Biology subjects

Harant, A.

Publications and source records attributed to Harant, A..

15 recordsLinked to original sources

bioRxiv2026

AI-guided discovery of atypical protein assemblies

Artificial intelligence (AI) systems such as AlphaFold have transformed structural biology by enabling accurate prediction of protein structures. However, their capacity to uncover new classes of macromolecular assemblies remains largely untapped. We developed the Structural Novelty Index (SNI), a quantitative framework for identifying protein complexes that diverge from canonical architectures. As one implementation of SNI, we developed SNINRC-Hexa, to identify unconventional resistosomes formed by nucleotide-binding, leucine-rich repeat immune receptors (NLRs). We used it to analyze AlphaFold 3 models of 637 non-redundant NRC proteins from 346 genomes representing 85 plant species. This analysis identified candidates with predicted architectures distinct from the canonical hexameric resistosomes of NRC proteins. Biochemical purification and negative-stain transmission electron microscopy of NRC7 orthologs from multiple species supported the SNI prediction and revealed an unexpected undecameric (11-mer) assembly. Our results establish SNI as a scalable approach for discovering atypical protein complexes.

bioinformatics↗

bioRxiv2025

A plant pathogen effector blocks stepwise assembly of a helper NLR resistosome

Helper NLRs function as central nodes in plant immune networks. Upon activation, they oligomerize into inflammasome-like resistosomes to initiate immune signaling, yet the dynamics of resistosome assembly remain poorly understood. Here, we show that the virulence effector AVRcap1b from the Irish potato famine pathogen Phytophthora infestans suppresses immune activation by directly engaging oligomerization intermediates of the tomato helper NLR SlNRC3. Cryo-EM structures of SlNRC3 in AVRcap1b-bound and unbound states reveal that AVRcap1b bridges multiple protomers, stabilizing a stalled intermediate and preventing formation of a functional resistosome. Leveraging AVRcap1b as a molecular tool, we also capture an additional SlNRC3 resistosome intermediate showing that assembly proceeds in a stepwise manner from dissociated monomers. These findings uncover a previously unrecognized vulnerability in NLR activation and reveal a pathogen strategy that disrupts immune complex assembly. This work advances mechanistic understanding of resistosome formation and uncovers a previously unrecognized facet of pathogen-plant coevolution.

plant biology↗

bioRxiv2025

The autoactivity of tomato helper NLR immune proteins of the NRC clade is unaltered in prf mutants of Nicotiana benthamiana

Nucleotide-binding domain and leucine-rich repeat immune receptors (NLRs) can function in networks of sensors and helpers to induce hypersensitive cell death and immunity against pathogens. The tomato sensor NLR Prf guards the Pto kinase from AvrPto and AvrPtoB effector perturbation and activates the downstream helpers NRC2 and NRC3. Prf is conserved across the Solanaceae and its ortholog in the model species Nicotiana benthamiana is also required for detection of AvrPto/AvrPtoB function on Pto. A recent study reported that cell death induction after transient expression of an autoactive mutant of tomato NRC3 is abolished upon RNAi silencing of Prf in N. benthamiana. Here we generated loss-of-function prf mutants in N. benthamiana and demonstrate that autoactive mutants of eight canonical tomato NRCs (NRC0, NRC1, NRC2, NRC3, NRC4a, NRC4b, NRC6, and NRC7) still induce hypersensitive cell death when expressed transiently in the prf mutant background. Autoactive tomato NRCs also triggered cell death when expressed in lettuce (Lactuca sativa), an Asteraceae plant that does not have a Prf ortholog. These results confirm a unidirectional dependency of sensors and helpers in the NRC network and underscore the value of the N. benthamiana and lettuce model systems for studying functional relationships between paired and networked NLRs.

plant biology↗

bioRxiv2024

Multiple horizontal mini-chromosome transfers drive genome evolution of clonal blast fungus lineages

Crop disease pandemics are often driven by clonal lineages of plant pathogens that reproduce asexually. How these clonal pathogens continuously adapt to their hosts despite harboring limited genetic variation, and in absence of sexual recombination remains elusive. Here, we reveal multiple instances of horizontal chromosome transfer within pandemic clonal lineages of the blast fungus Magnaporthe (Syn. Pyricularia) oryzae. We identified a horizontally transferred 1.2Mb supernumerary mini-chromosome which is remarkably conserved between M. oryzae isolates from both the rice blast fungus lineage and the lineage infecting Indian goosegrass (Eleusine indica), a wild grass that often grows in the proximity of cultivated cereal crops. Furthermore, we show that this mini-chromosome was horizontally acquired by clonal rice blast isolates through at least nine distinct transfer events over the past three centuries. These findings establish horizontal mini-chromosome transfer as a mechanism facilitating genetic exchange among different host-associated blast fungus lineages. We propose that blast fungus populations infecting wild grasses act as genetic reservoirs that drive genome evolution of pandemic clonal lineages that afflict cereal crops.

