Biology subjects

Zess, E. K.

Publications and source records attributed to Zess, E. K..

5 recordsLinked to original sources

bioRxiv2026

Comparative characterization of Cas12a2 orthologs identifies high-activity nucleases for programmable cell elimination

Cas12a2 CRISPR nucleases, including SuCas12a2, have been shown to have extensive collateral activity towards RNA, ssDNA, and dsDNA. This collateral activity results in targeted cell elimination and has applications across biotechnology, agriculture, and human health. We explored the natural genetic diversity of Cas12a2 nucleases and characterized nine novel orthologs in a DNA damage kinetic assay in E. coli. Three new Cas12a2 orthologs (RsCas12a2, SdCas12a2, and HmCas12a2) were shown to have high collateral activity towards DNA. These nucleases are highly divergent from SuCas12a2, have conserved core RuvC catalytic residues, and have sequence diversity in the previously reported aromatic clamp residues required for nucleic acid positioning in the active site. We defined PFS preferences and mismatch tolerance for each high-activity Cas12a2 nuclease, expanding the available Cas12a2 toolbox, and discovered functional differences with obvious impacts on downstream applications.

molecular biology↗

bioRxiv2021

Regressive evolution of an effector following a host jump in the Irish Potato Famine Pathogen Lineage

In order to infect a new host species, the pathogen must evolve to enhance infection and transmission in the novel environment. Although we often think of evolution as a process of accumulation, it is also a process of loss. Here, we document an example of regressive evolution in the Irish potato famine pathogen (Phytophthora infestans) lineage, providing evidence that a key sequence motif in the effector PexRD54 has degenerated following a host jump. We began by looking at PexRD54 and PexRD54-like sequences from across Phytophthora species. We found that PexRD54 emerged in the common ancestor of Phytophthora clade 1b and 1c species, and further sequence analysis showed that a key functional motif, the C-terminal ATG8-interacting motif (AIM), was also acquired at this point in the lineage. A closer analysis showed that the P. mirabilis PexRD54 (PmPexRD54) AIM appeared unusual, the otherwise-conserved central residue mutated from a glutamate to a lysine. We aimed to determine whether this PmPexRD54 AIM polymorphism represented an adaptation to the Mirabilis jalapa host environment. We began by characterizing the M. jalapa ATG8 family, finding that they have a unique evolutionary history compared to previously characterized ATG8s. Then, using co-immunoprecipitation and isothermal titration calorimetry assays, we showed that both full-length PmPexRD54 and the PmPexRD54 AIM peptide bind very weakly to the M. jalapa ATG8s. Through a combination of binding assays and structural modelling, we showed that the identity of the residue at the position of the PmPexRD54 AIM polymorphism can underpin high-affinity binding to plant ATG8s. Finally, we conclude that the functionality of the PexRD54 AIM was lost in the P. mirabilis lineage, perhaps owing to as-yet-unknown pressure on this effector in the new host environment. Author SummaryPathogens evolve in concert with their hosts. When a pathogen begins to infect a new host species, known as a "host jump," the pathogen must evolve to enhance infection and transmission. These evolutionary processes can involve both the gain and loss of genes, as well as dynamic changes in protein function. Here, we describe an example of a pathogen protein that lost a key functional domain following a host jump, a salient example of "regressive evolution." Specifically, we show that an effector protein from the plant pathogen Phytopthora mirabilis, a host-specific lineage closely related to the Irish potato famine pathogen Phytopthora infestans, has a derived amino acid polymorphism that results in a loss of interaction with certain host machinery.

plant biology↗

bioRxiv2018

N-terminal β-strand underpins biochemical specialization of an ATG8 isoform

ATG8 is a highly-conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and numerous proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins remains unclear. Here, we describe a comprehensive protein-protein interaction resource, obtained using in planta immunoprecipitation followed by mass spectrometry, to define the potato ATG8 interactome. We discovered that ATG8 isoforms bind distinct sets of plant proteins with varying degrees of overlap. This prompted us to define the biochemical basis of ATG8 specialization by comparing two potato ATG8 isoforms using both in vivo protein interaction assays and in vitro quantitative binding affinity analyses. These experiments revealed that the N-terminal {beta}-strand--and, in particular, a single amino acid polymorphism--underpins binding specificity to the substrate PexRD54 by shaping the hydrophobic pocket that accommodates this proteins ATG8 interacting motif. Additional proteomics experiments indicated that the N-terminal {beta}-strand shapes the ATG8 interactor profiles, defining interaction specificity with about 80 plant proteins. Our findings are consistent with the view that ATG8 isoforms comprise a layer of specificity in the regulation of selective autophagy pathways in plants.

plant biology↗

bioRxiv2018

The coming of age of EvoMPMI: evolutionary molecular plant-microbe interactions across multiple timescales

Plant-microbe interactions are great model systems to study co-evolutionary dynamics across multiple timescales, ranging from multimillion year macroevolution to extremely rapid evolutionary adaptations. However, mechanistic research on plant-microbe interactions has often been conducted with little consideration of the insights that can be gained from evolutionary concepts and methods. Conversely, evolutionary research has rarely integrated the diverse range of molecular mechanisms and models that continue to emerge from the molecular plant-microbe interactions field. These trends are changing. In recent years, the incipient field of evolutionary molecular plant-microbe interactions (EvoMPMI) has emerged to bridge the gap between mechanistic molecular research and evolutionary approaches. Here, we report on recent advances in EvoMPMI. In particular, we highlight new systems to study microbe interactions with early diverging land plants, and new findings from studies of adaptive evolution in pathogens and plants. By linking mechanistic and evolutionary research, EvoMPMI promises to add a new dimension to our understanding of plant-microbe interactions.

plant biology↗

bioRxiv2017

Lessons in effector and NLR biology of plant-microbe systems

A diversity of plant-associated organisms secrete effectors--proteins and metabolites that modulate plant physiology to favor host infection and colonization. However, effectors can also activate plant immune receptors, notably nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins, enabling plants to fight off invading organisms. This interplay between effectors, their host targets, and the matching immune receptors is shaped by intricate molecular mechanisms and exceptionally dynamic coevolution. In this article, we focus on three effectors, AVR-Pik, AVR-Pia, and AVR-Pii, from the rice blast fungus Magnaporthe oryzae (syn. Pyricularia oryzae), and their corresponding rice NLR immune receptors, Pik, Pia, and Pii, to highlight general concepts of plant-microbe interactions. We draw 12 lessons in effector and NLR biology that have emerged from studying these three little effectors and are broadly applicable to other plant-microbe systems.

plant biology↗