bioRxiv preprint doi: https://doi.org/10.1101/2020.11.15.383588; this version posted November 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 Development of a cricket paralysis virus-based system for inducing RNA interference-mediated 2 gene silencing in Diaphorina citri 3 4 Emilyn E. Matsumura1,a, Jared C. Nigg2, Elizabeth M. Henry1, Bryce W. Falk1* 5 6 1 Department of Plant Pathology, University of California, Davis, CA 95616, USA 7 2 Viruses and RNA Interference Unit, Institut Pasteur, UMR3569, CNRS, Paris, France 8 a Present address: Laboratory of Virology, Wageningen University and Research, 6700 AA 9 Wageningen, The Netherlands 10 * Corresponding author: Bryce W. Falk, [email protected] 11 12 Abstract 13 Diaphorina citri, the Asian citrus psyllid, is the insect vector of the phloem-limited bacterium 14 ‘Candidatus Liberibacter asiaticus’, which causes the most devastating citrus disease worldwide: 15 Huanglongbing (HLB). An efficient cure for HLB is still not available and the management of the 16 disease is restricted to the use of pesticides, antibiotics and eradication of infected plants. Plant- 17 and insect-infecting viruses have attracted increasing attention for their potential to manipulate 18 traits in insects, especially insect vectors of plant pathogens. However, so far there are no insect 19 virus-based vectors available for use in D. citri. Cricket paralysis virus (CrPV) is a well-studied 20 insect-infecting dicistrovirus with a wide host range and has been used as a model in previous 21 translational studies. In this work, we demonstrate for the first time that CrPV is infectious and 22 pathogenic to D. citri. We show that specific amino acid mutations in the CrPV primary cleavage 23 DvExNPGP motif resulted in a viral mutant that was attenuated compared to wild-type CrPV bioRxiv preprint doi: https://doi.org/10.1101/2020.11.15.383588; this version posted November 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 24 during infection of either Drosophila cells line or adult D. citri insects. This attenuated CrPV 25 mutant was then used as the backbone for engineering a recombinant CrPV-based vector to 26 specifically alter D. citri gene expression via the RNA interference (RNAi) pathway, a technology 27 called Virus Induced Gene Silencing (VIGS). As proof-of-concept, we engineered recombinant 28 CrPV-based vectors carrying nucleotide sequences derived from a previously reported D. citri 29 target gene: the inhibitor of apoptosis gene (IA). RT-qPCR analysis of insects either microinjected 30 or fed with the recombinant CrPV mutants showed decreased IA gene expression as soon as viral 31 replication was detected, indicating that the engineered CrPV-based VIGS system enables 32 functional gene silencing in D. citri. This novel insect virus-based tool is easily amenable to 33 genomic modification and represents a technical advance for understanding interactions between 34 insect virus-based VIGS systems and D. citri. 35 36 Introduction 37 Diaphorina citri Kuwayama (Hemiptera: Liviidae), also known as the Asian citrus psyllid, 38 is the insect vector of the phloem-limited bacterium ‘Candidatus Liberibacter asiaticus’ (CLas). 39 CLas is the causal agent of the most devastating citrus disease worldwide: Huanglongbing (HLB) 40 or Citrus Greening [1, 2]. In the absence of an effective cure for HLB, disease management 41 currently relies on insecticides, antibiotics and eradication of infected citrus plants [3-5]. However, 42 these current HLB management practices also create environmental and public health concerns. 43 Insecticides, for example, are not specific to the psyllid vector and might be harmful to beneficial 44 insects [6]. Likewise, previous works have demonstrated that the application of antibiotics in citrus 45 plants also alters the composition and reduces the diversity of the bacterial community present in 46 citrus leaves [2, 7]. In addition, insecticide resistance has been previously reported for D. citri bioRxiv preprint doi: https://doi.org/10.1101/2020.11.15.383588; this version posted November 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 47 populations in Brazil and in the United States [3, 8]. Thus, these concerns highlight the urgent need 48 for alternative psyllid management practices that facilitate reduced use of insecticides and 49 antibiotics and/or a rotation between chemical application and other management methods with 50 different modes of action [3]. 