Abstract
Citrus Greening or Huanglongbing (HLB) is a disease of citrus, causing high reduction in citrus production and is transmitted by the Asian citrus psyllid Diaphorina citri Kuwayama vectoring a phloem-limited bacterium Candidatus Liberibacter sp. We report research results using crowdsourcing challenge strategy identifying potential gene targets in D. citri to control the insect using RNA interference (RNAi). From 63 submitted sequences, 43 were selected and tested by feeding them to D. citri using artificial diet assays. After feeding on artificial diet, the three most effective dsRNAs causing 30% mortality above control silenced genes expressing iron-sulfur cluster subunit of the mitochondrial electron transport chain complex (Rieske), heme iron-binding terminal oxidase enzyme (Cytochrome P450) and tetrahydrobiopterin (BH4) pathway enzyme (Pterin 4α-Carbinolamine Dehydratase). These sequences were cloned into a citrus phloem-limited virus (Citrus tristeza virus, CTV T36) expressing dsRNA against these target genes in citrus. The use of a viral mediated “para-transgenic” citrus plant system caused higher mortality to adult D. citri than what was observed using artificial diet, reaching 100% when detached citrus leaves with the engineered CTV expressing dsRNA were fed to adult D. citri. Using this approach, a virus-induced gene silencing (VIGS) can be used to test future transgenic cultivars before genetically engineering citrus. RNA Seq analysis after feeding D. citri CTV-RIE on infected leaves identified transcriptionally modified genes located upstream and downstream of the targeted RIE gene. These genes were annotated showing that many are associated with the primary function of the Rieske gene that was targeted by VIGS.
Highlights
Citrus Greening or Huanglongbing (HLB) is a disease of citrus, first discovered in the state of Florida in 2005 (Stokstad, 2012), that is responsible for a greater than fifty percent reduction in citrus production and is present in other citrus producing states (National Agricultural and Statistics Service, 2005; Putnman et al, 2011)
Twenty-one diets of 43 tested dsRNAs, showed significant mortality when fed 48 ng/μL, only 3 dsRNA (Rieske, Cytochrome P450 4g15-like2 and Pterin 4α-Carbinolamine Dehydratase) showed significant mortalities at 48 ng/mL greater than 30% mortality above diet alone and significant mortalities at concentrations of 24 ng/μL and 12 ng/μL (Tables 1, 2)
We report an innovative approach to control D. citri an agricultural pest insect that threatens the citrus industry in the United States and beyond
Summary
Citrus Greening or Huanglongbing (HLB) is a disease of citrus, first discovered in the state of Florida in 2005 (Stokstad, 2012), that is responsible for a greater than fifty percent reduction in citrus production and is present in other citrus producing states (National Agricultural and Statistics Service, 2005; Putnman et al, 2011). The catalytic subunit of the RISC (a member of the Argonaut protein family with endonuclease activity) cleaves the mRNA rendering it non-functional (Bernstein et al, 2001; Lipardi et al, 2001). This effect can be redirected against the host cell by the exogenous introduction of dsRNAs having sequence identity for host genes, resulting in the reduction of host cell mRNA abundance and subsequent loss of function (Fire et al, 1998; Gregory et al, 2005). RNAi silencing of host genes has previously been demonstrated in eukaryotic organisms (including insects) following oral uptake of synthetic dsRNA molecules (Velásquez et al, 2009; Borgio, 2010; Gong et al, 2011; Pitino et al, 2011; Wuriyanghan et al, 2011; Galdeano et al, 2017; Yu et al, 2017; Yu and Killiny, 2018, 2020; Lu et al, 2019)
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