Species-wide genetic incompatibility analysis identifies immune genes as hot spots of deleterious epistasis.

Intraspecific genetic incompatibilities prevent the assembly of specific alleles into single genotypes and influence genome- and species-wide patterns of sequence variation. A common incompatibility in plants is hybrid necrosis, characterized by autoimmune responses due to epistatic interactions between natural genetic variants. By systematically testing thousands of F1 hybrids of Arabidopsis thaliana strains, we identified a small number of incompatibility hot spots in the genome, often in regions densely populated by nucleotide-binding domain and leucine-rich repeat (NLR) immune receptor genes. In several cases, these immune receptor loci interact with each other, suggestive of conflict within the immune system. A particularly dangerous locus is a highly variable cluster of NLR genes, DM2, which causes multiple independent incompatibilities with genes that encode a range of biochemical functions, including NLRs. Our findings suggest that deleterious interactions of immune receptors limit the combinations of favorable disease resistance alleles accessible to plant genomes.

Generating minimum set of gRNA to cover multiple targets in multiple genomes with MINORg.

MINORg is an offline gRNA design tool that generates the smallest possible combination of gRNA capable of covering all desired targets in multiple non-reference genomes. As interest in pangenomic research grows, so does the workload required for large screens in multiple individuals. MINORg aims to lessen this workload by capitalising on sequence homology to favour multi-target gRNA while simultaneously screening multiple genetic backgrounds in order to generate reusable gRNA panels. We demonstrated the practical application of MINORg by knocking out 11 homologous genes tandemly arrayed in a multi-gene cluster in two Arabidopsis thaliana lineages using three gRNA output by MINORg. We also described a new PCR-free modular cloning system for multiplexing gRNA, and used it to knockout three tandemly arrayed genes in another multi-gene cluster with gRNA designed by MINORg. Source code is freely available at https://github.com/rlrq/MINORg.

Knock-down of gene expression throughout meiosis and pollen formation by virus-induced gene silencing in Arabidopsis thaliana.

Through the inactivation of genes that act during meiosis it is possible to direct the genetic make-up of plants in subsequent generations and optimize breeding schemes. Offspring may show higher recombination of parental alleles resulting from elevated crossover (CO) incidence, or by omission of meiotic divisions, offspring may become polyploid. However, stable mutations in genes essential for recombination, or for either one of the two meiotic divisions, can have pleiotropic effects on plant morphology and line stability, for instance by causing lower fertility. Therefore, it is often favorable to temporarily change gene expression during meiosis rather than relying on stable null mutants. It was previously shown that virus-induced gene silencing (VIGS) can be used to transiently reduce CO frequencies. We asked if VIGS could also be used to modify other processes throughout meiosis and during pollen formation in Arabidopsis thaliana. Here, we show that VIGS-mediated knock-down of FIGL1, RECQ4A/B, OSD1 and QRT2 can induce (i) an increase in chiasma numbers, (ii) unreduced gametes and (iii) pollen tetrads. We further show that VIGS can target both sexes and different genetic backgrounds and can simultaneously silence different gene copies. The successful knock-down of these genes in A. thaliana suggests that VIGS can be exploited to manipulate any process during or shortly after meiosis. Hence, the transient induction of changes in inheritance patterns can be used as a powerful tool for applied research and biotechnological applications.

Oxidative stress response and programmed cell death guided by NAC013 modulate pithiness in radish taproots.

Radish, Raphanus sativus L., is an important root crop that is cultivated worldwide. Owing to its evolutionary proximity to Arabidopsis thaliana, radish can be used as a model root crop in research on the molecular basis of agronomic traits. Pithiness is a significant defect that reduces the production of radish with commercial value; however, traditional breeding to eliminate this trait has thus far been unsuccessful. Here, we performed transcriptomics and genotype-by-sequencing (GBS)-based quantitative trait locus (QTL) analyses of radish inbred lines to understand the molecular basis of pithiness in radish roots. The transcriptome data indicated that pithiness likely stems from the response to oxidative stress, leading to cell death of the xylem parenchyma during the root-thickening process. Subsequently, we narrowed down a list of candidates responsible for pithiness near a major QTL and found polymorphisms in a radish homologue of Arabidopsis ANAC013 (RsNAC013), an endoplasmic reticulum bound NAC transcription factor that is targeted to the nucleus to mediate the mitochondrial retrograde signal. We analysed the effects of polymorphisms in RsNAC013 using Arabidopsis transgenic lines overexpressing RsNAC013 alleles as well as in radish inbred lines bearing these alleles. This analysis indicated that non-synonymous variations within the coding sequence result in different levels of RsNAC013 activities, thereby providing a genetic condition for root pithiness. The elevated oxidative stress or hypoxia that activates RsNAC013 for mitochondrial signalling enhances this process. Collectively, this study serves as an exemplary case of translational research taking advantage of the extensive information available from a model organism.

