Antibacterial FANA oligonucleotides as a novel approach for managing the Huanglongbing pathosystem

Abstract

Candidatus Liberibacter asiaticus (CLas), a bacterium transmitted by the Asian citrus psyllid, Diaphorina citri, is the causal agent of citrus greening disease, or Huanglongbng (HLB). Currently, vector population suppression with insecticides and tree removal are the most effective strategies for managing the HLB pathosystem. In this study, we assessed the bactericidal capabilities of 2′-deoxy-2′-fluoro-d-arabinonucleic acid antisense oligonucleotides (FANA ASO) both in vitro and in vivo by (1) confirming their capacity to penetrate insect cells, (2) silencing bacterial essential genes, and (3) quantifying reductions in bacterial titer and D. citri transmission. We confirmed that FANA ASO are able to penetrate insect cells without the use of a delivery agent. Expression of an essential gene in the D. citri endosymbiont, Wolbachia (wDi), significantly decreased by 30% following incubation with a wDi-specific FANA ASO. Viability of isolated wDi cells also decreased in response to the FANA ASO treatment. Delivery of a CLas-specific FANA ASO to infected adult D. citri in feeding assays resulted in significant silencing of a CLas essential gene. CLas relative density and transmission were significantly lower among D. citri fed FANA ASO in diet compared to untreated insects. Root infusions of a CLas-specific FANA ASO into infected Citrus trees significantly reduced CLas titer during a 30-day trial. Our results suggest that FANA ASO targeting insect-transmitted plant bacteria or insect endosymbionts may be useful tool for integrated management of agricultural pathogens.

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

“…One key trait is the gymnotic, "self-delivery" property of FANA oligos comes from the modifications of the phosphorothioate backbone, and modifications that produce a high affinity for proteins on the cell surface membrane promoting increase adsorptive endocytosis (Lebedeva et al, 2000;Souleimanian et al, 2012;Alves Ferreira-Bravo et al, 2015;Rose et al, 2019;Scoles et al, 2019;Ochoa and Milam, 2020). FANA are readily absorbed by the roots of citrus trees, potato, tomato, and okra, basil, and ornamental plants (sunflower, periwinkle, Orange jasmine, Murraya paniculata) and grapevines (see text footnote 1) (Hunter and Aishwarya, 2020;Pelz-Stelinski et al, 2020;Sandoval-Mojica et al, 2021). The movement of the FANA ASO through the plant xylem and phloem enables targeting and suppression of plant-infecting bacterial pathogens, like phloem-limited CLas in citrus trees; and CLso in potato and tomato plants (Hunter et al, 2017a(Hunter et al, ,b, 2018b(Hunter et al, , 2019bPelz-Stelinski et al, 2017;Sandoval-Mojica et al, 2021).…”

“…FANA are readily absorbed by the roots of citrus trees, potato, tomato, and okra, basil, and ornamental plants (sunflower, periwinkle, Orange jasmine, Murraya paniculata) and grapevines (see text footnote 1) (Hunter and Aishwarya, 2020;Pelz-Stelinski et al, 2020;Sandoval-Mojica et al, 2021). The movement of the FANA ASO through the plant xylem and phloem enables targeting and suppression of plant-infecting bacterial pathogens, like phloem-limited CLas in citrus trees; and CLso in potato and tomato plants (Hunter et al, 2017a(Hunter et al, ,b, 2018b(Hunter et al, , 2019bPelz-Stelinski et al, 2017;Sandoval-Mojica et al, 2021). The reduction of the CLso bacterial titers with FANA soil applied treatments in infected potted potato and tomato plants decreased the foliar symptom severity and spud browning in potato, resulting in improved plant growth.…”

“…CLas DNA Ligase-A, Insect Feeding Assay CLas-infected adult psyllids fed on FANA DNA Ligase-A treated citrus leaves produced significant reduction of CLas titer. Four FANA_DNA Ligase-A (CLIBASIA-1,-2,-3,-4) designed to different regions were evaluated were evaluated in a citrus leaf feeding assay, as described in Sandoval-Mojica et al (2021). Ingestion of FANA ASOs targeting the DNA Ligase-A, reduced CLas titers in infected-psyllid given a 6-day feeding access period on FANA treated citrus leaves (Figure 3).…”

“…Therefore, antisense oligo modifications permit targeting of bacteria, like CLas, that cannot be suppressed with dsRNA strategies (Scott et al, 2013). The unique properties of the FANA designs make them more stable, enhance binding efficiency and increase specificity to the targeted organism (Gruenke et al, 2020;Ochoa and Milam, 2020), including the psyllid vectors (Hunter et al, 2017a,b;Pelz-Stelinski et al, 2017;Sandoval-Mojica et al, 2021). In the current study, FANA ASO were evaluated for plant delivery and systemic movement, insect delivery and activity to suppress psyllid and several bacteria: CLas, CLso, and psyllid endosymbionts.…”

“…The study demonstrated successful delivery of FANA with subsequent movement into target tissues in plants and psyllid, with reductions in CLas, and CLso titers within their perspective hosts (citrus, potato, tomato) and psyllid vector. Ultimately, the decreased bacteria titer, CLas in citrus and in the vector, ACP, translated into a biological significance of reduced CLas transmission by the vector, ACP 1 (Pelz-Stelinski et al, 2017;Brown et al, 2020;Sandoval-Mojica et al, 2021).…”