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Analysis of Engineered Tobacco Mosaic Virus and Potato Virus X Nanoparticles as Carriers for Biocatalysts.

ABSTRACT: Plant virus nanoparticles are promising candidates for the development of novel materials, including nanocomposites and scaffolds/carriers for functional molecules such as enzymes. Their advantages for enzyme immobilization include a modular organization, a robust and programmable structure, and a simple, cost-effective production. However, the activity of many enzymes relies on posttranslational modification and most plant viruses replicate in the cytoplasm, so functional enzymes cannot be displayed on the virus surface by direct coat protein fusions. An alternative display system to present the Trichoderma reesei endoglucanase Cel12A on potato virus X (PVX) using SpyTag/SpyCatcher (ST/SC) technology was recently developed by the authors, which allows the carrier and enzyme to be produced separately before isopeptide conjugation. Although kinetic analysis clearly indicated efficient biocatalyst activity, the PVX carrier interfered with substrate binding. To overcome this, the suitability of tobacco mosaic virus (TMV) was tested, which can also accommodate a larger number of ST peptides. We produced TMV particles displaying ST as a new platform for the immobilization of enzymes such as Cel12A, and compared its performance to the established PVX-ST platform in terms of catalytic efficiency. Although more enzyme molecules were immobilized on the TMV-ST particles, we found that the rigid scaffold and helical spacing significantly affected enzyme activity.

SUBMITTER: Schuphan J

PROVIDER: S-EPMC8377429 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The potato/tomato psyllid, Bactericera cockerelli (B. cockerelli), is an important plant pest and the vector of the phloem-limited bacterium Candidatus Liberibacter psyllaurous (solanacearum), which is associated with the zebra chip disease of potatoes. Previously, we reported induction of RNA interference effects in B. cockerelli via in vitro-prepared dsRNA/siRNAs after intrathoracic injection, and after feeding of artificial diets containing these effector RNAs. In order to deliver RNAi effectors via plant hosts and to rapidly identify effective target sequences in plant-feeding B. cockerelli, here we developed a plant virus vector-based in planta system for evaluating candidate sequences. We show that recombinant Tobacco mosaic virus (TMV) containing B. cockerelli sequences can efficiently infect and generate small interfering RNAs in tomato (Solanum lycopersicum), tomatillo (Physalis philadelphica) and tobacco (Nicotiana tabacum) plants, and more importantly delivery of interfering sequences via TMV induces RNAi effects, as measured by actin and V-ATPase mRNA reductions, in B. cockerelli feeding on these plants. RNAi effects were primarily detected in the B. cockerelli guts. In contrast to our results with TMV, recombinant Potato virus X (PVX) and Tobacco rattle virus (TRV) did not give robust infections in all plants and did not induce detectable RNAi effects in B. cockerelli. The greatest RNA interference effects were observed when B. cockerelli nymphs were allowed to feed on leaf discs collected from inoculated or lower expanded leaves from corresponding TMV-infected plants. Tomatillo plants infected with recombinant TMV containing B. cockerelli actin or V-ATPase sequences also showed phenotypic effects resulting in decreased B. cockerelli progeny production as compared to plants infected by recombinant TMV containing GFP. These results showed that RNAi effects can be achieved in plants against the phloem feeder, B. cockerelli, and the TMV-plant system will provide a faster and more convenient method for screening of suitable RNAi target sequences in planta.

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Analysis of Engineered Tobacco Mosaic Virus and Potato Virus X Nanoparticles as Carriers for Biocatalysts.

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