Synthetic Peptide Nucleic Acids in Action:In VivoInterference of thegroELGene from Pea Aphid EndosymbiontBuchnera aphidicola

Abstract

The unculturable nature of intracellular obligate symbionts presents a significant challenge for elucidating gene functionality, necessitating the development of gene manipulation techniques. One of the best-studied obligate symbioses is that between aphids and the bacterial endosymbiontBuchnera aphidicola. Given the extensive genome reduction observed inBuchnera, the remaining genes are crucial for understanding the host-symbiont relationship, but a lack of tools for manipulating gene function in the endosymbiont has significantly impeded the exploration of the molecular mechanisms underlying this mutualism. In this study, we introduced a novel gene manipulation technique employing synthetic single-stranded peptide nucleic acids (PNAs). We targeted the criticalBuchnera groELusing specially designed antisense PNAs conjugated to an arginine-rich cell-penetrating peptide (CPP). Within 24 h of PNA administration via microinjection, we observed a significant reduction ingroELexpression andBuchneracell count. Notably, the interference ofgroELled to profound morphological malformations inBuchnera, indicative of impaired cellular integrity. The gene knockdown technique developed in this study, involving the microinjection of CPP-conjugated antisense PNAs, provides a potent approach forin vivogene manipulation of unculturable intracellular symbionts, offering valuable insights into their biology and interactions with hosts.