Biology subjects

Falk, B. W.

Publications and source records attributed to Falk, B. W..

3 recordsLinked to original sources

bioRxiv2020

Development of a cricket paralysis virus-based system for inducing RNA interference-mediated gene silencing in Diaphorina citri

Diaphorina citri, the Asian citrus psyllid, is the insect vector of the phloem-limited bacterium Candidatus Liberibacter asiaticus, which causes the most devastating citrus disease worldwide: Huanglongbing (HLB). An efficient cure for HLB is still not available and the management of the disease is restricted to the use of pesticides, antibiotics and eradication of infected plants. Plant- and insect-infecting viruses have attracted increasing attention for their potential to manipulate traits in insects, especially insect vectors of plant pathogens. However, so far there are no insect virus-based vectors available for use in D. citri. Cricket paralysis virus (CrPV) is a well-studied insect-infecting dicistrovirus with a wide host range and has been used as a model in previous translational studies. In this work, we demonstrate for the first time that CrPV is infectious and pathogenic to D. citri. We show that specific amino acid mutations in the CrPV primary cleavage DvExNPGP motif resulted in a viral mutant that was attenuated compared to wild-type CrPV during infection of either Drosophila cells line or adult D. citri insects. This attenuated CrPV mutant was then used as the backbone for engineering a recombinant CrPV-based vector to specifically alter D. citri gene expression via the RNA interference (RNAi) pathway, a technology called Virus Induced Gene Silencing (VIGS). As proof-of-concept, we engineered recombinant CrPV-based vectors carrying nucleotide sequences derived from a previously reported D. citri target gene: the inhibitor of apoptosis gene (IA). RT-qPCR analysis of insects either microinjected or fed with the recombinant CrPV mutants showed decreased IA gene expression as soon as viral replication was detected, indicating that the engineered CrPV-based VIGS system enables functional gene silencing in D. citri. This novel insect virus-based tool is easily amenable to genomic modification and represents a technical advance for understanding interactions between insect virus-based VIGS systems and D. citri.

molecular biology↗

bioRxiv2020

Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus

Partial integrations of DNA and non-retroviral RNA virus genomes, termed endogenous viral elements (EVEs), are abundant in arthropod genomes and often produce PIWI-interacting RNAs (piRNAs) speculated to target cognate viruses through the ping-pong cycle, a post-transcriptional RNA silencing mechanism. Here we describe a Diaphorina citri densovirus (DcDV)-derived EVE in the genome of Diaphorina citri. We found that this EVE gives rise to DcDV-specific primary piRNAs and is unevenly distributed among D. citri populations. Unexpectedly, we found that DcDV is targeted by ping-pong-dependent viral piRNAs (vpiRNAs) in D. citri lacking the DcDV-derived EVE, while four naturally infecting RNA viruses of D. citri are not targeted by vpiRNAs. Furthermore, a recombinant Cricket paralysis virus containing a portion of the DcDV genome corresponding to the DcDV-derived EVE was not targeted by vpiRNAs during infection in D. citri harboring the EVE. These results represent the first report of ping-pong-dependent vpiRNAs outside of mosquitoes.

microbiology↗

bioRxiv2018

Endogenous viral elements are widespread in arthropod genomes and commonly give rise to piRNAs

Arthropod genomes contain sequences derived from integrations of DNA and non-retroviral RNA viruses. These sequences, known as endogenous viral elements (EVEs), have been acquired over the course of evolution and have been proposed to serve as a record of past viral infection. Recent evidence indicates that EVEs can function as templates for the biogenesis of PIWI-interacting RNAs (piRNAs) in some mosquito species and cell lines, raising the possibility that EVEs may function as a source of immunological memory in these organisms. However, whether EVEs are capable of acting as templates for piRNA production in other arthropod species is unknown. Here we used publically available genome assemblies and small RNA sequencing datasets to characterize the repertoire and function of EVEs across 48 arthropod genomes. We found that EVEs are widespread in arthropod genomes and primarily correspond to unclassified ssRNA viruses and viruses belonging to the Rhabdoviridae and Parvoviridae families. Additionally, EVEs were enriched in piRNA clusters in a majority of species and we found that production of primary piRNAs from EVEs is common, particularly for EVEs located within piRNA clusters. While we found evidence suggesting that piRNAs mapping to a number of EVEs are produced via the ping-pong cycle, potentially pointing towards a role for EVE-derived piRNAs during viral infection, limited nucleotide identity between currently described viruses and EVEs identified here likely limits the extent to which this process plays a role during infection with known viruses.

microbiology↗