High allelic diversity in Arabidopsis NLRs is associated with distinct genomic features.

Plants rely on Nucleotide-binding, Leucine-rich repeat Receptors (NLRs) for pathogen recognition. Highly variable NLRs (hvNLRs) show remarkable intraspecies diversity, while their low-variability paralogs (non-hvNLRs) are conserved between ecotypes. At a population level, hvNLRs provide new pathogen-recognition specificities, but the association between allelic diversity and genomic and epigenomic features has not been established. Our investigation of NLRs in Arabidopsis Col-0 has revealed that hvNLRs show higher expression, less gene body cytosine methylation, and closer proximity to transposable elements than non-hvNLRs. hvNLRs show elevated synonymous and nonsynonymous nucleotide diversity and are in chromatin states associated with an increased probability of mutation. Diversifying selection maintains variability at a subset of codons of hvNLRs, while purifying selection maintains conservation at non-hvNLRs. How these features are established and maintained, and whether they contribute to the observed diversity of hvNLRs is key to understanding the evolution of plant innate immune receptors.

Multiple wheat genomes reveal global variation in modern breeding.

Advances in genomics have expedited the improvement of several agriculturally important crops but similar efforts in wheat (Triticum spp.) have been more challenging. This is largely owing to the size and complexity of the wheat genome1, and the lack of genome-assembly data for multiple wheat lines2,3. Here we generated ten chromosome pseudomolecule and five scaffold assemblies of hexaploid wheat to explore the genomic diversity among wheat lines from global breeding programs. Comparative analysis revealed extensive structural rearrangements, introgressions from wild relatives and differences in gene content resulting from complex breeding histories aimed at improving adaptation to diverse environments, grain yield and quality, and resistance to stresses4,5. We provide examples outlining the utility of these genomes, including a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm16, a gene associated with insect resistance. These genome assemblies will provide a basis for functional gene discovery and breeding to deliver the next generation of modern wheat cultivars.

Majority of the highly variable NLRs in maize share genomic location and contain additional target-binding domains.

Nucleotide-binding, Leucine Rich Repeat proteins (NLRs) are a major class of immune receptors in plants. NLRs include both conserved and rapidly evolving members, however their evolutionary trajectory in crops remains understudied. Availability of crop pan-genomes enables analysis of the recent events in the evolution of this highly complex gene family within domesticated species. Here, we investigated the NLR complement of 26 nested association mapping (NAM) founder lines of maize. We found that maize has just four main subfamilies containing rapidly evolving highly variable NLR (hvNLR) receptors. Curiously, three of these phylogenetically distinct hvNLR lineages are located in adjacent clusters on chromosome 10. Members of the same hvNLR clade show variable expression and methylation across lines and tissues, consistent with their rapid evolution. By combining sequence diversity analysis and AlphaFold2 computational structure prediction we predicted ligand binding sites in the hvNLRs. We also observed novel insertion domains in the LRR regions of two hvNLR subfamilies that likely contribute to target recogniton. To make this analysis accessible, we created NLRCladeFinder, a Google Colaboratory notebook, that accepts any newly identified NLR sequence, places it in the evolutionary context of the maize pan-NLRome, and provides an updated clade alignment, phylogenetic tree, and sequence diversity information for the gene of interest.

Novel Secreted Effectors Conserved Among Smut Fungi Contribute to the Virulence of Ustilago maydis.

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. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome and time-of-day transcriptome of Wolffia australiana link morphological minimization with gene loss and less growth control.

