Resurrection of the Family Grateloupiaceae Emend. (Halymeniales, Rhodophyta) Based on a Multigene Phylogeny and Comparative Reproductive Morphology

The marine red algal order Halymeniales currently includes two families, the Halymeniaceae and Tsengiaceae, and consist of 38 genera and about 358 species. Phylogenetic analyses on specific taxa of the order are common, but not comprehensive, leaving the many intra-ordinal relationships within the Halymeniales unresolved. To reassess the phylogeny of the Halymeniales, we conducted extensive phylogenetic analyses based on 207 rbcL sequences and multigene analyses (rbcL, psaA, psbA, cox1, and LSU) using 47 taxa from the order. The combined data set fully supports the monophyly of the Grateloupia sensu lato clade. Phylogenetic assessment of the reproductive structures in the order using the type of auxiliary cell ampullae, pericarp origin, and tetrasporangial development characters, supports a Grateloupia sensu lato clade distinct from the Halymeniaceae exemplified by the generitype Halymenia. As a result, we propose to reinstate the family Grateloupiaceae Schmitz based on the Grateloupia sensu lato clade and including Grateloupia and eight other genera: Dermocorynus, Mariaramirezia, Neorubra, Pachymeniopsis, Kintokiocolax, Phyllymenia, Prionitis, and Yonagunia. The emended Grateloupiaceae is distinguished from the Halymeniaceae by the following three characteristics; (i) simple unbranched and unilateral type of auxiliary cell ampullae, (ii) pericarp formed densely by the fusion of secondary medullary filaments from subcortical cells and lateral ampullary filaments from a fusion cell complex, (iii) tetrasporangia originating laterally from the outer cortex. The Halymeniales comprises the monophyletic Grateloupiaceae, Halymeniaceae sensu lato (which requires further study), and the Tsengiaceae.

Modulating the influenza A virus – target membrane fusion interface with synthetic DNA-lipid receptors

Influenza A virus (IAV) binds to sialylated glycans on the cell membrane before endocytosis and fusion. Cell surface glycans are highly heterogenous in length and glycosylation density, which leads to variation in the distance and rigidity with which IAV is held away from the cell membrane. To gain mechanistic insight into how receptor length and rigidity impact the mechanism of IAV entry, we employed synthetic DNA-lipids as highly tunable surrogate receptors. We tethered IAV to target membranes with a panel of DNA-lipids to investigate the effects of the distance and tether flexibility between virions and target membranes on the kinetics of IAV binding and fusion. Tether length and the presence of a flexible linker led to higher rates of IAV binding, while the efficiencies of lipid and content mixing were typically lower for longer and more rigid DNA tethers. For all DNA tether modifications, we found that the rates of IAV lipid and content mixing were unchanged. These results suggest that variations in the interface between IAV and a target membrane do not significantly impact the rate-limiting step of fusion, or the low-pH triggered engagement of viral fusion peptides with the target membrane. However, our results imply that the flexibility of the viral receptor is important for ensuring that hemifusion events are able to successfully proceed to pore formation.

Modulating the influenza A virus – target membrane fusion interface with synthetic DNA-lipid receptors

Influenza A virus (IAV) binds to sialylated glycans on the cell membrane before endocytosis and fusion. Cell surface glycans are highly heterogenous in length and glycosylation density, which leads to variation in the distance and rigidity with which IAV is held away from the cell membrane. To gain mechanistic insight into how receptor length and rigidity impact the mechanism of IAV entry, we employed synthetic DNA-lipids as highly tunable surrogate receptors. We tethered IAV to target membranes with a panel of DNA-lipids to investigate the effects of the distance and tether flexibility between virions and target membranes on the kinetics of IAV binding and fusion. Tether length and the presence of a flexible linker led to higher rates of IAV binding, while the efficiencies of lipid and content mixing were typically lower for longer and more rigid DNA tethers. For all DNA tether modifications, we found that the rates of IAV lipid and content mixing were unchanged. These results suggest that variations in the interface between IAV and a target membrane do not significantly impact the rate-limiting step of fusion, or the low-pH triggered engagement of viral fusion peptides with the target membrane. However, our results imply that the flexibility of the viral receptor is important for ensuring that hemifusion events are able to successfully proceed to pore formation.

