Biology subjects

Perreau, J.

Publications and source records attributed to Perreau, J..

3 recordsLinked to original sources

bioRxiv2022

Addressing the challenges of symbiont-mediated RNAi in aphids

Because aphids are global agricultural pests and models for bacterial endosymbiosis, there is a need for reliable methods to study and control their gene function. However, current methods available for aphid gene knockout and knockdown of gene expression are often unreliable and time consuming. Techniques like CRISPR-Cas genome editing can take several months to achieve a single gene knockout because they rely on aphids going through a cycle of sexual reproduction, and aphids often lack strong, consistent levels of knockdown when fed or injected with molecules that induce an RNA interference (RNAi) response. In the hopes of addressing these challenges, we attempted to adapt a new method called symbiont-mediated RNAi (smRNAi) for use in aphids. smRNAi involves engineering a bacterial symbiont of the insect to continuously supply dsRNA inside the insect body. This approach has been successful in thrips, kissing bugs, and honeybees. We engineered the laboratory E. coli strain HT115 and the native aphid symbiont S. symbiotica CWBI-2.3T to produce dsRNA inside the gut of the pea aphid (Acyrthosiphon pisum) targeting salivary effector protein (C002) or ecdysone receptor genes. For C002 assays, we also tested co-knockdown with an aphid nuclease (Nuc1) to reduce RNA degradation. However, we found that smRNAi was not a reliable method for aphid gene knockdown under our conditions. We were unable to consistently achieve the expected phenotypic changes with either target. However, we did see indications that elements of the RNAi pathway were modestly upregulated, and expression of some targeted genes appeared to be somewhat reduced in some trials. We conclude with a discussion of the possible avenues through which smRNAi, and aphid RNAi in general, could be improved in the future.

microbiology↗

bioRxiv2020

Engineering a culturable Serratia symbiotica strain for aphid paratransgenesis

Aphids are global agricultural pests and important models for bacterial symbiosis. To date, none of the native symbionts of aphids have been genetically manipulated, which limits our understanding of how they interact with their hosts. Serratia symbiotica CWBI-2.3T is a culturable, gut-associated bacterium isolated from the black bean aphid. Closely related Serratia symbiotica strains are facultative aphid endosymbionts that are vertically transmitted from mother to offspring during embryogenesis. We demonstrate that CWBI-2.3T can be genetically engineered using a variety of techniques, plasmids, and gene expression parts. Then, we use fluorescent protein expression to track the dynamics with which CWBI-2.3T colonizes the guts of multiple aphid species, and we measure how this bacterium affects aphid fitness. Finally, we show that we can induce heterologous gene expression from engineered CWBI-2.3T in living aphids. These results inform the development of CWBI-2.3T for aphid paratransgenesis, which could be used to study aphid biology and enable future agricultural technologies. IMPORTANCEInsects have remarkably diverse and integral roles in global ecosystems. Many harbor symbiotic bacteria, but very few of these bacteria have been genetically engineered. Aphids are major agricultural pests and an important model system for the study of symbiosis. This work describes methods for engineering a culturable aphid symbiont, Serratia symbiotica CWBI-2.3T. These approaches and genetic tools could be used in the future to implement new paradigms for the biological study and control of aphids.

synthetic biology↗

bioRxiv2020

Vertical transmission at the pathogen-symbiont interface: Serratia symbiotica and aphids

Many insects possess beneficial bacterial symbionts that occupy specialized host cells and are maternally transmitted. As a consequence of their host-restricted lifestyle, these symbionts often possess reduced genomes and cannot be cultured outside hosts, limiting their study. The bacterial species Serratia symbiotica was originally described by noncultured strains that live as mutualistic symbionts of aphids and are vertically transmitted through transovarial endocytosis within the mothers body. More recently, culturable strains of S. symbiotica were discovered that retain a larger set of ancestral Serratia genes, are gut pathogens in aphid hosts, and are principally transmitted via a fecal-oral route. We find that these culturable strains, when injected into pea aphids, replicate in the hemolymph and are pathogenic. Unexpectedly, they are also capable of maternal transmission via transovarial endocytosis: using GFP-tagged strains, we observe that pathogenic S. symbiotica, but not Escherichia coli, are endocytosed into early embryos. Furthermore, pathogenic S. symbiotica strains are compartmentalized into specialized aphid cells in a similar fashion to mutualistic S. symbiotica strains during later stages of embryonic development. Thus, cultured, pathogenic strains of S. symbiotica have the latent capacity to transition to lifestyles as mutualistic symbionts of aphid hosts. This capacity is blocked by pathogenicity: their hosts die before infected progeny are born. To transition into stably inherited symbionts, culturable S. symbiotica strains may need to adapt to regulate their titer, limit their pathogenicity, and/or provide benefits to aphids that outweigh their cost. ImportanceInsects have evolved various mechanisms to reliably transmit their beneficial bacterial symbionts to the next generation. Sap-sucking insects, including aphids, transmit symbionts by endocytosis of the symbiont into cells of the early embryo within the mothers body. Experimental studies of this process are hampered by the inability to culture or genetically manipulate host-restricted, symbiotic bacteria. Serratia symbiotica is a bacterial species that includes strains ranging from obligate, heritable symbionts to culturable gut pathogens. We demonstrate that culturable S. symbiotica strains, that are aphid gut pathogens, can be maternally transmitted by endocytosis. Cultured S. symbiotica therefore possess a latent capacity for evolving a host-restricted lifestyle and can be used to understand the transition from pathogenicity to beneficial symbiosis.

microbiology↗