Hunter et al. · Frontiers in Agronomy · 2021

Improving Suppression of Hemipteran Vectors and Bacterial Pathogens of Citrus and Solanaceous Plants: Advances in Antisense Oligonucleotides (FANA)

Hunter WB, Cooper WR, Sandoval-Mojica AF, McCollum G, Aishwarya V, Pelz-Stelinski KS

The study

What was asked, and what was found

Hunter et al., Frontiers in Agronomy, 2021 took self-delivering antisense chemistry out of the culture dish and into orchards and potato fields. The problem is huanglongbing, the bacterial disease that is destroying citrus production worldwide, and its relative that causes zebra chip in potato. Both are caused by Candidatus Liberibacter bacteria living inside the plant, and both are carried between plants by psyllids. Nothing sprayed on a leaf reaches a bacterium in the phloem, which is why the disease has been so hard to treat.

The study asked whether AUMsilence sdASOs could get there instead, and it answered that first with pictures. A labelled oligonucleotide put into a detached citrus leaf was found in the xylem and the phloem. When psyllids fed on treated growth, the same label turned up in the insect's midgut within 48 hours and, by 7 days, in the fat body, the brain and vesicles throughout the body. Nothing carried it. More than 160 sequences were made, averaging 22 nucleotides, roughly four per target, against bacterial genes for DNA ligase, gyrase A, a carboxylase subunit and a replicative helicase, and against the psyllid's own trehalase.

The effects followed. Infected psyllids that fed on treated citrus leaves carried less of the pathogen, and less pathogen meant less onward transmission. Targeting gyrase A in Profftella armatura, a bacterium the psyllid depends on, killed the insects: 76% mortality at 8 days and 87% at 12, against 16% and 21% in controls. Treated potato and tomato plants showed reduced or delayed disease, and fewer tubers with the zebra chip defect. Feeding on treated diet lowered the psyllid's own trehalase message. The paper is equally clear about what did not work: two of four sequences worked against one bacterial gene, one of four against another, two of five against the insect gene, and the authors attribute the misses to target RNA folding. A single trunk injection raised the proportion of dead bacteria at 3 weeks but the titres were back to baseline by 4 months, so repeat or heavier dosing would be needed. AUM BioTech co-authored the study and the conflict of interest is declared in the paper.

Key findings

A fluorescently labelled AUMsilence sdASO put into a citrus leaf reached the xylem and the phloem, and after psyllids fed on treated growth the same label turned up in the fat body, the brain and vesicles through the insect body. That is systemic movement through a plant and then into the insect that feeds on it, with no carrier of any kind.(Results, the citrus cutting feeding assay subsection using the labelled probe, Fig. 2)
Silencing a gene in the bacterial partner that the psyllid depends on killed the insects: 87% mortality by day 12 against 21% in the controls, with a very large effect size.(Results, the gyrase A subsection targeting the Diaphorina citri endosymbiont Profftella armatura, Fig. 6)
Oligonucleotides eaten by infected psyllids in treated leaf tissue lowered the titre of the plant pathogen inside the insect, and lowering it lowered onward transmission.(Abstract)
Treating infected potato and tomato plants reduced or delayed the disease: severity scores differed significantly, and the tuber defect that gives the disease its name was reduced against untreated plants.(Results, the section on treating CLso infected potato and tomato with the gyrase A oligonucleotides, Fig. 8A)
Sequence choice decided everything, and the paper reports the failures beside the successes: two of four sequences worked against one bacterial gene, one of four against another, and two of five against the insect gene, with the authors attributing the misses to target RNA folding.(Results, CLas Replicative Helicase, DNA-B)
A single trunk injection did not hold. Dead bacteria rose at 3 weeks, then titres were back to where they started by 4 months, and the authors say plainly that repeat dosing or a higher dose would be needed.(Results, Tree Trunk Injection Into CLas-Infected Citrus Trees)
Feeding on treated diet lowered a psyllid's own trehalase message, so the same reagents silenced an insect gene as well as bacterial ones.(Results, the subsection on targeting Diaphorina citri trehalase, Fig. 7)

