Garcia, A. G. | Search bioRxiv

bioRxiv2025

Enabling the Study of Gene Function in Gymnosperms: VIGS in Ephedra tweedieana

PremiseAs the sister clade to angiosperms, gymnosperms are key to enabling the reconstruction of ancestral gene regulatory networks for seed plants. However, tools to rapidly and efficiently investigate gene function in gymnosperms remain limited due to the challenges of long life cycles and large genome sizes. Species within the xerophytic genus Ephedra (Gnetales) have comparatively smaller genomes and shrubby growth habits with shorter life spans, making them better suited for greenhouse cultivation and laboratory experiments. Methods and ResultsHere, we implement Virus-Induced Gene Silencing (VIGS) to manipulate gene expression in Ephedra tweedieana. Agrobacterium-mediated vacuum infiltration of Tobacco Rattle Virus (TRV2 and TRV1) in seedlings resulted in highly efficient silencing of the E. tweedieana PHYTOENE DESATURASE ortholog EtwPDS. The expected photobleaching phenotype was observed as early as two weeks. It lasted at least three months, in stems, shoot tips, leaves, axillary meristems, and lateral branches of treated plants. ConclusionsThis first report of transient transformation and targeted gene silencing in a gymnosperm will further enable functional studies of the genetic mechanisms underpinning adaptations in this important and underrepresented lineage of seed plants.

plant biology↗

bioRxiv2024

Identification of a large cohort of Enterobacter jumbo phages with broad host ranges across pathogenic Gammaproteobacteria

ESKAPE pathogens cause most hospital-acquired infections globally and often carry antibiotic resistance. Many of them have been the target of bacteriophage therapies. Enterobacter is an ESKAPE pathogen but is less frequently a target for phage therapy due to a relative lack of available phages. We isolated eight jumbo phages with genomes ranging from 223 to 366 kbp targeting Enterobacter spp. and found that they belonged to separate phage clades. Six of them formed nucleus-like structures confirmed by DAPI-staining, and were phylogenetically related to Chimalliviridae. Two jumbo phages did not form nucleus-like structures and did not cluster with Chimalliviridae. Although these jumbo phages were found on Enterobacter, many were closely related to phages with non-Enterobacter hosts. To test whether these phages may have had expanded host ranges, we examined 14 pathogenic Gammaproteobacteria and found that these phages were capable of creating plaques on 8 of them. These species included Escherichia coli, Klebsiella aerogenes, Serratia marcescens, Salmonella spp., Shigella spp., Providencia spp., Citrobacter spp., and Cronobacter sakazakii. We verified that there was phage amplification in these microbes rather than lysis from without by performing qPCR to confirm DNA replication in each species. Phages typically have narrow host ranges, a benefit for microbiome-sparing compared to antibiotics. However, the broad host ranges of these Gammaproteobacteria jumbo phages suggests that not all phages have the same risk/benefit ratios. While this broad range could aid their development as antibiotic alternatives, further study is needed to assess potential microbiome disruption. SignificanceWith the growing threat of antibiotic resistant bacteria, alternative treatments like bacteriophages have emerged. Bacteriophages typically have narrow host ranges, a disadvantage compared to antibiotics. We discovered eight jumbo phages that kill antibiotic resistant Enterobacter, but were diverse phylogenetically. Six of them formed nucleus-like structures and were members of the Chimalliviridae family and the other two did not form nucleus-like structures. We confirmed that each phage had broad host ranges capable of lysing at least eight different human Gammaproteobacteria including pathogens such as E. coli, K. aerogenes, and S. marcescens. By identifying broad host range jumbo phages that attack pathogens, we may have identified phages with spectrums of activity more similar to antibiotics than have been traditionally attributed to phages.

microbiology↗