Phloem-restricted, independently mobile RNA gene silencing system for mitigating Citrus Greening (HLB) by targeting Liberibacter asiaticus and citrus gene expression

Research by: Dr. Anne Simon, Neena Mitter, Shunyuan Xiao, Silvec Biologics

Article written by:  Anne Simon
Article edited by:  Ed Stover,  Peggy G. Lemaux


What is the technique?

Bacterial, fungal and viral pathogens are causing the deaths of billions of trees and vines every year.  It is widely accepted that RNA and peptide therapies offer solutions to controlling plant pathogens and the insects that deliver them, and such therapies are also hailed as the future of human medicine.  Tiny RNAs known as siRNAs, which are naturally generated by all plants, get taken up by invading pathogens and pests where they target essential genes, thereby significantly reducing harmful pathogen effects.  Numerous studies have demonstrated that plant siRNAs, whether natural or designed, are harmless when consumed by animals. 

How is that procedure used in this research?

Although siRNAs offer solutions to many agricultural problems, the problem of getting designer, protective siRNAs into a tree and spread throughout the canopy (without genetically      engineering the tree) has been a major impediment for applying this technology to citrus. Although spraying and trunk injecting the siRNAs are possible, applications need to be repeated every few weeks making such treatments unsustainable, especially for long-lived trees. Viruses, however, are well known to be excellent vehicles (vectors) for generating copious amounts of designer siRNAs (and antibacterial peptides) throughout a tree, a technique known as virus-induced gene silencing (VIGS).  While laboratory usage of VIGS in short-lived annuals has been routine for decades, VIGS vectors have not been stable, with most rapidly losing the ability to make designer siRNAs and peptides, significantly complicating applying this technology to trees.  

We developed a natural citrus virus as a VIGS vector that is symptomless, has no discernable effect on fruit quality, and cannot be transmitted from tree-to-tree through pollen or seeds.  Most importantly, we have discovered how to make our VIGS vector stable, allowing the protective effect to last a long time, hopefully for the life of a tree with a single application.  For this project, we are modifying our VIGS vector to generate siRNAs that can target Candidatus Liberibacter asiaticus (CLas), and other funding is being used to target citrus pathogenic fungi, nematodes and other disease agents (Figure 1). 

Who is working on this project?

The Simon lab at the University of Maryland leads the project, but collaborations with other labs have contributed technologies to enhance progress. While our VIGS vector can easily be introduced into seedlings (or bearing trees) through grafting, for more rapid dissemination in mature trees we are working with Dr. Neena Mitter, University of Queensland, on using her novel nanoparticle nucleic acid delivery method to introduce the vector as a stable spray throughout a tree’s canopy.  The team also includes Dr. Shunyuan Xiao (IBBR/UMd), who is developing the anti-CLas siRNAs using the closely related Liberibacter crescens as a surrogate host, Michelle Heck (Cornell/USDA), who will test the VIGS vector in infected citrus trees, and Silvec Biologics, a company founded to commercialize VIGS vectors, and which has licensed the technology from UMd.

What are the challenges being facing?

We are also engineering the VIGS vector to make an anti-bacterial peptide, as it is not known yet whether we can reach the levels of siRNAs needed to target bacteria.  Several such peptides have already been developed that are extremely good at targeting CLas.

Funding source:  USDA-NIFA ECDR      2022-70029-38492

Figure 1. VIGS vector CY1 with anti-fungal siRNA showing excellent protection in laboratory plants against the fungus, Botrytis cinerea