Biology subjects

Attah, V.

Publications and source records attributed to Attah, V..

5 recordsLinked to original sources

bioRxiv2026

Stable transformation of Micractinium conductrix SAG 241.80: New tools for exploring photosymbiotic interactions

Endosymbiosis played a key role in the evolution of cellular complexity, but mechanisms underpinning establishment and maintenance remain unclear. Paramecium bursaria harbours Chlorellaceae algal endosymbionts and is a valuable model system for understanding photosymbiosis. However, there are a lack of molecular tools available for exploring the association. Here we report stable transformation of the P. bursaria endosymbiont Micractinium conductrix SAG 241.80. Bioluminescent reporter assays were used to identify endogenous regulatory sequences for transgene expression and to refine a protocol for delivery of exogenous DNA. The bleomycin resistance gene Shble was then identified as an effective selectable marker for isolating transformed cell lines. We demonstrate that endogenous introns enhance transgene expression and that isolation of transformants with desirable characteristics can be achieved without extensive screening by using a 2A peptide to couple expression of an upstream non-selectable transgene to expression of the Shble selectable marker. Finally, we demonstrate that M. conductrix SAG 241.80 transformants expressing fluorescent proteins can be introduced into host cells and observed in the absence of selection, enabling distinct endosymbiont genotypes to be unambiguously identified, compared and monitored within the host environment. The development of molecular tools reported here opens new avenues for addressing unresolved questions in photosymbiosis. Significance StatementO_LIParamecium bursaria is a single-celled, mixotrophic ciliate which forms photosymbiotic interactions with Chlorellaceae green algae and is a popular model system for symbiosis research. C_LIO_LIWe established a stable transformation protocol for the P. bursaria algal endosymbiont M. conductrix SAG 241.80 and identified properties of synthetic gene constructs which facilitate isolation of transformants with robust transgene expression that can be detected within the host environment. C_LIO_LIThis marks significant progress in model system development and opens new avenues for exploring molecular and cellular mechanisms underpinning photosymbiotic interactions. C_LI

evolutionary biology↗

bioRxiv2024

Immune-like glycan-sensing and horizontally-acquired glycan-processing orchestrate host control in a microbial endosymbiosis

Endosymbiosis was a key factor in the evolution of eukaryotic cellular complexity. Yet the mechanisms that allow host regulation of intracellular symbionts, a pre-requisite for stable endosymbiosis and subsequent organelle evolution, are largely unknown. Here, we describe an immune-like glycan-sensing/processing network, partly assembled through horizontal gene-transfers (HGTs), that enables Paramecium bursaria to control its algal endosymbionts. Using phylogenetics, RNA-interference (RNAi), and metabolite exposure experiments, we show that P. bursaria regulates endosymbiont destruction using glycan-sensing/processing - a system that includes a eukaryotic-wide chitin-binding chitinase-like protein (CLP) localized to the host phago-lysosome. RNAi of CLP alters expression of eight glycan-processing genes, including two prokaryote-derived HGTs, during endosymbiont destruction. Furthermore, glycan-sensing/processing dynamically regulates endosymbiont number in P. bursaria, plasticity crucial to maximize host fitness across ecological conditions. CLP is homologous to a human phagocyte-associated innate immune factor, revealing how immune functions can be alternatively adapted and expanded, partly through HGT, enabling endosymbiotic control. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/613017v2_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@13aeaaorg.highwire.dtl.DTLVardef@302e6dorg.highwire.dtl.DTLVardef@138224forg.highwire.dtl.DTLVardef@54aca8_HPS_FORMAT_FIGEXP M_FIG C_FIG

evolutionary biology↗

bioRxiv2023

Duplication and neofunctionalization of a horizontally-transferred xyloglucanase as a facet of the red queen co-evolutionary dynamic

