Synthetic Peptide Nucleic Acids in Action: In Vivo Interference of the groEL Gene from Pea Aphid Endosymbiont Buchnera aphidicola

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 endosymbiont Buchnera aphidicola . Given the extensive genome reduction observed in Buchnera , 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 critical Buchnera groEL using 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 in groEL expression and Buchnera cell count. Notably, the interference of groEL led to profound morphological malformations in Buchnera , 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 for in vivo gene manipulation of unculturable intracellular symbionts, offering valuable insights into their biology and interactions with hosts. ### Competing Interest Statement The authors have declared no competing interest.