Kyungyong Seong

Summary: Triticum turgidum cv Kronos is a tetraploid wheat cultivar that underpins one of the most widely used community platforms for functional genomics. Over the past decade, researchers have generated c . 3000 exome‐capture (EC) and promoter‐capture (PC) datasets linked to mutagenized seed stocks, along with extensive transcriptomic and phenotypic resources. However, the absence of a reference genome has constrained their full utility. We assembled a chromosome‐scale reference genome for Kronos, with high‐confidence annotations, including manual curation of over 1000 disease resistance (nucleotide‐binding leucine‐rich repeat (NLR)) genes and genome‐wide identification of microRNAs and phasiRNAs. We additionally reanalyzed EC and PC data to capture mutational landscapes across ethyl methane sulphonate‐mutagenized Kronos populations. We revealed previously hidden NLR diversity and resolved their genomic organization at chromosomal ends. Re‐analysis of capture datasets enabled high‐resolution mutation discovery in genes and regulatory regions, providing a more comprehensive view of the variations detectable in the Kronos mutant populations. Collectively, these resources provide a reference‐quality genomic framework for Kronos and position it as a versatile platform for functional and translational wheat research.

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.

Introducing and characterizing variation through mutagenesis plus functional genomics can accelerate resistance breeding as well as our understanding of crop plant immunity. To reveal new germplasm resources for fungal disease resistance breeding in elite durum wheat, we challenged the diverse alleles in a sequenced and cataloged ethyl methanesulfonate mutagenized population of elite tetraploid wheat Triticum turgidum subsp. durum cv ‘Kronos’ with stripe rust. We screened 2,000 mutant lines and identified sixteen enhanced disease resistance (EDR) lines with persistent resistance to stripe rust over four years of field testing. To find broad-spectrum resistance, we challenged these lines with other major biotrophic and necrotrophic pathogens, including those causing Septoria tritici blotch, tan spot, Fusarium head blight and leaf rust. Enhanced resistance to multiple fungi was found in 13 of 16 EDR lines. Five EDR lines showed spontaneous lesion formation in the absence of pathogens, providing new mutant resources to study plant stress response in the absence of the confounding effects of pathogen infection. We mapped exome capture sequencing data of the EDR lines to a recently released long-read Kronos genome to aid in the identification of causal mutations. We located an EDR resistance locus to an 175 Mb interval on chromosome 1B. Importantly, these phenotypically characterized EDR lines are newly described durum germplasm coupled with improved functional genomics resources that are readily available for both wheat fungal resistance breeding and basic plant immunity research.

Plants rely on germline-encoded, innate immune receptors to sense pathogens and initiate the defense response. The exponential increase in quality and quantity of genomes, RNA-seq datasets, and protein structures has underscored the incredible biodiversity of plant immunity. Arabidopsis continues to serve as a valuable model and theoretical foundation of our understanding of wild plant diversity of immune receptors, while expansion of study into agricultural crops has also revealed distinct evolutionary trajectories and challenges. Here, we provide the classical context for study of both intracellular nucleotide-binding, leucine-rich repeat receptors and surface-localized pattern recognition receptors at the levels of DNA sequences, transcriptional regulation, and protein structures. We then examine how recent technology has shaped our understanding of immune receptor evolution and informed our ability to efficiently engineer resistance. We summarize current literature and provide an outlook on how researchers take inspiration from natural diversity in bioengineering efforts for disease resistance from Arabidopsis and other model systems to crops.

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 AvrSr50 ^QCMJC , 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 AvrSr50 ^QCMJC recognition. Enhancing Sr50 ^K711D 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 AvrSr50 ^QCMJC in both Nicotiana benthamiana and wheat protoplasts. Notably, the K711D substitution was essential and synergistic with the additional substitutions for AvrSr50 ^QCMJC recognition, demonstrating protein epistasis. Furthermore, this single substitution altered AlphaFold 2 predictions, enabling accurate modeling of the Sr50 ^K711D –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.

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.

Fungal pathogens deploy a set of molecules (proteins, specialized metabolites, and sRNAs), so-called effectors, to aid the infection process. In comparison to other plant pathogens, smut fungi have small genomes and secretomes of 20 Mb and around 500 proteins, respectively. Previous comparative genomic studies have shown that many secreted effector proteins without known domains, i.e., novel, are conserved only in the Ustilaginaceae family. By analyzing the secretomes of 11 species within Ustilaginaceae, we identified 53 core homologous groups commonly present in this lineage. By collecting existing mutants and generating additional ones, we gathered 44 Ustilago maydis strains lacking single core effectors as well as 9 strains containing multiple deletions of core effector gene families. Pathogenicity assays revealed that 20 of these 53 mutant strains were affected in virulence. Among the 33 mutants that had no obvious phenotypic changes, 13 carried additional, sequence-divergent, structurally similar paralogs. We report a virulence contribution of seven previously uncharacterized single core effectors and of one effector family. Our results help to prioritize effectors for understanding U. maydis virulence and provide genetic resources for further characterization.[ ] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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. We explored stomatal developmental genetics as a mechanism for improving drought resilience in rice while maintaining yield under climate stress. CRISPR/Cas9-mediated knockouts of the positive regulator of stomatal development STOMAGEN and its paralog EPIDERMAL PATTERNING FACTOR-LIKE10 (EPFL10) yielded lines with ∼25% and 80% of wild-type stomatal density, respectively. epfl10 lines with moderate reductions in stomatal density were able to conserve water to similar extents as stomagen lines but did not suffer from the concomitant reductions in stomatal conductance, carbon assimilation, or thermoregulation observed in stomagen knockouts. Moderate reductions in stomatal density achieved by editing EPFL10 present a climate-adaptive approach for safeguarding yield in rice. Editing the paralog of STOMAGEN in other species may provide a means for tuning stomatal density in agriculturally important crops beyond rice.

Elucidating the similarity and diversity of pathogen effectors is critical to understand their evolution across fungal phytopathogens. However, rapid divergence that diminishes sequence similarities between putatively homologous effectors has largely concealed the roots of effector evolution. Here we modelled the structures of 26,653 secreted proteins from 14 agriculturally important fungal phytopathogens, six non-pathogenic fungi and one oomycete with AlphaFold 2. With 18,000 successfully predicted folds, we performed structure-guided comparative analyses on two aspects of effector evolution: uniquely expanded sequence-unrelated structurally similar (SUSS) effector families and common folds present across the fungal species. Extreme expansion of lineage-specific SUSS effector families was found only in several obligate biotrophs,Blumeria graminisandPuccinia graminis. The highly expanded effector families were the source of conserved sequence motifs, such as the Y/F/WxC motif. We identified new classes of SUSS effector families that include known virulence factors, such as AvrSr35, AvrSr50 and Tin2. Structural comparisons revealed that the expanded structural folds further diversify through domain duplications and fusion with disordered stretches. Putatively sub- and neo-functionalized SUSS effectors could 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 conserved across fungi. Collectively, our study highlights diverse effector evolution mechanisms and supports divergent evolution as a major force in driving SUSS effector evolution from ancestral proteins.