Horn, T. | Search bioRxiv

bioRxiv2022

Establishing a reproducible approach for the controllable deposition and maintenance of plants cells with 3D bioprinting

Capturing cell-to-cell and cell-to-environment signals in a defined 3 dimensional (3D) microenvironment is key to study cellular functions, including cellular reprogramming towards tissue regeneration. A major challenge in current culturing methods is that these methods cannot accurately capture this multicellular 3D microenvironment. In this study, we established the framework of 3D bioprinting with plant cells to study cell viability, cell division, and cell identity. We established long-term cell viability for bioprinted Arabidopsis root cells and soybean meristematic cells. To analyze the large image datasets generated during these long-term viability studies, we developed an open source high-throughput image analysis pipeline. Furthermore, we showed the cell cycle re-entry of the isolated Arabidopsis and soybean cells leading to the formation of microcalli. Finally, we showed that the identity of isolated cells of Arabidopsis roots expressing endodermal markers maintained longer periods of time. The framework established in this study paves the way for a general use of 3D bioprinting for studying cellular reprogramming and cell cycle re-entry towards tissue regeneration.

plant biology↗

bioRxiv2021

Persistent parental RNAi in the beetle Tribolium castaneum involves maternal transmission of long double-stranded RNA

Parental RNA interference (pRNAi) is a powerful and widely used method for gene-specific knockdown. Yet in insects its efficacy varies between species, and how the systemic RNAi response is transmitted from mother to offspring remains elusive. Using the flour beetle Tribolium castaneum, we report an RT-qPCR strategy to unmask the presence of double-stranded RNA (dsRNA) distinct from endogenous mRNA. We find that the injected dsRNA is directly transmitted into the egg and persists throughout embryogenesis. Despite this depletion of dsRNA from the mother, we show that strong pRNAi can persist for months before waning at strain-specific rates. In seeking the receptor proteins for cellular uptake of long dsRNA into the egg, we lastly present a phylogenomics profiling approach to ascertain macroevolutionary distributions of candidate proteins. We demonstrate a visualization strategy based on taxonomically hierarchical assessment of orthology clustering data to rapidly assess gene age and copy number changes, refined by several lines of sequence-based evidence. We use this approach to document repeated losses of SID-1-like channel proteins in the arthropods, including wholesale loss in the Heteroptera (true bugs), which are nonetheless highly sensitive to pRNAi. Overall, we elucidate practical considerations for insect pRNAi against a backdrop of outstanding questions on the molecular mechanism of dsRNA transmission to achieve long-term, systemic knockdown.

genetics↗