UV‐LASER adjuvant–surfactant‐facilitated delivery of mobile dsRNA to tomato plant vasculature and evidence of biological activity by gene knockdown in the potato psyllid

• Published in: Pest management science Vol. 80; no. 4; pp. 2141 - 2153
• Main Authors: Thakre, Neha, Carver, Megan, Paredes‐Montero, Jorge R, Mondal, Mosharrof, Hu, Jiahuai, Saberi, Esmaeil, Ponvert, Nathaniel, Qureshi, Jawwad A, Brown, Judith K
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.04.2024; Wiley Subscription Services, Inc
• Subjects: Adjuvants; Amplification; Bacteria; Biological activity; Biological effects; Candidatus Liberibacter; Cdc42 protein; citrus greening disease; Confocal microscopy; Double-stranded RNA; Fluorimetry; Fluorometry; foliar uptake; Gelsolin; gene knockdown; Gene silencing; Host plants; Ingestion; Insects; Laser radiation; Lasers; Leaves; Localization; Organosilicon compounds; Pest control; Pesticides; Polymerase chain reaction; Potatoes; Reverse transcription; Surfactants; Tomatoes; Ultraviolet radiation; UV‐LASER; Vectors; Wounding; foliar uptake; surfactants; citrus greening disease; double-stranded RNA; UV-LASER; gene knockdown
• ISSN: 1526-498X; 1526-4998
• DOI: 10.1002/ps.7952

BACKGROUND Double‐stranded RNA (dsRNA) biopesticides are of interest for the abatement of insect vectors of pathogenic bacteria such as ‘Candidatus Liberibacter’, which infects both its psyllid and plant hosts. Silencing of genes essential for psyllids, or for Liberibacter, is anticipated to lead to mortality or impeded bacterial multiplication. Foliar delivery is preferred for biopesticide application; however, the cuticle impedes dsRNA penetration into the vasculature. Here, conditions were established for wounding tomato leaves using ultraviolet light amplification by stimulated emissions of radiation (UV‐LASER) to promote dsRNA penetration into leaves and vasculature. RESULTS UV‐LASER treatment with application of select adjuvants/surfactants resulted in vascular delivery of 100‐, 300‐ and 600‐bp dsRNAs that, in general, were correlated with size. The 100‐bp dsRNA required no pretreatment, whereas 300‐ and 600‐bp dsRNAs entered the vasculature after UV‐LASER treatment only and UV‐LASER adjuvant/surfactant treatment, respectively. Of six adjuvant/surfactants evaluated, plant‐derived oil combined with an anionic organosilicon compound performed most optimally. Localization of dsRNAs in the tomato vasculature was documented using fluorometry and fluorescence confocal microscopy. The biological activity of in planta‐delivered dsRNA (200–250 bp) was determined by feeding third‐instar psyllids on tomato leaves post UV‐LASER adjuvant/surfactant treatment, with or without psyllid cdc42‐ and gelsolin dsRNAs. Gene knockdown was quantified by quantitative, real‐time polymerase chain reaction with reverse transcription (RT–qPCR) amplification. At 10 days post the ingestion‐access period, knockdown of cdc42 and gelsolin expression was 61% and 56%, respectively, indicating that the dsRNAs delivered to the tomato vasculature were mobile and biologically active. CONCLUSION Results indicated that UV‐LASER adjuvant/surfactant treatments facilitated the delivery of mobile, biologically active dsRNA molecules to the plant vasculature. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. Ultraviolet laser wounding and adjuvant/surfactant application is optimized for the penetration of biologically active, RNA molecules into tomato leaf vasculature for RNA interference‐based genetic regulation of the ‘Candidatus Liberibacter’ vector Bactericera cockerelli after feeding on treated leaves. The penetration, localization and biological activity of supplied double‐stranded RNA is demonstrated using fluorometry, confocal microscopy and quantitative, real‐time polymerase chain reaction with reverse transcription.