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.
Subject(s)
Gene Expression Regulation, Bacterial/drug effects , Hemiptera/microbiology , Oligonucleotides, Antisense/administration & dosage , Plant Diseases/prevention & control , Rhizobiaceae/drug effects , Animals , Arabinonucleotides/administration & dosage , Arabinonucleotides/genetics , Cell Line , Citrus/microbiology , Drosophila , Gene Silencing , Hemiptera/drug effects , Insect Vectors/drug effects , Insect Vectors/microbiology , Oligonucleotides, Antisense/genetics , Plant Diseases/microbiology , Rhizobiaceae/genetics , Rhizobiaceae/pathogenicity , Symbiosis/drug effects , Symbiosis/geneticsABSTRACT
We have investigated, for the first time, short interfering duplexes containing arabinonucleotides (ANA; the 2'-stereoisomer of RNA), as well as combinations of ANA with RNA, and their 2'-fluorinated derivatives 2F-ANA and/or 2'F-RNA. The results show that ANA is especially well accommodated in the sense strand of small interfering RNA (siRNA) duplexes, which can be extensively modified with little effect on potency. Furthermore, combining ANA with RNA and 2'F-ANA in siRNA passenger strands, particularly in patterns that bias duplex thermal stability, produces duplexes with similar (and sometimes enhanced) potency compared with native siRNA. Effective patterns of modification were identified against firefly luciferase screens in HeLa cells and then applied to knockdown of down-regulated in renal cell carcinoma (DRR), a novel and clinically tractable target for the treatment of glioblastoma.