Targeted disruption of the cls gene in Buchnera aphidicola impairs membrane integrity and host symbiont dynamics.

The obligate symbiosis between pea aphids (Acyrthosiphon pisum) and Buchnera aphidicola represents metabolic interdependence between the host insect and its bacterial symbiont. Buchnera has a highly reduced genome that has lost nearly all phospholipid synthesis genes except cls, encoding a cardiolipin synthase homologue. We employed in vivo antisense, cell-penetrating peptide (CPP)-conjugated synthetic peptide nucleic acids (PNAs) to knock down cls in Buchnera. This intervention resulted in significant downregulation of cls expression, lowered Buchnera titers, pronounced morphological distortions, and reduced aphid reproduction. Notably, Buchnera cells were often detected in the aphid gut following anti-cls PNAs treatment, deviating from their typical intracellular niche within bacteriocytes. Collectively, the cls gene is critical for maintaining Buchnera integrity, proper cellular localization, and symbiont-host interactions. Given that the retention of cls is a common feature among many obligate endosymbionts despite massive gene loss, our findings offer key insights into the evolutionary principles shaping symbiotic relationships involving membrane biology.