Soukupova, H. | bioRxiv Science

bioRxiv2026

Division of labor between seed plant RAB GDI paralogs: insights from genetic analysis in Arabidopsis thaliana

BackgroundRAB Guanine Nucleotide Dissociation Inhibitors (RAB GDIs) are important vesicle transport regulators in eukaryotes, participating in the functional cycle of RAB GTPases by stabilizing their non-active GDP-conformation. AimsWe address the importance of the three Arabidopsis thaliana RAB GDI paralogs by genetic and developmental analyses and put these results into the seed plants evolution context. MethodsWe use methods of genetics, microscopy and phylogenetics. ResultsOur genetic analyses of Arabidopsis T-DNA insertional mutants confirm recent CRISPR alleles data indicating lethality of double gdi1 gdi2 mutants, and our microscopic data point to embryo development arrest in double mutant seeds. We also confirm the involvement of GDI2 and GDI3 in pollen tube growth. Moreover, our data show that GDI1 also contributes to proper pollen function. Our phylogenetic analysis reveals independent diversification of RAB GDIs in Gymnosperms and Angiosperms, with early specialization of an Angiosperm reproduction-and gametophyte-related clade. ConclusionsIn Arabidopsis, RAB GDI1 and 2 are important for the vegetative growth while RAB GDI2 and 3 are vital for reproduction. Evolution of the RAB GDI family reflects the evolution of seed plants. HighlightsRAB GDIs are vital for plant growth and reproduction and act redundantly. Even the low-transcribed RAB GDI1 isoform contributes to the proper pollen function. Two RAB GDI clades evolved in early Angiosperms.

plant biology↗

bioRxiv2024

Evaluating Bacterial Viability in Faecal Microbiota Transplantation: A Comparative Analysis of In Vitro Cultivation and Membrane Integrity Methods

BackgroundFaecal microbiota transplantation (FMT) is a developing therapy for disorders related to gut dysbiosis. Despite its growing application, standardized protocols for FMT filtrate preparation and quality assessment remain undeveloped. The viability of bacteria in the filtrate is crucial for FMTs efficacy and for validating protocol execution. We compared two methods--in vitro cultivation and membrane integrity assessment--for their accuracy, reproducibility, and clinical applicability in measuring bacterial viability in frozen FMT stool filtrate. MethodsBacterial viability in stool filtrate was evaluated using (i) membrane integrity through fluorescent DNA staining with SYTO9 and propidium iodide, followed by flow cytometry; and (ii) culturable bacteria counts (colony-forming units, CFU) under aerobic or anaerobic conditions. ResultsWe refined the bacterial DNA staining protocol integrated with flow cytometry for stool samples. Both the membrane integrity-based and cultivation-based methods exhibited significant variability in bacterial viability across different FMT filtrates, without correlation. The cultivation-based method showed a mean coefficient of variance of 17%, ranging from 5.3% to 52.9%. Conversely, the membrane integrity approach yielded highly reproducible results, with a median coefficient of variance for viable cells of 0.9%, ranging from 8.5% to 0.04%. ConclusionBacterial viability assessment using cultivation-dependent methods produces inconsistent outcomes. In contrast, the membrane integrity method offers robust and precise data, making it a viable option for routine faecal material evaluation in FMT.

microbiology↗