A Truncated Singleton NLR Causes Hybrid Necrosis in Arabidopsis thaliana.

Hybrid necrosis in plants arises from conflict between divergent alleles of immunity genes contributed by different parents, resulting in autoimmunity. We investigate a severe hybrid necrosis case in Arabidopsis thaliana, where the hybrid does not develop past the cotyledon stage and dies 3 weeks after sowing. Massive transcriptional changes take place in the hybrid, including the upregulation of most NLR (nucleotide-binding site leucine-rich repeat) disease-resistance genes. This is due to an incompatible interaction between the singleton TIR-NLR gene DANGEROUS MIX 10 (DM10), which was recently relocated from a larger NLR cluster, and an unlinked locus, DANGEROUS MIX 11 (DM11). There are multiple DM10 allelic variants in the global A. thaliana population, several of which have premature stop codons. One of these, which has a truncated LRR-PL (leucine-rich repeat [LRR]-post-LRR) region, corresponds to the DM10 risk allele. The DM10 locus and the adjacent genomic region in the risk allele carriers are highly differentiated from those in the nonrisk carriers in the global A. thaliana population, suggesting that this allele became geographically widespread only relatively recently. The DM11 risk allele is much rarer and found only in two accessions from southwestern Spain-a region from which the DM10 risk haplotype is absent-indicating that the ranges of DM10 and DM11 risk alleles may be nonoverlapping.

RPW8/HR repeats control NLR activation in Arabidopsis thaliana.

In many plant species, conflicts between divergent elements of the immune system, especially nucleotide-binding oligomerization domain-like receptors (NLR), can lead to hybrid necrosis. Here, we report deleterious allele-specific interactions between an NLR and a non-NLR gene cluster, resulting in not one, but multiple hybrid necrosis cases in Arabidopsis thaliana. The NLR cluster is RESISTANCE TO PERONOSPORA PARASITICA 7 (RPP7), which can confer strain-specific resistance to oomycetes. The non-NLR cluster is RESISTANCE TO POWDERY MILDEW 8 (RPW8) / HOMOLOG OF RPW8 (HR), which can confer broad-spectrum resistance to both fungi and oomycetes. RPW8/HR proteins contain at the N-terminus a potential transmembrane domain, followed by a specific coiled-coil (CC) domain that is similar to a domain found in pore-forming toxins MLKL and HET-S from mammals and fungi. C-terminal to the CC domain is a variable number of 21- or 14-amino acid repeats, reminiscent of regulatory 21-amino acid repeats in fungal HET-S. The number of repeats in different RPW8/HR proteins along with the sequence of a short C-terminal tail predicts their ability to activate immunity in combination with specific RPP7 partners. Whether a larger or smaller number of repeats is more dangerous depends on the specific RPW8/HR autoimmune risk variant.

Genetics of autoimmunity in plants: an evolutionary genetics perspective.

Autoimmunity in plants has been found in numerous hybrids as a form of hybrid necrosis and mutant panels. Uncontrolled cell death is a main cellular outcome of autoimmunity, which negatively impacts growth. Its occurrence highlights the vulnerable nature of the plant immune system. Genetic investigation of autoimmunity in hybrid plants revealed that extreme variation in the immune receptor repertoire is a major contributor, reflecting an evolutionary conundrum that plants face in nature. In this review, we discuss natural variation in the plant immune system and its contribution to fitness. The value of autoimmunity genetics lies in its ability to identify combinations of a natural immune receptor and its partner that are predisposed to triggering autoimmunity. The network of immune components for autoimmunity becomes instrumental in revealing mechanistic details of how immune receptors recognize cellular invasion and activate signaling. The list of autoimmunity-risk variants also allows us to infer evolutionary processes contributing to their maintenance in the natural population. Our approach to autoimmunity, which integrates mechanistic understanding and evolutionary genetics, has the potential to serve as a prognosis tool to optimize immunity in crops.

Modulation of ACD6 dependent hyperimmunity by natural alleles of an Arabidopsis thaliana NLR resistance gene.

