Citrus tristeza virus-based RNAi in citrus plants induces gene silencing in Diaphorina citri, a phloem-sap sucking insect vector of citrus greening disease (Huanglongbing)

Abstract

A transient expression vector based on Citrus tristeza virus (CTV) is unusually stable. Because of its stability it is being considered for use in the field to control Huanglongbing (HLB), which is caused by Candidatus Liberibacter asiaticus (CLas) and vectored by Asian citrus psyllid, Diaphorina citri. In the absence of effective control strategies for CLas, emphasis has been on control of D. citri. Coincident cohabitation in phloem tissue by CLas, D. citri and CTV was exploited to develop a novel method to mitigate HLB through RNA interference (RNAi). Since CTV has three RNA silencing suppressors, it was not known if CTV-based vector could induce RNAi in citrus. Yet, expression of sequences targeting citrus phytoene desaturase gene by CTV-RNAi resulted in photo-bleaching phenotype. CTV-RNAi vector, engineered with truncated abnormal wing disc (Awd) gene of D. citri, induced altered Awd expression when silencing triggers ingested by feeding D. citri nymphs. Decreased Awd in nymphs resulted in malformed-wing phenotype in adults and increased adult mortality. This impaired ability of D. citri to fly would potentially limit the successful vectoring of CLas bacteria between citrus trees in the grove. CTV-RNAi vector would be relevant for fast-track screening of candidate sequences for RNAi-mediated pest control.

Grapevine Leafroll Disease and Associated Viruses: A Unique Pathosystem

“…Recently, development of the next generation of CTV vectors has proved to be a significant step forward in the practical application of virus-based vectors to fight invading viruses and diseases in perennial crops such as citrus (45,60). Studies with GLRaV-2-based VIGS vectors exemplify the potential use of GLRaVs for RNAi-mediated strategies in a variety of applications (40,77).…”

Emerging strategies for RNA interference (RNAi) applications in insects

“…Citrus tristeza virus (CTV) engineered with truncated abnormal wing disc (Awd) RNA sequence of D. citri triggered degradation of the Awd mRNA after D. citri nymphs fed on these plants. 87 Furthermore, wing malformation and mortality of adult insects also was observed and attributed to RNAi. 87 This result is a very promising result, suggesting that even in woody perennial plants like citrus, such an approach could have practical application potential.…”

“…87 Furthermore, wing malformation and mortality of adult insects also was observed and attributed to RNAi. 87 This result is a very promising result, suggesting that even in woody perennial plants like citrus, such an approach could have practical application potential.…”

“…Therefore, RNAi inducers must be available in the phloem, but evidence already shows that phloem feeding hemipterans can be targeted by this approach. 50,51,87 And, if viruses are to be used in agriculture, they must not induce a negative phenotype (disease) in the plant, they must be engineered to not spread beyond the treated plants but must still be able to move in phloem of treated plants, and the recombinant virus must retain and express the RNAi inducing sequence while infecting the plant. Although this approach offers great potential, clearly more studies are needed.…”

“…87 Depending on the insect stage, in some cases it was possible to see phenotypic effects on the target insects and in others it was only possible to see target mRNA reductions. For example, the results from feeding tests with A. pisum showed that reduced expression of the hunchback (hb) mRNA encoded by a gap gene, (zinc finger transcription factor), which is a key player for insect segmentation, led to pea aphid lethality.…”