth of alfalfa. She has published more than 150 articles on this topic, but when she understood that the extensive research that her lab had been doing for decades could help solve the citrus greening – a disease that devastates crops. citrus fruit – she was encouraged to take a new direction.

"I am only two generations off a farm and I read that citrus growers are losing their livelihood and their land, as well as generations of family," said Long, who is William C. Steere, Jr. – Pfizer Inc. Professor of Biology at the Faculty of Humanities. "We decided to redirect our efforts to work on this problem because we wanted to make a difference."

This risk has been rewarded by a new way of finding potential treatments for the disease and a short list of 130 compounds to explore further. The details of the system and their projections were published on August 19 in the Proceedings of the National Academy of Sciences.

"What we have completed is only a small part of what remains to be done," said Melanie Barnett, senior researcher at Long Lab and lead author of the paper. "It's not our expertise to pursue these discoveries to the level needed for real-world application, but it's a good place for researchers to take these next steps."

Decades of knowledge

The citrus greenery has devastated the citrus industry in Florida and is found in many citrus producing areas of the country. Even with high surveillance, the disease spreads and, by the time the symptoms of the deadly bacterial infection appear, it is too late: the plants, bearing marbled leaves and ugly fruits with unalterable bitter juice, must be ripped and destroyed.

An increasingly common treatment for infection is to spray antibiotics into whole orchards, a risky procedure that could allow drug-resistant bacteria to grow and spread.

Despite its ravages, citrus greening has been difficult for researchers to study. The bacterium causing the disease – Liberibacter asiaticus – does not develop in a laboratory and the study of infected plants is only possible in a few highly protected and sealed sites in the United States. Some researchers have turned to a less dangerous bacterial close relative. to find answers. But Long Lab realized that it could tackle the problem by focusing on a more distant relative, Sinorhizobium meliloti, which associates with certain plants to allow them to get together. develop without additional nitrogen fertilizer.

"We have been working on this bacterium for 40 years and have developed tools that enable us to conduct finely detailed genetic studies," said Long. "This provides an impossible experimental platform by working directly on this pathogen or even its relatives."

The researchers began by introducing genes from the citrus greening bacteria into their familiar cell of S. meliloti. These genes each code for a protein that, according to scientists, regulates aspects of infection.

Then they designed the bacteria so that when these critical proteins for infection were active, the bacteria would become green in a certain light. With this configuration, if the bacteria were exposed to a protein-inhibiting chemical – and perhaps also reduced the bacterium's ability to infect citrus – the cell would become visibly less green.

This visual signal screened more than 120,000 different compounds with the help of the Stanford High Throughput Bioscience Center. This screen identified 130 compounds that attenuated the green glow of cells without affecting its growth.

"Our system has allowed us to find very specific inhibitors that do not harm beneficial bacteria," Long said. "Such inhibitors would be a great improvement over the environmental spray of general antibiotics."

Beyond the study of the 130 compounds, the group said that other researchers could now test other chemicals with the system they designed or look at different genes.

"With this system, any gene for this pathogen or related pathogens can be tested in a very controlled and highly effective manner," said Barnett. "The years of research that have been devoted to the study and use of Sinorhizobium can now save years that others would have spent developing such a system from scratch."

Make the difference

For Long and Barnett, this effort had a personal significance. Long is from Texas, whose grandparents grew up on farms and whose aunt came from a city in the Rio Grande Valley known for its citrus industry. She financed the first months of this research with her own savings. Before moving to California, two of Barnett's great-grandparents owned a wheat farm in Colfax, Washington. Another group left Arkansas to settle in California in 1920 and worked as a migrant farm worker throughout the state, often in the citrus sector. Barnett grew up in Southern California, where citrus greening disease was first discovered in 2012.

"Being able to provide for one's needs is rewarding – it is also important for our well-being and for our national security," Barnett said. "Most citrus fruit eaten from my hand comes from California and I feel a lot of pride and gratitude for a state that feeds the country."

In addition to encouraging future work on their candidate compounds and screening system, Long and Barnett completed their presentation by calling for action. They urge other researchers to follow their example and step out of their comfort zone to contribute with all their might to the citrus greening crisis. The team donated all information and materials to the public domain to encourage as many researchers as possible to use the system.