Plants defend themselves against pathogens by activating an array of immune responses. Unfortunately, immunity programs may also cause unintended collateral damage to the plant itself. The quantitative disease resistance gene ACCELERATED CELL DEATH 6 (ACD6) serves to balance growth and pathogen resistance in natural populations of Arabidopsis thaliana. An autoimmune allele, ACD6-Est, which strongly reduces growth under specific laboratory conditions, is found in over 10% of wild strains. There is, however, extensive variation in the strength of the autoimmune phenotype expressed by strains with an ACD6-Est allele, indicative of genetic modifiers. Quantitative genetic analysis suggests that ACD6 activity can be modulated in diverse ways, with different strains often carrying different large-effect modifiers. One modifier is SUPPRESSOR OF NPR1-1, CONSTITUTIVE 1 (SNC1), located in a highly polymorphic cluster of nucleotide-binding domain and leucine-rich repeat (NLR) immune receptor genes, which are prototypes for qualitative disease resistance genes. Allelic variation at SNC1 correlates with ACD6-Est activity in multiple accessions, and a common structural variant affecting the NL linker sequence can explain differences in SNC1 activity. Taken together, we find that an NLR gene can mask the activity of an ACD6 autoimmune allele in natural A. thaliana populations, thereby linking different arms of the plant immune system.

Mechanisms to Mitigate the Trade-Off between Growth and Defense.

Plants have evolved an array of defenses against pathogens. However, mounting a defense response frequently comes with the cost of a reduction in growth and reproduction, carrying critical implications for natural and agricultural populations. This review focuses on how costs are generated and whether and how they can be mitigated. Most well-characterized growth-defense trade-offs stem from antagonistic crosstalk among hormones rather than an identified metabolic expenditure. A primary way plants mitigate such costs is through restricted expression of resistance; this can be achieved through inducible expression of defense genes or by the concentration of defense to particular times or tissues. Defense pathways can be primed for more effective induction, and primed states can be transmitted to offspring. We examine the resistance (R) genes as a case study of how the toll of defense can be generated and ameliorated. The fine-scale regulation of R genes is critical to alleviate the burden of their expression, and the genomic organization of R genes into coregulatory modules reduces costs. Plants can also recruit protection from other species. Exciting new evidence indicates that a plant's genotype influences the microbiome composition, lending credence to the hypothesis that plants shape their microbiome to enhance defense.

Transmission ratio distortion is frequent in Arabidopsis thaliana controlled crosses.

The equal probability of transmission of alleles from either parent during sexual reproduction is a central tenet of genetics and evolutionary biology. Yet, there are many cases where this rule is violated. The preferential transmission of alleles or genotypes is termed transmission ratio distortion (TRD). Examples of TRD have been identified in many species, implying that they are universal, but the resolution of species-wide studies of TRD are limited. We have performed a species-wide screen for TRD in over 500 segregating F2 populations of Arabidopsis thaliana using pooled reduced-representation genome sequencing. TRD was evident in up to a quarter of surveyed populations. Most populations exhibited distortion at only one genomic region, with some regions being repeatedly affected in multiple populations. Our results begin to elucidate the species-level architecture of biased transmission of genetic material in A. thaliana, and serve as a springboard for future studies into the biological basis of TRD in this species.

Genetic architecture of nonadditive inheritance in Arabidopsis thaliana hybrids.

The ubiquity of nonparental hybrid phenotypes, such as hybrid vigor and hybrid inferiority, has interested biologists for over a century and is of considerable agricultural importance. Although examples of both phenomena have been subject to intense investigation, no general model for the molecular basis of nonadditive genetic variance has emerged, and prediction of hybrid phenotypes from parental information continues to be a challenge. Here we explore the genetics of hybrid phenotype in 435 Arabidopsis thaliana individuals derived from intercrosses of 30 parents in a half diallel mating scheme. We find that nonadditive genetic effects are a major component of genetic variation in this population and that the genetic basis of hybrid phenotype can be mapped using genome-wide association (GWA) techniques. Significant loci together can explain as much as 20% of phenotypic variation in the surveyed population and include examples that have both classical dominant and overdominant effects. One candidate region inherited dominantly in the half diallel contains the gene for the MADS-box transcription factor AGAMOUS-LIKE 50 (AGL50), which we show directly to alter flowering time in the predicted manner. Our study not only illustrates the promise of GWA approaches to dissect the genetic architecture underpinning hybrid performance but also demonstrates the contribution of classical dominance to genetic variance.

