Biology subjects

Seong, K.

Publications and source records attributed to Seong, K..

10 recordsLinked to original sources

bioRxiv2025

The Annotated Blueprint: Integrated Functional Genomic Resources for a Model Tetraploid Wheat Triticum turgidum cv. Kronos

Triticum turgidum cv. Kronos is a tetraploid wheat cultivar that underpins one of the richest community platforms for functional genomics. Over the past decade, about 3,000 exome- and promoter-capture datasets, linked to mutagenized seed stocks, and transcriptomic and phenotypic resources have accumulated, yet the absence of a reference genome has constrained their impact. Here, we present a chromosome-scale reference genome of Kronos with high-confidence annotations, including manual curation of over 1,000 disease resistance (NLR) genes. This reference revealed previously hidden NLR diversity and clarified their genomic organization at chromosomal ends. Re-analysis of exome- and promoter-capture datasets enabled high-resolution mutation discovery in genes and regulatory regions that were previously inaccessible, uncovering the full standing variation present in Kronos mutant lines. We further re-curated transcriptomic and small RNA datasets, generating improved, genome-wide maps of microRNAs and phasiRNAs important for wheat development. Collectively, these resources elevate Kronos to reference quality and establish it as a versatile platform for functional and translational wheat research.

plant biology↗

bioRxiv2024

Engineering the plant intracellular immune receptor Sr50 to restore recognition of the AvrSr50 escape mutant

Pathogen-driven plant diseases cause significant crop losses worldwide. The introgression of intracellular nucleotide-binding leucine-rich repeat receptor (NLR) genes into elite crop cultivars is a common strategy for disease control, yet pathogens rapidly evolve to evade NLR-mediated immunity. The NLR gene Sr50 protects wheat against stem rust, a devastating disease caused by the fungal pathogen Puccinia graminis f. sp. tritici (Pgt). However, mutations in AvrSr50 allowed Pgt to evade Sr50 recognition, leading to resistance breakdown. Advances in protein structure modeling can enable targeted NLR engineering to restore recognition of escaped effectors. Here, we combined iterative computational structural analyses and site-directed mutagenesis to engineer Sr50 recognition of AvrSr50QCMJC, a Pgt effector variant that evades wild-type Sr50 detection. Derived by molecular docking, our initial structural model identified the K711D substitution in Sr50, which partially restored AvrSr50QCMJC recognition. Enhancing Sr50K711D expression via strong promoters compensated for weak recognition and restored robust immune responses. Further structural refinements led to the generation of five double and two triple receptor mutants. These engineered mutants, absent in nature, showed robust dual recognition for AvrSr50 and AvrSr50QCMJC in both Nicotiana benthamiana and wheat protoplasts. Notably, the K711D substitution was essential and synergistic with the additional substitutions for AvrSr50QCMJC recognition, demonstrating protein epistasis. Furthermore, this single substitution altered AlphaFold 2 predictions, enabling accurate modeling of the Sr50K711D-AvrSr50 complex structure, consistent with our final structural hypothesis. Collectively, this study outlines a framework for NLR engineering to counteract pathogen adaptation and provides novel Sr50 variants with potential for stem rust resistance.

plant biology↗

bioRxiv2024

Wheat Enhanced Disease Resistance EMS-Mutants Include Lesion-mimics With Adult Plant Resistance to Stripe Rust

Tetraploid durum wheat Triticum turgidum subsp. durum cv Kronos has extensive genetic variation resources, including a sequenced and cataloged ethyl methanesulfonate (EMS) mutagenized population. To utilize this allelic diversity, we screened over 2,000 mutant lines and identified over 30 enhanced disease resistance (EDR) mutants in a forward genetic field screen against stripe rust. Sixteen of the EDR lines have persistent resistance to stripe rust after four years, and several mutants showed differential disease responses against other fungal pathogens, indicating that the lines possess diverse alleles that affect multiple routes of pathogen suppression. Five of these 16 lines showed spontaneous lesion formation in the absence of pathogens. Only one showed a reduction in kernel weight under pathogen pressure, a testimony to the high mutational density that wheat can tolerate. Phenotypic selection for resistance at the adult stage identified useful EMS alleles for stripe rust resistance. The mutations in the 16 EDR lines were newly mapped to a recently released long-read Kronos genome to enhance their utility in molecular breeding for fungal resistance and for fundamental studies of plant-pathogen interactions.