Oomycetes are heterotrophic protists that share phenotypic similarities with fungi, including the ability to cause plant diseases, but branch in a separate and distant region of the eukaryotic tree of life. It has been suggested that multiple horizontal gene transfers (HGTs) from fungi-to-oomycetes contributed to the evolution of plant-pathogenic traits. These HGTs are predicted to include secreted proteins that degrade plant cell walls. This is a key trait in the pathology of many oomycetes, as the plant cell wall represents a primary barrier to pathogen invasion and a rich source of carbohydrates. Many of the HGT gene families identified have undergone multiple rounds of duplication. Using a combination of phylogenomic analysis and functional assays, we investigate the diversification of a horizontally-transferred xyloglucanase gene family in the model oomycete species Phytophthora sojae. Our analyses detect 11 genes retained in P. sojae among a complex pattern of gene duplications and losses. Using a phenotype assay, based on heterologous expression in yeast, we show that eight of these paralogs have xyloglucanase function, including variants with distinct protein characteristics, such as a long-disordered C-terminal extension that can increase xyloglucanase activity. The functional xyloglucanase variants analysed subtend an ancestral node close to the fungi-oomycetes gene transfer, suggesting the horizontally-transferred gene was a bona fide xyloglucanase. Expression of xyloglucanase paralogs in Nicotiana benthamiana triggers distinct patterns of reactive oxygen species (ROS) generation, demonstrating that enzyme variants differentially stimulate pattern-triggered immunity in plants. Mass spectrometry of detectable enzymatic products demonstrates that some paralogs catalyze production of variant breakdown profiles, suggesting that secretion of multiple xyloglucanase variants increases efficiency of xyloglucan breakdown, as well as potentially diversifying the range of Damage-Associated Molecular Patterns (DAMPs) released during pathogen attack. We suggest that such patterns of protein neofunctionalization, and variant host responses, represent an aspect of the Red Queen host-pathogen co-evolutionary dynamic. Significance StatementThe oomycetes are a diverse group of eukaryotic microbes that include some of the most devastating pathogens of plants. Oomycetes perceive, invade, and colonize plants in similar ways to fungi, in part because they acquired the genes to attack and feed on plants from fungi. These genes are predicted to be useful to oomycete plant pathogens because they have undergone multiple rounds of gene duplication. One key enzyme for attacking plant cell wall structures is called xyloglucanase. Xyloglucanase in the oomycetes has undergone multiple rounds of gene duplication, leading to variants including an enzyme with a C-terminal extension that increases activity. Some xyloglucanase variants trigger unique patterns of reactive oxygen species (ROS) in planta, and generate different profiles of cell wall breakdown products - such outcomes could act to mystify and increase the workload of the plant immune system, allowing successful pathogens to proliferate.

evolutionary biology↗

bioRxiv2023

Glycoproteomic landscape and structural dynamics of TIM family immune checkpoints enabled by mucinase SmE

Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of biological functions. In particular, the T cell immunoglobulin and mucin-domain containing family of proteins (TIM-1, -3, -4) decorate immune cells and act as key checkpoint inhibitors in cancer. However, their dense O-glycosylation remains enigmatic both in terms of glycoproteomic landscape and structural dynamics, primarily due to the challenges associated with studying mucin domains. Here, we present a mucinase (SmE) and demonstrate its ability to selectively cleave along the mucin glycoprotein backbone, similar to others of its kind. Unlike other mucinases, though, SmE harbors the unique ability to cleave at residues bearing extremely complex glycans which enabled improved mass spectrometric analysis of several mucins, including the entire TIM family. With this information in-hand, we performed molecular dynamics (MD) simulations of TIM-3 and -4 to demonstrate how glycosylation affects structural features of these proteins. Overall, we present a powerful workflow to better understand the detailed molecular structures of the mucinome.

cancer biology↗

bioRxiv2019

The Zymoseptoria tritici ORFeome: a functional genomics community resource

Libraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for Zymoseptoria tritici, which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, cloned into the Gateway(R) Entry Vector pDONR207, and sequenced, providing a total of 3022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors (n = 410) and secondary metabolite biosynthetic proteins (n = 171), in addition to genes residing at dispensable chromosomes (n= 122), or those that are preferentially expressed during plant infection (n = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway(R) Destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The Z. tritici ORFeome constitutes a powerful resource for functional genomics, and offers unparalleled opportunities to understand the biology of Z. tritici.

systems biology↗