Rootless plants in the genus Wolffia are some of the fastest growing known plants on Earth. Wolffia have a reduced body plan, primarily multiplying through a budding type of asexual reproduction. Here, we generated draft reference genomes for Wolffia australiana (Benth.) Hartog & Plas, which has the smallest genome size in the genus at 357 Mb and has a reduced set of predicted protein-coding genes at about 15,000. Comparison between multiple high-quality draft genome sequences from W. australiana clones confirmed loss of several hundred genes that are highly conserved among flowering plants, including genes involved in root developmental and light signaling pathways. Wolffia has also lost most of the conserved nucleotide-binding leucine-rich repeat (NLR) genes that are known to be involved in innate immunity, as well as those involved in terpene biosynthesis, while having a significant overrepresentation of genes in the sphingolipid pathways that may signify an alternative defense system. Diurnal expression analysis revealed that only 13% of Wolffia genes are expressed in a time-of-day (TOD) fashion, which is less than the typical ∼40% found in several model plants under the same condition. In contrast to the model plants Arabidopsis and rice, many of the pathways associated with multicellular and developmental processes are not under TOD control in W. australiana, where genes that cycle the conditions tested predominantly have carbon processing and chloroplast-related functions. The Wolffia genome and TOD expression data set thus provide insight into the interplay between a streamlined plant body plan and optimized growth.

Variation in plant Toll/Interleukin-1 receptor domain protein dependence on ENHANCED DISEASE SUSCEPTIBILITY 1.

Toll/Interleukin-1 receptor (TIR) domains are integral to immune systems across all kingdoms. In plants, TIRs are present in nucleotide-binding leucine-rich repeat (NLR) immune receptors, NLR-like, and TIR-only proteins. Although TIR-NLR and TIR signaling in plants require the ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) protein family, TIRs persist in species that have no EDS1 members. To assess whether particular TIR groups evolved with EDS1, we searched for TIR-EDS1 co-occurrence patterns. Using a large-scale phylogenetic analysis of TIR domains from 39 algal and land plant species, we identified 4 TIR families that are shared by several plant orders. One group occurred in TIR-NLRs of eudicots and another in TIR-NLRs across eudicots and magnoliids. Two further groups were more widespread. A conserved TIR-only group co-occurred with EDS1 and members of this group elicit EDS1-dependent cell death. In contrast, a maize (Zea mays) representative of TIR proteins with tetratricopeptide repeats was also present in species without EDS1 and induced EDS1-independent cell death. Our data provide a phylogeny-based plant TIR classification and identify TIRs that appear to have evolved with and are dependent on EDS1, while others have EDS1-independent activity.

Analysis of intraspecies diversity reveals a subset of highly variable plant immune receptors and predicts their binding sites.

The evolution of recognition specificities by the immune system depends on the generation of receptor diversity and on connecting the binding of new antigens with the initiation of downstream signaling. In plant immunity, the innate Nucleotide-Binding Leucine-Rich Repeat (NLR) receptor family enables antigen binding and immune signaling. In this study, we surveyed the NLR complements of 62 ecotypes of Arabidopsis thaliana and 54 lines of Brachypodium distachyon and identified a limited number of NLR subfamilies that show high allelic diversity. We show that the predicted specificity-determining residues cluster on the surfaces of Leucine-Rich Repeat domains, but the locations of the clusters vary among NLR subfamilies. By comparing NLR phylogeny, allelic diversity, and known functions of the Arabidopsis NLRs, we formulate a hypothesis for the emergence of direct and indirect pathogen-sensing receptors and of the autoimmune NLRs. These findings reveal the recurring patterns of evolution of innate immunity and can inform NLR engineering efforts.

Altering Specificity and Autoactivity of Plant Immune Receptors Sr33 and Sr50 Via a Rational Engineering Approach.