51 RNA interference (RNAi)-based technology has shown to be an effective approach in 52 controlling agriculture pests [9]. The RNAi pathway in insects is well documented [10-13], and 53 most of the RNAi-based strategies use double-stranded RNA (dsRNA) to trigger RNAi responses 54 in the target insects [14-21]. Several previous works have already demonstrated successful use of 55 RNAi technology to specifically alter D. citri gene expression [4, 22-24], mostly by oral delivery 56 of in vitro synthesized dsRNAs specific to D. citri genes [4, 23, 25-29]. However, the use of 57 dsRNA as an inducer of RNAi in insects has shown some limitations due to its low stability in the 58 natural environment and for eliciting an RNAi-response that is constrained to the insect gut [30]. 59 Additionally, the choice of the strategy employed for delivering dsRNA into the target insect has 60 also been a challenge [13]. 61 A promising alternative is the use of insect viruses as vectors to deliver dsRNAs to target 62 insects, a technology called Virus Induced Gene Silencing (VIGS) [31-34]. Upon virus infection, 63 RNAi-mediated antiviral defense is induced in the insects, which targets both viral RNA and any 64 transgenic RNA inserted within the virus genome [31-34]. In attempting to explore insect viruses 65 as potential VIGS vectors in D. citri, several D. citri-specific viruses were discovered through next 66 generation sequencing [35]. However, established infectious clones for any of these previously 67 described D. citri-specific virus are still unavailable. 68 Cricket paralysis virus (CrPV) is a well-studied insect virus that has a wide host range [36- 69 41] and established infectious clones are available [42]. CrPV belongs to the family bioRxiv preprint doi: https://doi.org/10.1101/2020.11.15.383588; this version posted November 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 70 Dicistroviridae and has a positive single-stranded RNA genome containing two open reading 71 frames (ORFs), both translated from internal ribosome entry sites (IRESs) [43-45]. In this work, 72 we show that CrPV also infects D. citri. We used a previously constructed CrPV infectious clone 73 (a gift from Dr. Shou-Wei Ding) to develop a recombinant CrPV-based VIGS system carrying 74 transgene sequences that specifically target D. citri through RNAi-mediated gene silencing. This 75 is a valuable insect virus-based tool that can be used for several applications, such as (1) to study 76 the interactions between an insect virus-based VIGS systems and D. citri, (2) to screen for D. citri 77 or even CLas target genes, and (3) to establish an efficient delivery method for the VIGS system 78 to other target insects. Additionally, the knowledge obtained from studies using the CrPV-based 79 VIGS system in D. citri could be translated to naturally-infecting viruses of D. citri (Nouri et al., 80 2016) in attempts to develop specific, and perhaps field-applicable virus-based tools to 81 complement HLB management. 82 83 Results 84 85 CrPV is infectious and pathogenic to D. citri 86 To assess the infectivity of CrPV in D. citri, CrPV virions were produced in Drosophila 87 melanogaster Schneider line 2 (S2) cells after transfection with in vitro-transcribed RNAs (Fig 88 S1A) derived from a previously constructed CrPV infectious clone (kindly provided by Dr. Shou- 89 Wei Ding). The accumulation of CrPV RNA in transfected S2 cells due to viral replication was 90 demonstrated by northern blot analysis (Figure S1B); and purified CrPV virions were examined 91 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Figure S1C) and 92 transmission electron microscopy (TEM, Figure S1D). The infectivity of purified CrPV virions bioRxiv preprint doi: https://doi.org/10.1101/2020.11.15.383588; this version posted November 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 93 was further confirmed by inoculating S2 cells with purified virions and checking for CrPV-induced 94 cytopathic symptoms (not shown). 95 The purified CrPV virions were used for microinjection of adult D. citri insects (~ 20 days- 3 96 old). Insects were intrathoracically microinjected with either ~ 300 nL of a 10 TCID50 units/µL 97 virus suspension in 10 mM tris buffer (pH 7.4) or virus-free buffer as control. Microinjected insects 98 were maintained in a single citrus leaf system and monitored daily for mortality or symptom 99 development (Figure 1A).