Improving Suppression of Hemipteran Vectors and Bacterial Pathogens of Citrus and Solanaceous Plants: Advances in Antisense Oligonucleotides (FANA)

Abstract

We report on the development, evaluation, and efficient delivery of antisense oligonucleotide FANA (2′-deoxy-2′-fluoro-arabinonucleotide) RNA-targeting technology into citrus trees and potato plants for management of bacterial pathogens and arthropod pests. The FANA ASO technology is a single nucleotide strand of 20–24 nt in length that incorporates 2′F- chemically modifications of nucleotides, along with a phosphorothioate backbone and modified flanking nucleotides, in their structure called “gapmers,” produced by AUM LifeTech., Inc. These unique modified structures of FANA “triggers” enables gymnotic activity that self-delivers into cells, moving systemically in treated plants and insects, with significant suppression of their RNA targets. Reported is the FANA suppression of two plant-infecting bacterium Candidatus Liberibacter asiaticus, CLas (in citrus trees), and C. Liberibacter solanacearum, CLso (in potato and tomato). The CLas pathogen is associated with huanglongbing (a.k.a. Citrus Greening Disease), which causes severe loss of citrus trees, threatening global citrus production. The CLas bacterium is transmitted during feeding by the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae). CLso causes Zebra-Chip disease in potato and is transmitted by the potato psyllid, Bactericera cockerelli (Hemiptera: Triozidae). Infected citrus trees or potato plants were treated with aqueous FANA solutions applied as a soil drench, root-infusion, topical spray, tree trunk injection or by absorption into cuttings, detached leaves, and leaf disks. Plants showed significant reduction of each pathogen or symptom development in response to FANA treatments. Similarly, ingestion of FANA solutions designed specifically to CLas by insects via artificial diets produced significant titer reductions in infected citrus psyllid adults that resulted in reduction of CLas transmission. The unique properties of FANA ASO solves many of the problems of stability, cell entry, and binding affinity that plagues exogenous RNAi strategies. Breakthroughs in production methods are reducing costs enabling these ASO to expand beyond medical applications into agricultural treatments. Thus, FANA ASO may provide viable treatments in the response to crop pandemics, like huanglongbing in citrus that threatens global food production.

Optimizing Efficient RNAi-Mediated Control of Hemipteran Pests (Psyllids, Leafhoppers, Whitefly): Modified Pyrimidines in dsRNA Triggers

“…For dug development, these benefits in activity led to the use of 2 -F modifications extensively on the primary guide strand [83,189,216,217]. While there are many reports on modification strategies to stabilize aptamers and dsRNA for therapeutics in drug development [83,85,104] there are very few evaluations on modifications specific for increasing activity in insects and arthropods [34,84,[86][87][88]91,218]. Since RNAi biopesticides are built on the biological foundation of a natural RNA processing system [73], understanding the molecular mechanism of RNA processing in cells, along with DNA and protein processing, provides new information to develop more innovative treatments that will have greater activity in modulating gene expression [100,219].…”

“…The future of RNAi biopesticides will incorporate one or more types of modifications to produce a stable product with consistent activity. The incorporation of a wide variety of modified pyrimidines and/or purines [59,62,84,100], along with modifications in shape designs [218,223] have the potential to produce many different types of effective biopesticides. While 2 -F (fluorine) is just one type of modification that has been widely used, in nature there are a myriad of chemical moieties that encompass ribonucleoside modifications for each nucleoside: adenosine, guanosine, cytidine or uridine [224].…”

“…Combine this with the current success of RNA-technologies that produced the COVID vaccines (https://www.nature.com/articles/d41586-020-03626-1, accessed 22 August 2021), and the massive investments made by chemical industries to increase production of chemically modified nucleosides at lower production costs [228], produces a promising future for biopesticides. Biopesticides are an emerging market that can now produce synthesized modified RNA 'triggers' at a competitive price approaching that of current commercial chemical insecticides [49,55,57,59,73,84,[228][229][230]. Simpler, cost effective approaches have also been reported for producing siRNAs with 2 F-and other natural modifications [52,192,231].…”

“…Other types of RNA targeting aptamers, which have also used modified nucleotides include antisense oligonucleotides such as FANA's [83][84][85][86], morpholino's [34,[87][88][89][90][91][92][93] and other aptamers (i.e., microRNA, single-stranded RNA, etc.) used for medical and agricultural applications [49,85,[94][95][96][97][98][99][100][101][102][103][104].…”

CRISPR/Cas- and Topical RNAi-Based Technologies for Crop Management and Improvement: Reviewing the Risk Assessment and Challenges Towards a More Sustainable Agriculture

“…The advantages of topically-applied dsRNA and its potential as a biopesticide commercial product are still hindered by technical issues, including molecule uptake and stability, delivery methods, inconsistent activity of the dsRNA trigger, and activity level of RNAi suppression ( Hunter et al, 2021 ). Hence, one of the first aspects that should be addressed when thinking about topical RNAi-based technology is the uptake efficiency of dsRNAs and/or siRNAs/miRNAs either by the pathogen or plants, depending on the adopted strategy.…”