Forever Young: The Role of Ubiquitin Receptor DA1 and E3 Ligase BIG BROTHER in Controlling Leaf Growth and Development.

The final size of plant organs is determined by a combination of cell proliferation and cell expansion. Leaves account for a large part of above-ground biomass and provide energy to complete the plant's life cycle. Although the final size of leaves is remarkably constant under fixed environmental conditions, several genes have been described to enhance leaf growth when their expression is modulated. In Arabidopsis (Arabidopsis thaliana), mutations in DA1 and BB increase leaf size, an effect that is synergistically enhanced in the double mutant. Here, we show that overexpression of a dominant-negative version of DA1 enhances leaf size in a broad range of natural accessions of this species, indicating a highly conserved role of this protein in controlling organ size. We also found that during early stages of development, leaves of da1-1 and bb/eod1-2 mutants were already larger than the isogenic Col-0 wild type, but this phenotype was triggered by different cellular mechanisms. Later during development, da1-1 and bb/eod1-2 leaves showed a prolonged longevity, which was enhanced in the double mutant. Conversely, ectopic expression of DA1 or BB restricted growth and promoted leaf senescence. In concert, shortly upon induction of DA1 and BB expression, several marker genes for the transition from proliferation to expansion were highly up-regulated. Additionally, multiple genes involved in maintaining the mitotic cell cycle were rapidly down-regulated and senescence genes were strongly up-regulated, particularly upon BB induction. With these results, we demonstrate that DA1 and BB restrict leaf size and promote senescence through converging and different mechanisms.

Natural Variation of Molecular and Morphological Gibberellin Responses.

Although phytohormones such as gibberellins are essential for many conserved aspects of plant physiology and development, plants vary greatly in their responses to these regulatory compounds. Here, we use genetic perturbation of endogenous gibberellin levels to probe the extent of intraspecific variation in gibberellin responses in natural accessions of Arabidopsis (Arabidopsis thaliana). We find that these accessions vary greatly in their ability to buffer the effects of overexpression of GA20ox1, encoding a rate-limiting enzyme for gibberellin biosynthesis, with substantial differences in bioactive gibberellin concentrations as well as transcriptomes and growth trajectories. These findings demonstrate a surprising level of flexibility in the wiring of regulatory networks underlying hormone metabolism and signaling.

Novel allelic variants in ACD6 cause hybrid necrosis in local collection of Arabidopsis thaliana.

Hybrid necrosis is a common type of hybrid incompatibility in plants. This phenomenon is caused by deleterious epistatic interactions, resulting in spontaneous activation of plant defenses associated with leaf necrosis, stunted growth and reduced fertility in hybrids. Specific combinations of alleles of ACCELERATED CELL DEATH 6 (ACD6) have been shown to be a common cause of hybrid necrosis in Arabidopsis thaliana. Increased ACD6 activity confers broad-spectrum resistance against biotrophic pathogens but reduces biomass production. We generated 996 crosses among individuals derived from a single collection area around Tübingen (Germany) and screened them for hybrid necrosis. Necrotic hybrids were further investigated by genetic linkage, amiRNA silencing, genomic complementation and metabolic profiling. Restriction site associated DNA (RAD)-sequencing was used to understand genetic diversity in the collection sites containing necrosis-inducing alleles. Novel combinations of ACD6 alleles found in neighbouring stands were found to activate the A. thaliana immune system. In contrast to what we observed in controlled conditions, necrotic hybrids did not show reduced fitness in the field. Metabolic profiling revealed changes associated with the activation of the immune system in ACD6-dependent hybrid necrosis. This study expands our current understanding of the active role of ACD6 in mediating trade-offs between defense responses and growth in A.  thaliana.

Analysis of a plant complex resistance gene locus underlying immune-related hybrid incompatibility and its occurrence in nature.