Product: AUMsilence sdASO
Targets: DNA gyrase subunit A (GyrA) · Candidatus Liberibacter asiaticus, Candidatus Liberibacter solanacearum, Wolbachia of Diaphorina citri, and Profftella armatura · bacterial mRNANAD-dependent DNA ligase A (LigA, CLIBASIA), gyrase A (GyrA) and acetyl-CoA carboxylase biotin carboxylase subunit (BTIN) · Candidatus Liberibacter asiaticus · bacterial mRNAreplicative helicase DNA-B · Candidatus Liberibacter asiaticus · bacterial mRNAtrehalase · Diaphorina citri, the Asian citrus psyllid · insect mRNA
Models: citrus seedlings and trees, sweet orange Madam vinous, infected with Candidatus Liberibacter asiaticus · in vivo, whole plantpotato and tomato plants inoculated with Candidatus Liberibacter solanacearum by infected potato psyllids · in vivo, whole plantadult Asian citrus psyllid Diaphorina citri, carrying Candidatus Liberibacter asiaticus, fed on treated citrus leaves or on artificial diet · in vivo, insectpotato psyllid Bactericera cockerelli carrying Candidatus Liberibacter solanacearum haplotype B · in vivo, insect
Delivery: self-delivering, taken up without any transfection or carrier and moving systemically: absorbed into cuttings and detached leaves, applied as a soil drench, root infusion or topical spray, injected into the trunk, or eaten by insects in treated leaf tissue or artificial diet
Readouts: RT-qPCR for target gene expression, quantitative PCR for bacterial titre and copy number, confocal microscopy of fluorophore-tagged oligonucleotide in plant and insect tissue, insect mortality counts, foliar symptom severity scoring, tuber zebra chip symptom scoring, live and dead bacterial staining
Measured: size of the design campaign behind the study: over 160 oligonucleotides, averaging 22 nt, with about four sequences per target and up to 8 for some insect targets (bacterial and insect RNA targets)psyllid mortality when the bacterial endosymbiont Profftella armatura was targeted: 76.3% (±SE 1.07) at 8 days and 87.2% (±SE 0.89) at 12 days, against 15.7% (±SE 0.48) and 21.1% (±SE 0.38) in controls (adult psyllids feeding on treated potted citrus seedlings, effect size eta squared 0.92)reduction in bacterial titre in infected psyllids targeting the acetyl-CoA carboxylase subunit: 3.5- to 3.7-fold less bacteria for two of the four sequences; the other two were not different from control (6-day feeding access period on treated citrus leaf disks, one-way ANOVA F(3,32) = 8.52, p = 0.0003)reduction in bacterial titre targeting the replicative helicase: a drop of 4.32 equivalents for the best sequence, against 4.8 equivalents in untreated controls, with the other three sequences not significant (6-day feeding on treated citrus leaf tissue that had absorbed 300 μL at 1 μM)concentration absorbed by treated citrus leaf tissue in the helicase experiment: 300 μL at 1 μM (citrus leaf tissue fed to infected adult psyllids)knockdown of an insect gene by feeding on treated artificial diet: 0.73 log fold and 0.55 log fold less total trehalase mRNA for two of five sequences (psyllids at 48 h of feeding, the other three sequences not significant at any time point)dose used to visualise uptake into detached citrus leaves: 6.93 nmol in 500 μL water, imaged 7 days after treatment (fluorophore-tagged oligonucleotide, confocal imaging of the leaf midrib)duration of suppression after a single trunk injection: an average of 65% dead bacteria at 3 weeks against 48.2% in untreated infected trees, with titres back to pretreatment levels by 4 months (single injection into infected citrus seedlings, no repeat dose)
Figures: Figure 1 The labelled oligonucleotide inside the xylem and phloem of a citrus leaf midrib, 7 days after a detached leaf absorbed it.Figure 2 The same labelled oligonucleotide inside the psyllid after it fed on treated growth, in the midgut at 48 hours and through the body by 7 days.Figure 6 Psyllid mortality after feeding on seedlings treated against the insect's bacterial endosymbiont, against untreated controls, on days 2, 5, 8 and 12.Figure 7 Trehalase message in psyllids after feeding on artificial diet carrying each of the five oligonucleotides.
Citation: Hunter et al. Improving Suppression of Hemipteran Vectors and Bacterial Pathogens of Citrus and Solanaceous Plants: Advances in Antisense Oligonucleotides (FANA). Frontiers in Agronomy 2021;3:675247. doi:10.3389/fagro.2021.675247

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