Novel tools for antimicrobial testing and discovery of new HLB therapies

Article written by Kranthi Mandadi, Sonia Irigoyen, Manikandan Ramasamy, Peggy G. Lemaux, Lukasz Stelinski, Madison Sankovitz, Monique Rivera

What is the technique?

A consensus study by the U.S. National Academy of Sciences on citrus greening research development concluded that there is no single effective therapy to fight the disease and suggested that a combination of therapies and management practices will likely be most successful in managing the disease. To discover new anti-CLas therapies, we developed an innovative CLas-citrus hairy root system that enables high throughput screening and discovery of antimicrobial peptides, small molecules, defense-related genes, and gene editing (CRISPR) targets for HLB management1.

What are the challenges and opportunities?

Challenges in finding new HLB therapies include the inability to grow the causal pathogen of HLB, Candidatus Liberibacter asiaticus (CLas), and the long developmental period of perennial citrus trees. Standard antimicrobial testing relies on whole plant assays and laborious and time-consuming plant transformations - not suited for screening large numbers of samples. Some researchers have used distantly related microbes and annual plants as substitutes. However, results obtained from such substitutes may not represent the situation with CLas-infected citrus due to differences in disease expression and severity. We recently showed that citrus shoots can be triggered to produce hairy roots1 (Fig. 1). Because the transport vessels in hairy roots mimic the natural environment of CLas, they support the growth of the bacterium. We showed that citrus hairy roots can be used for high throughput screening of potential new HLB therapies.  A key advantage to hairy root assays is they can be performed ~3-4 times faster than whole plant assays - critical for accelerating the discovery of therapies. Using this system, we screened candidates and discovered new antimicrobial peptides, immune regulators, and small molecules, which inhibited CLas in citrus root tissues. One or more of these candidates, currently being evaluated in plants, might be developed for HLB management. 

Additionally, we have extended the technology to multiple public- and private-sector collaborators and stakeholders to maximize the impact of these discoveries and to speed deployment of new HLB control measures.

Who is working on this project?

Kranthi Mandadi, Sonia Irigoyen, Mani Ramasamy, San Fernando (Texas A&M AgriLife/Texas A&M University); Veronica Ancona, Mamoudou Setamou (Texas A&M-Kingsville); James Borneman (University of California, Riverside); William Dawson, Choaa El-Mohtar (University of Florida); Mike Irey (Southern Gardens Citrus).

Funding: USDA NIFA, FFAR, Southern Gardens Citrus and Texas A&M AgriLife Research

References        

1   Irigoyen, S. et al. Plant hairy roots enable high throughput identification of new antimicrobials against Candidatus Liberibacter spp. Nature Commun. 11, 5802 (2020).

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