Mechanisms underlying speciation in plants include detrimental (incompatible) genetic interactions between parental alleles that incur a fitness cost in hybrids. We reported on recessive hybrid incompatibility between an Arabidopsis thaliana strain from Poland, Landsberg erecta (Ler), and many Central Asian A. thaliana strains. The incompatible interaction is determined by a polymorphic cluster of Toll/interleukin-1 receptor-nucleotide binding-leucine rich repeat (TNL) RPP1 (Recognition of Peronospora parasitica1)-like genes in Ler and alleles of the receptor-like kinase Strubbelig Receptor Family 3 (SRF3) in Central Asian strains Kas-2 or Kond, causing temperature-dependent autoimmunity and loss of growth and reproductive fitness. Here, we genetically dissected the RPP1-like Ler locus to determine contributions of individual RPP1-like Ler (R1-R8) genes to the incompatibility. In a neutral background, expression of most RPP1-like Ler genes, except R3, has no effect on growth or pathogen resistance. Incompatibility involves increased R3 expression and engineered R3 overexpression in a neutral background induces dwarfism and sterility. However, no individual RPP1-like Ler gene is sufficient for incompatibility between Ler and Kas-2 or Kond, suggesting that co-action of at least two RPP1-like members underlies this epistatic interaction. We find that the RPP1-like Ler haplotype is frequent and occurs with other Ler RPP1-like alleles in a local population in Gorzów Wielkopolski (Poland). Only Gorzów individuals carrying the RPP1-like Ler haplotype are incompatible with Kas-2 and Kond, whereas other RPP1-like alleles in the population are compatible. Therefore, the RPP1-like Ler haplotype has been maintained in genetically different individuals at a single site, allowing exploration of forces shaping the evolution of RPP1-like genes at local and regional population scales.

Activation of the Arabidopsis thaliana immune system by combinations of common ACD6 alleles.

A fundamental question in biology is how multicellular organisms distinguish self and non-self. The ability to make this distinction allows animals and plants to detect and respond to pathogens without triggering immune reactions directed against their own cells. In plants, inappropriate self-recognition results in the autonomous activation of the immune system, causing affected individuals to grow less well. These plants also suffer from spontaneous cell death, but are at the same time more resistant to pathogens. Known causes for such autonomous activation of the immune system are hyperactive alleles of immune regulators, or epistatic interactions between immune regulators and unlinked genes. We have discovered a third class, in which the Arabidopsis thaliana immune system is activated by interactions between natural alleles at a single locus, ACCELERATED CELL DEATH 6 (ACD6). There are two main types of these interacting alleles, one of which has evolved recently by partial resurrection of a pseudogene, and each type includes multiple functional variants. Most previously studies hybrid necrosis cases involve rare alleles found in geographically unrelated populations. These two types of ACD6 alleles instead occur at low frequency throughout the range of the species, and have risen to high frequency in the Northeast of Spain, suggesting a role in local adaptation. In addition, such hybrids occur in these populations in the wild. The extensive functional variation among ACD6 alleles points to a central role of this locus in fine-tuning pathogen defenses in natural populations.

All-organic transparent plant e-skin for noninvasive phenotyping.

Real-time in situ monitoring of plant physiology is essential for establishing a phenotyping platform for precision agriculture. A key enabler for this monitoring is a device that can be noninvasively attached to plants and transduce their physiological status into digital data. Here, we report an all-organic transparent plant e-skin by micropatterning poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) on polydimethylsiloxane (PDMS) substrate. This plant e-skin is optically and mechanically invisible to plants with no observable adverse effects to plant health. We demonstrate the capabilities of our plant e-skins as strain and temperature sensors, with the application to Brassica rapa leaves for collecting corresponding parameters under normal and abiotic stress conditions. Strains imposed on the leaf surface during growth as well as diurnal fluctuation of surface temperature were captured. We further present a digital-twin interface to visualize real-time plant surface environment, providing an intuitive and vivid platform for plant phenotyping.

Long-Lasting Unruptured Large Intracranial Aneurysm in Bilateral Moyamoya Disease: Case Report.

Introduction: The rupture risk of intracranial aneurysms in patients with moyamoya disease is higher than that in the general population. We report a confirmed case of moyamoya disease with bilateral middle cerebral artery (MCA) occlusion with a large and long-lasting aneurysm. Case: A 71-year-old woman visited the clinic with a large intracranial aneurysm. The patient was diagnosed with an ischemic stroke 2 months ago. She exhibited weakness in the left upper and lower extremities and dysarthria and was taking aspirin. The brain magnetic resonance imaging showed complete occlusion in the bilateral MCA proximal (M1) and a large 11 × 11 mm nonruptured cerebral aneurysm in the A3 segment of the left anterior cerebral artery. On transfemoral cerebral angiography, the patient was diagnosed with Suzuki grade VI moyamoya disease with bilateral MCA occlusion. After 7 years, the cerebral aneurysm size further increased, but it remained unruptured. Conclusions: Here, the patient had moyamoya disease with a large aneurysm, but aneurysmal rupture did not occur even after 7 years. Our case report might help in understanding the mechanisms of cerebral aneurysm occurrence and rupture in moyamoya patients.