A New Workflow to Generate Monoclonal Antibodies against Microorganisms

Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research and diagnostic applications. Nevertheless, the specific targeting of complex antigens such as whole microorganisms remains a challenge. To provide a comprehensive workflow, we combined bioinformatic analyses with novel immunization and selection tools to design monoclonal antibodies for the detection of whole microorganisms. In our initial study, we used the human pathogenic strain E. coli O157:H7 as a model target and identified 53 potential protein candidates by using reverse vaccinology methodology. Five different peptide epitopes were selected for immunization using epitope-engineered viral proteins. The identification of antibody-producing hybridomas was performed by using a novel screening technology based on transgenic fusion cell lines. Using an artificial cell surface receptor expressed by all hybridomas, the desired antigen-specific cells can be sorted fast and efficiently out of the fusion cell pool. Selected antibody candidates were characterized and showed strong binding to the target strain E. coli O157:H7 with minor or no cross-reactivity to other relevant microorganisms such as Legionella pneumophila and Bacillus ssp. This approach could be useful as a highly efficient workflow for the generation of antibodies against microorganisms.

Transfer of the monospecific genus Nienburgella (Delesseriaceae, Rhodophyta) to Phycodrys, based on morphological and molecular evidence

The genus Phycodrys is characterized by having a Phycodrys-type apical organization, a well-developed central midrib with paired lateral veins, monostromatic areas between the veins, four-celled carpogonial branches with two groups of sterile cells, and a cystocarp with a large fusion cell and short-catenated carposporangia. In contrast, the monospecific genus Nienburgella has a faint midrib, a mostly polystromatic blade without lateral veins, bilaterally isometric second-order cell row length, and Phycodrys-like apical growth and cystocarp. Nothing is known about Nienburgella’s procarp. We collected several specimens of Nienburgella angusta from the East Sea of South Korea and noticed that they resemble Phycodrys radicosa. We compared these two species using morphological and molecular phylogenetic analyses. Our results revealed that both species are closely related to one another than to other taxa in the tribe Phycodryeae, and that Nienburgella angusta procarp developmental pattern is the same as the one observed in Phycodrys rubens, the generitype species of Phycodrys. Thallus structure (mono- or polystromatic) was species-delimiting but not genus-delimiting. Bilateral second-order cell row isometry occurs in other Phycodrys species, including the type species. Therefore, we propose transferring the species Nienburgella angusta as well as the monospecific genus Nienburgella to Phycodrys.

Programmed Cell Death in Neurospora crassa Is Controlled by the Allorecognition Determinant rcd-1

Nonself recognition following cell fusion between genetically distinct individuals of the same species in filamentous fungi often results in a programmed cell death (PCD) reaction, where the heterokaryotic fusion cell is compartmentalized and rapidly killed. The allorecognition process plays a key role as a defense mechanism that restricts genome exploitation, resource plundering, and the spread of deleterious senescence plasmids and mycoviruses. Although a number of incompatibility systems have been described that function in mature hyphae, less is known about the PCD pathways in asexual spores, which represent the main infectious unit in various human and plant fungal pathogens. Here, we report the identification of regulator of cell death-1 (rcd-1), a novel allorecognition gene, controlling PCD in germinating asexual spores of Neurospora crassa; rcd-1 is one of the most polymorphic genes in the genomes of wild N. crassa isolates. The coexpression of two antagonistic rcd-1-1 and rcd-1-2 alleles was necessary and sufficient to trigger cell death in fused germlings and in hyphae. Based on analysis of wild populations of N. crassa and N. discreta, rcd-1 alleles appeared to be under balancing selection and associated with trans-species polymorphisms. We shed light on genomic rearrangements that could have led to the emergence of the incompatibility system in Neurospora and show that rcd-1 belongs to a much larger gene family in fungi. Overall, our work contributes toward a better understanding of allorecognition and PCD in an underexplored developmental stage of filamentous fungi.