Many resistance genes deployed against pathogens in crops are intracellular nucleotide-binding (NB) leucine-rich repeat (LRR) receptors (NLRs). The ability to rationally engineer the specificity of NLRs will be crucial in the response to newly emerging crop diseases. Successful attempts to modify NLR recognition have been limited to untargeted approaches or depended on previously available structural information or knowledge of pathogen-effector targets. However, this information is not available for most NLR-effector pairs. Here, we demonstrate the precise prediction and subsequent transfer of residues involved in effector recognition between two closely related NLRs without their experimentally determined structure or detailed knowledge about their pathogen effector targets. By combining phylogenetics, allele diversity analysis, and structural modeling, we successfully predicted residues mediating interaction of Sr50 with its cognate effector AvrSr50 and transferred recognition specificity of Sr50 to the closely related NLR Sr33. We created synthetic versions of Sr33 that contain amino acids from Sr50, including Sr33syn, which gained the ability to recognize AvrSr50 with 12 amino-acid substitutions. Furthermore, we discovered that sites in the LRR domain needed to transfer recognition specificity to Sr33 also influence autoactivity in Sr50. Structural modeling suggests these residues interact with a part of the NB-ARC domain, which we named the NB-ARC latch, to possibly maintain the inactive state of the receptor. Our approach demonstrates rational modifications of NLRs, which could be useful to enhance existing elite crop germplasm. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Convergent Loss of an EDS1/PAD4 Signaling Pathway in Several Plant Lineages Reveals Coevolved Components of Plant Immunity and Drought Response.

Plant innate immunity relies on nucleotide binding leucine-rich repeat receptors (NLRs) that recognize pathogen-derived molecules and activate downstream signaling pathways. We analyzed the variation in NLR gene copy number and identified plants with a low number of NLR genes relative to sister species. We specifically focused on four plants from two distinct lineages, one monocot lineage (Alismatales) and one eudicot lineage (Lentibulariaceae). In these lineages, the loss of NLR genes coincides with loss of the well-known downstream immune signaling complex ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1)/PHYTOALEXIN DEFICIENT 4 (PAD4). We expanded our analysis across whole proteomes and found that other characterized immune genes were absent only in Lentibulariaceae and Alismatales. Additionally, we identified genes of unknown function that were convergently lost together with EDS1/PAD4 in five plant species. Gene expression analyses in Arabidopsis (Arabidopsis thaliana) and Oryza sativa revealed that several homologs of the candidates are differentially expressed during pathogen infection, drought, and abscisic acid treatment. Our analysis provides evolutionary evidence for the rewiring of plant immunity in some plant lineages, as well as the coevolution of the EDS1/PAD4 pathway and drought responses.

Genomic innovation for crop improvement.

Crop production needs to increase to secure future food supplies, while reducing its impact on ecosystems. Detailed characterization of plant genomes and genetic diversity is crucial for meeting these challenges. Advances in genome sequencing and assembly are being used to access the large and complex genomes of crops and their wild relatives. These have helped to identify a wide spectrum of genetic variation and permitted the association of genetic diversity with diverse agronomic phenotypes. In combination with improved and automated phenotyping assays and functional genomic studies, genomics is providing new foundations for crop-breeding systems.

The extrachromosomal circular DNAs of the rice blast pathogen Magnaporthe oryzae contain a wide variety of LTR retrotransposons, genes, and effectors.

BACKGROUND: One of the ways genomes respond to stress is by producing extrachromosomal circular DNAs (eccDNAs). EccDNAs can contain genes and dramatically increase their copy number. They can also reinsert into the genome, generating structural variation. They have been shown to provide a source of phenotypic and genotypic plasticity in several species. However, whole circularome studies have so far been limited to a few model organisms. Fungal plant pathogens are a serious threat to global food security in part because of their rapid adaptation to disease prevention strategies. Understanding the mechanisms fungal pathogens use to escape disease control is paramount to curbing their threat. RESULTS: We present a whole circularome sequencing study of the rice blast pathogen, Magnaporthe oryzae. We find that M. oryzae has a highly diverse circularome that contains many genes and shows evidence of large LTR retrotransposon activity. We find that genes enriched on eccDNAs in M. oryzae occur in genomic regions prone to presence-absence variation and that disease-associated genes are frequently on eccDNAs. Finally, we find that a subset of genes is never present on eccDNAs in our data, which indicates that the presence of these genes on eccDNAs is selected against. CONCLUSIONS: Our study paves the way to understanding how eccDNAs contribute to adaptation in M. oryzae. Our analysis also reveals how M. oryzae eccDNAs differ from those of other species and highlights the need for further comparative characterization of eccDNAs across species to gain a better understanding of these molecules.