plant biology↗

bioRxiv2024

An array of Zymoseptoria tritici effectors suppress plant immune responses

Zymoseptoria tritici is the most economically significant fungal pathogen of wheat in Europe. However, despite the importance of this pathogen, the molecular interactions between pathogen and host during infection are not well understood. Herein, we describe the use of two libraries of cloned Z. tritici effectors that were screened to identify effector candidates with putative pathogen associated molecular pattern (PAMP) triggered immunity (PTI)-suppressing activity. The effectors from each library were transiently expressed in Nicotiana benthamiana, and expressing leaves were treated with bacterial or fungal PAMPs to assess the effectors ability to suppress reactive oxygen species (ROS) production. From these screens, numerous effectors were identified with PTI-suppressing activity. In addition, some effectors were able to suppress cell death responses induced by other Z. tritici secreted proteins. We used structural prediction tools to predict the putative structures of all of the Z. tritici effectors, and used these predictions to examine whether there was enrichment of specific structural signatures among the PTI-suppressing effectors. From among the libraries, multiple members of the killer protein-like 4 (KP4) and killer protein-like 6 (KP6) effector families were identified as PTI-suppressors. This observation is intriguing, as these protein families were previously associated with antimicrobial activity rather than virulence or host manipulation. This data provides mechanistic insight into immune suppression by Z. tritici during infection, and suggests that similar to biotrophic pathogens, this fungus relies on a battery of secreted effectors to suppress host immunity during early phases of colonisation.

plant biology↗

bioRxiv2022

Comparative computational structural genomics highlights divergent evolution of fungal effectors

Elucidating the evolution of pathogen effector molecules is critical to understand infection mechanisms of fungal phytopathogens and ensure crop security. However, rapid diversification that diminishes sequence similarities between homologous effectors has largely concealed the roots of effector evolution. We predicted the folds of 26,653 secreted proteins from 21 species with AlphaFold and performed structure-guided comparative analyses on two aspects of effector evolution: uniquely expanded effector families and common folds present across the fungal species. Extreme expansion of nearly lineage-specific effector families was found only in several obligate biotrophs, Blumeria graminis and Puccinia graminis. The highly expanded effector families were the source of conserved sequence motifs, such as the Y/F/WxC motif. We identified additional lineage-specific effector families that include known (a)virulence factors, such as AvrSr35, AvrSr50 and Tin2. These families represented new classes of sequence-unrelated structurally similar effectors. Structural comparisons revealed that the expanded structural folds further diversify through domain duplications and fusion with disordered stretches. Sub-and neo-functionalized structurally similar effectors reconverge on regulation, expanding the functional pools of effectors in the pathogen infection cycle. We also found evidence that many effector families could have originated from ancestral folds that have been conserved across fungi. Collectively, our study highlights diverse mechanisms of effector evolution and supports divergent evolution of effectors that leads to emergence of pathogen virulence from ancestral proteins.

microbiology↗

bioRxiv2022

Engineering of Sr33 and Sr50 plant immune receptors to alter recognition specificity and autoactivity

Plants possess cytoplasmic immune receptors called nucleotide-binding leucine-rich repeat receptors (NLRs) that recognize the presence of a pathogen through a range of mechanisms: direct binding of effectors or indirect recognition of effector actions. The direct binding of effectors has been shown to be mediated through the NLRs leucine-rich repeat (LRR) domain. Accurate prediction of amino acids involved in these direct interactions can greatly enhance understanding of effector recognition and inform efforts to engineer new resistance. In this study, we utilized two homologous NLR resistance genes from wheat, Sr33 and Sr50, that recognize distinct effectors by directly binding to them through their LRR domain. While the effector recognized by Sr50 is known and described as AvrSr50, the effector recognized by Sr33 remains unknown. Through a combination of phylogenetics, allele diversity analysis in the LRR and structural modeling, we identified the amino acids in Sr50 likely to physically interact with its effector. Mutation of these sites helped identify 12 amino acids we hypothesized to be sufficient to mediate effector binding in Sr50. Changing these 12 corresponding amino acids in Sr33, we showed AvrSr50-dependent initiation of cell death in wheat protoplasts and Nicotiana benthamiana. Furthermore, we were able to pinpoint and change amino acid residues that govern autoactivity of Sr50 in the wheat protoplast cell death assay. These findings are a major advance towards the successful engineering of new effector recognition specificities in direct binder NLRs.

plant biology↗

bioRxiv2022

A draft genome assembly for the heterozygous wild tomato Solanum habrochaites highlights haplotypic structural variations of intracellular immune receptors