Characterization of defense responses against bacterial pathogens in duckweeds lacking EDS1.

ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) mediates the induction of defense responses against pathogens in most angiosperms. However, it has recently been shown that a few species have lost EDS1. It is unknown how defense against disease unfolds and evolves in the absence of EDS1. We utilize duckweeds; a collection of aquatic species that lack EDS1, to investigate this question. We established duckweed-Pseudomonas pathosystems and used growth curves and microscopy to characterize pathogen-induced responses. Through comparative genomics and transcriptomics, we show that the copy number of infection-associated genes and the infection-induced transcriptional responses of duckweeds differ from other model species. Pathogen defense in duckweeds has evolved along different trajectories than in other plants, including genomic and transcriptional reprogramming. Specifically, the miAMP1 domain-containing proteins, which are absent in Arabidopsis, showed pathogen responsive upregulation in duckweeds. Despite such divergence between Arabidopsis and duckweed species, we found conservation of upregulation of certain genes and the role of hormones in response to disease. Our work highlights the importance of expanding the pool of model species to study defense responses that have evolved in the plant kingdom independent of EDS1.

Computational Structural Genomics Unravels Common Folds and Novel Families in the Secretome of Fungal Phytopathogen Magnaporthe oryzae.

Structural biology has the potential to illuminate the evolution of pathogen effectors and their commonalities that cannot be readily detected at the primary sequence level. Recent breakthroughs in protein structure modeling have demonstrated the feasibility to predict the protein folds without depending on homologous templates. These advances enabled a genome-wide computational structural biology approach to help understand proteins based on their predicted folds. In this study, we employed structure prediction methods on the secretome of the destructive fungal pathogen Magnaporthe oryzae. Out of 1,854 secreted proteins, we predicted the folds of 1,295 proteins (70%). We showed that template-free modeling by TrRosetta captured 514 folds missed by homology modeling, including many known effectors and virulence factors, and that TrRosetta generally produced higher quality models for secreted proteins. Along with sensitive homology search, we employed structure-based clustering, defining not only homologous groups with divergent members but also sequence-unrelated structurally analogous groups. We demonstrate that this approach can reveal new putative members of structurally similar MAX effectors and novel analogous effector families present in M. oryzae and possibly in other phytopathogens. We also investigated the evolution of expanded putative ADP-ribose transferases with predicted structures. We suggest that the loss of catalytic activities of the enzymes might have led them to new evolutionary trajectories to be specialized as protein binders. Collectively, we propose that computational structural genomics approaches can be an integral part of studying effector biology and provide valuable resources that were inaccessible before the advent of machine learning-based structure prediction.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The NLR-Annotator Tool Enables Annotation of the Intracellular Immune Receptor Repertoire.

Disease resistance genes encoding nucleotide-binding and leucine-rich repeat (NLR) intracellular immune receptor proteins detect pathogens by the presence of pathogen effectors. Plant genomes typically contain hundreds of NLR-encoding genes. The availability of the hexaploid wheat (Triticum aestivum) cultivar Chinese Spring reference genome allows a detailed study of its NLR complement. However, low NLR expression and high intrafamily sequence homology hinder their accurate annotation. Here, we developed NLR-Annotator, a software tool for in silico NLR identification independent of transcript support. Although developed for wheat, we demonstrate the universal applicability of NLR-Annotator across diverse plant taxa. We applied our tool to wheat and combined it with a transcript-validated subset of genes from the reference gene annotation to characterize the structure, phylogeny, and expression profile of the NLR gene family. We detected 3,400 full-length NLR loci, of which 1,560 were confirmed as expressed genes with intact open reading frames. NLRs with integrated domains mostly group in specific subclades. Members of another subclade predominantly locate in close physical proximity to NLRs carrying integrated domains, suggesting a paired helper function. Most NLRs (88%) display low basal expression (in the lower 10 percentile of transcripts). In young leaves subjected to biotic stress, we found up-regulation of 266 of the NLRs To illustrate the utility of our tool for the positional cloning of resistance genes, we estimated the number of NLR genes within the intervals of mapped rust resistance genes. Our study will support the identification of functional resistance genes in wheat to accelerate the breeding and engineering of disease-resistant varieties.

An improved assembly and annotation of the allohexaploid wheat genome identifies complete families of agronomic genes and provides genomic evidence for chromosomal translocations.

Advances in genome sequencing and assembly technologies are generating many high-quality genome sequences, but assemblies of large, repeat-rich polyploid genomes, such as that of bread wheat, remain fragmented and incomplete. We have generated a new wheat whole-genome shotgun sequence assembly using a combination of optimized data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents >78% of the genome with a scaffold N50 of 88.8 kb that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNA-seq and Pacific Biosciences (PacBio) full-length cDNAs to identify 104,091 high-confidence protein-coding genes and 10,156 noncoding RNA genes. We confirmed three known and identified one novel genome rearrangements. Our approach enables the rapid and scalable assembly of wheat genomes, the identification of structural variants, and the definition of complete gene models, all powerful resources for trait analysis and breeding of this key global crop.

Uncovering hidden variation in polyploid wheat.

Comprehensive reverse genetic resources, which have been key to understanding gene function in diploid model organisms, are missing in many polyploid crops. Young polyploid species such as wheat, which was domesticated less than 10,000 y ago, have high levels of sequence identity among subgenomes that mask the effects of recessive alleles. Such redundancy reduces the probability of selection of favorable mutations during natural or human selection, but also allows wheat to tolerate high densities of induced mutations. Here we exploited this property to sequence and catalog more than 10 million mutations in the protein-coding regions of 2,735 mutant lines of tetraploid and hexaploid wheat. We detected, on average, 2,705 and 5,351 mutations per tetraploid and hexaploid line, respectively, which resulted in 35-40 mutations per kb in each population. With these mutation densities, we identified an average of 23-24 missense and truncation alleles per gene, with at least one truncation or deleterious missense mutation in more than 90% of the captured wheat genes per population. This public collection of mutant seed stocks and sequence data enables rapid identification of mutations in the different copies of the wheat genes, which can be combined to uncover previously hidden variation. Polyploidy is a central phenomenon in plant evolution, and many crop species have undergone recent genome duplication events. Therefore, the general strategy and methods developed herein can benefit other polyploid crops.

From plant immunity to food security: an interview with Ksenia Krasileva.

Ksenia Krasileva is an Assistant Professor at UC Berkley, studying innate immunity in plants. Ksenia's work combines plant genomics and plant-microbe interactions with new technologies, spanning basic studies and translational research in agriculture. In this interview Ksenia shares her experience with research and leading a lab, as well as thoughts on innovations in publishing.

Efficient Genome-Wide Detection and Cataloging of EMS-Induced Mutations Using Exome Capture and Next-Generation Sequencing.