Solanum habrochaites LA1353 is a self-incompatible, highly heterozygous wild tomato that is a useful germplasm resource for the study of metabolism, reproduction and disease resistance. We generated a draft genome assembly with PacBio HiFi reads and genome annotations, which underscored the expansion of gene families associated with metabolite-production, self-incompatibility, DNA regulation and immunity. After manually curating intracellular nucleotide-binding leucine-rich repeat immune receptors (NLRs), we found that S. habrochaites LA1353 has a larger NLR inventory than other wild tomato species. A great number of heterozygous local copy number variations (CNVs) driven by haplotypic structural variations further expands the inventory, both enhancing NLR diversity and providing more opportunities for sequence evolution. The NLRs associated with local CNVs predominantly appear in the helper NLR (NRC)-related phylogenetic clades and are concentrated in a few physical NLR gene clusters. Synteny analysis points out that these genomic regions correspond to the known NLR clusters from which experimentally validated, functional NLRs, such as Hero, Mi-1.2 and Rpi-amr1, have been identified. Producing and incorporating Resistance Gene Enrichment Sequencing (RenSeq) data across wild tomato species, we reveal that the regions with local CNVs might have been shaped nearly equally by recent NLR gains and losses, along with enhanced sequence diversification that diminishes one-to-one orthology between heterozygous alleles. Our analysis suggests that these genomic regions may have accelerated evolutionary dynamics for NLR diversity generation in S. habrochaites LA1353.

plant biology↗

bioRxiv2021

CRISPR/Cas9 knockout of EPFL10 reduces stomatal density while maintaining photosynthesis and enhancing water conservation in rice

Rice (Oryza sativa) is of paramount importance for global nutrition, supplying at least 20% of global calories. However, water scarcity and increased drought severity are anticipated to reduce rice yields globally. Rice stomatal developmental genetics were explored as a mechanism to improve drought resilience while maintaining yield under climate stress. CRISPR/Cas9-mediated knockouts of EPFL10 and STOMAGEN yielded lines with c. 80% and 25% of wild-type stomatal density, respectively. epfl10 lines with moderate reductions in stomatal densities are able to conserve water to similar extents as stomagen lines, but do not suffer from the concomitant reductions in stomatal conductance, carbon assimilation, or thermoregulation observed in stomagen knockouts. Moderate reductions in stomatal densities achieved by editing EPFL10 may be a climate-adaptive approach in rice that can safeguard yield. Editing the paralog of STOMAGEN in other species may provide a means to tune stomatal density in agriculturally important crops beyond rice.

plant biology↗

bioRxiv2021

Computational structural genomics unravels common folds and predicted functions in the secretome of fungal phytopathogen Magnaporthe oryzae

Magnaporthe oryzae relies on a diverse collection of secreted effector proteins to reprogram the host metabolic and immune responses for the pathogens benefit. Characterization of the effectors is thus critical for understanding the biology and host infection mechanisms of this phytopathogen. In rapid, divergent effector evolution, structural information has the potential to illuminate the unknown aspects of effectors that sequence analyses alone cannot reveal. It has recently become feasible to reliably predict the protein structures without depending on homologous templates. In this study, we tested structure modeling on 1854 secreted proteins from M. oryzae and evaluated success and obstacles involved in effector structure prediction. With sensitive homology search and structure-based clustering, we defined both distantly related homologous groups and structurally related analogous groups. With this dataset, we propose sequence-unrelated, structurally similar effectors are a common theme in M. oryzae and possibly in other phytopathogens. We incorporated the predicted models for structure-based annotations, molecular docking and evolutionary analyses to demonstrate how the predicted structures can deepen our understanding of effector biology. We also provide new experimentally testable structure-derived hypotheses of effector functions. Collectively, we propose that computational structural genomic approaches can now be an integral part of studying effector biology and provide valuable resources that were inaccessible before the advent of reliable, machine learning-based structure prediction.

microbiology↗

bioRxiv2019

Evolution of NLR resistance genes with non-canonical N-terminal domains in wild tomato species

BackgroundNucleotide-binding and leucine-rich repeat immune receptors (NLRs) are an important component of plant immunity that provides resistance against diverse pathogens. NLRs often exist as large gene families, the members of which display diverse multi-domain architectures (MDAs) and evolve through various mechanisms of duplications and selections.\n\nResultsWe conducted resistance gene enrichment sequencing (RenSeq) with single-molecule real time (SMRT) sequencing of PacBio for 18 accessions in Solanaceae including 15 wild tomatoes. We demonstrate what was previously known as Solanaceae Domain (SD) not only is more diverse in structure and function but also far anciently originated from the most recent common ancestor (MRCA) between Asterids and Amaranthaceae. In tomato, NLRs with the extended N-terminus displayed distinct patterns of evolution based on phylogenetic clades by proliferation, continuous elongation and domain losses.\n\nConclusionsOur study provides high quality gene models of NLRs that can serve as resources for future studies for crop engineering and elucidates greater evolutionary dynamics of the extended NLRs than previously assumed.

plant biology↗