Chemical mutagenesis efficiently generates phenotypic variation in otherwise homogeneous genetic backgrounds, enabling functional analysis of genes. Advances in mutation detection have brought the utility of induced mutant populations on par with those produced by insertional mutagenesis, but systematic cataloguing of mutations would further increase their utility. We examined the suitability of multiplexed global exome capture and sequencing coupled with custom-developed bioinformatics tools to identify mutations in well-characterized mutant populations of rice (Oryza sativa) and wheat (Triticum aestivum). In rice, we identified ∼18,000 induced mutations from 72 independent M2 individuals. Functional evaluation indicated the recovery of potentially deleterious mutations for >2600 genes. We further observed that specific sequence and cytosine methylation patterns surrounding the targeted guanine residues strongly affect their probability to be alkylated by ethyl methanesulfonate. Application of these methods to six independent M2 lines of tetraploid wheat demonstrated that our bioinformatics pipeline is applicable to polyploids. In conclusion, we provide a method for developing large-scale induced mutation resources with relatively small investments that is applicable to resource-poor organisms. Furthermore, our results demonstrate that large libraries of sequenced mutations can be readily generated, providing enhanced opportunities to study gene function and assess the effect of sequence and chromatin context on mutations.

Comparative analysis of plant immune receptor architectures uncovers host proteins likely targeted by pathogens.

BACKGROUND: Plants deploy immune receptors to detect pathogen-derived molecules and initiate defense responses. Intracellular plant immune receptors called nucleotide-binding leucine-rich repeat (NLR) proteins contain a central nucleotide-binding (NB) domain followed by a series of leucine-rich repeats (LRRs), and are key initiators of plant defense responses. However, recent studies demonstrated that NLRs with non-canonical domain architectures play an important role in plant immunity. These composite immune receptors are thought to arise from fusions between NLRs and additional domains that serve as "baits" for the pathogen-derived effector proteins, thus enabling pathogen recognition. Several names have been proposed to describe these proteins, including "integrated decoys" and "integrated sensors". We adopt and argue for "integrated domains" or NLR-IDs, which describes the product of the fusion without assigning a universal mode of action. RESULTS: We have scanned available plant genome sequences for the full spectrum of NLR-IDs to evaluate the diversity of integrations of potential sensor/decoy domains across flowering plants, including 19 crop species. We manually curated wheat and brassicas and experimentally validated a subset of NLR-IDs in wild and cultivated wheat varieties. We have examined NLR fusions that occur in multiple plant families and identified that some domains show re-occurring integration across lineages. Domains fused to NLRs overlap with previously identified pathogen targets confirming that they act as baits for the pathogen. While some of the integrated domains have been previously implicated in disease resistance, others provide new targets for engineering durable resistance to plant pathogens. CONCLUSIONS: We have built a robust reproducible pipeline for detecting variable domain architectures in plant immune receptors across species. We hypothesize that NLR-IDs that we revealed provide clues to the host proteins targeted by pathogens, and that this information can be deployed to discover new sources of disease resistance.

Computational prediction and molecular characterization of an oomycete effector and the cognate Arabidopsis resistance gene.

Hyaloperonospora arabidopsidis (Hpa) is an obligate biotroph oomycete pathogen of the model plant Arabidopsis thaliana and contains a large set of effector proteins that are translocated to the host to exert virulence functions or trigger immune responses. These effectors are characterized by conserved amino-terminal translocation sequences and highly divergent carboxyl-terminal functional domains. The availability of the Hpa genome sequence allowed the computational prediction of effectors and the development of effector delivery systems enabled validation of the predicted effectors in Arabidopsis. In this study, we identified a novel effector ATR39-1 by computational methods, which was found to trigger a resistance response in the Arabidopsis ecotype Weiningen (Wei-0). The allelic variant of this effector, ATR39-2, is not recognized, and two amino acid residues were identified and shown to be critical for this loss of recognition. The resistance protein responsible for recognition of the ATR39-1 effector in Arabidopsis is RPP39 and was identified by map-based cloning. RPP39 is a member of the CC-NBS-LRR family of resistance proteins and requires the signaling gene NDR1 for full activity. Recognition of ATR39-1 in Wei-0 does not inhibit growth of Hpa strains expressing the effector, suggesting complex mechanisms of pathogen evasion of recognition, and is similar to what has been shown in several other cases of plant-oomycete interactions. Identification of this resistance gene/effector pair adds to our knowledge of plant resistance mechanisms and provides the basis for further functional analyses.