evolutionary biology↗

bioRxiv2024

Bioengineering a plant NLR immune receptor with a robust binding interface towards a conserved fungal pathogen effector

Bioengineering of plant immune receptors has emerged as a key strategy for generating novel disease resistance traits to counteract the expanding threat of plant pathogens to global food security. However, current approaches are limited by rapid evolution of plant pathogens in the field and may lack durability when deployed. Here, we show that the rice nucleotide-binding, leucine-rich repeat (NLR) immune receptor Pik-1 can be engineered to respond to a conserved family of effectors from the multihost blast fungus pathogen Magnaporthe oryzae. We switched the effector binding and response profile of the Pik NLR from its cognate rice blast effector AVR-Pik to the host-determining factor Pwl2 by installing a putative host target, OsHIPP43, in place of the native integrated HMA domain (generating Pikm-1OsHIPP43). This chimeric receptor also responded to other PWL alleles from diverse blast isolates. The crystal structure of the Pwl2/OsHIPP43 complex revealed a multifaceted, robust interface that cannot be easily disrupted by mutagenesis, and may therefore provide durable, broad resistance to blast isolates carrying PWL effectors in the field. Our findings highlight how the host targets of pathogen effectors can be used to bioengineer new recognition specificities that have more robust properties compared to naturally evolved disease resistance genes.

plant biology↗

bioRxiv2023

A root-specific NLR network confers resistance to plant parasitic nematodes

Nucleotide-binding domain and leucine-rich repeat immune receptors (NLRs) confer disease resistance to a multitude of foliar and root parasites of plants. However, the extent to which NLR immunity is expressed differentially between plant organs is poorly known. Here, we show that a large cluster of tomato genes, which encodes the cyst and root-knot nematode disease resistance proteins Hero and MeR1 as well as the NLR-helper NRC6, exhibits nearly exclusive expression in the roots. This root-specific gene cluster emerged in Solanum species about 21 million years ago through gene duplication from the ancient NRC network of asterid plants. NLR-sensors in this gene cluster exclusively signal through NRC6 helpers to trigger the hypersensitive cell death immune response. These findings indicate that the NRC6 gene cluster has sub-functionalized from the larger NRC network to specialize for resistance against root pathogens, including cyst and root-knot nematodes. We propose that NLR gene clusters and networks have evolved organ-specific gene expression as an adaptation to particular parasites and to reduce the risk of autoimmunity.

plant biology↗

bioRxiv2022

Resurrection of plant disease resistance proteins via helper NLR bioengineering

Parasites counteract host immunity by suppressing helper NLR proteins that function as central nodes in immune receptor networks. Understanding the mechanisms of immunosuppression can lead to strategies for bioengineering disease resistance. Here, we show that a cyst nematode virulence effector binds and inhibits oligomerization of the helper NLR protein NRC2 by physically preventing intramolecular rearrangements required for activation. A single amino acid polymorphism at the binding interface between NRC2 and the inhibitor is sufficient for this helper NLR to evade immune suppression, thereby restoring the activity of multiple disease resistance genes. This points to a novel strategy for resurrecting disease resistance in crop genomes. One sentence summaryA helper NLR is mutated to evade inhibition by a parasite effector.

plant biology↗

bioRxiv2022

Wild grass isolates of Magnaporthe (Syn. Pyricularia) spp. from Germany can cause blast disease on cereal crops

Pathogens that cause destructive crop diseases often infect wild host plants. However, surveys of major plant pathogens tend to be skewed towards cultivated crops and often neglect the wild hosts. Here, we report an emerging disease threat generated by the blast fungus Magnaporthe (Syn. Pyricularia) spp. in central Europe. We found that this notoriously devastating plant pathogen infects the wild grasses foxtail millet (Setaria spp.) and crabgrass (Digitaria spp.) in south-western Germany, a region previously deemed unfavorable for blast disease. Using phenotypic characterization and genomic analyses, we determined that the observed disease symptoms are associated with the Setaria spp.-infecting lineage of M. oryzae and its sister species Magnaporthe grisea. We showed that M. oryzae isolates can infect barley and wheat, thus highlighting the risk of pathogen spread to crops. In addition, M. oryzae isolates which co-occur in natural populations display compatible mating types and variable candidate effector gene content, which may enhance the pathogens adaptive potential. Our findings stress the risk of blast fungus infections expanding into European cereal crops through migration and host jumps. This underlines the importance for pathogen surveillance not only on cultivated crops, but also on wild host plants. Author SummaryWild plant species are often overlooked during pathogen virulence surveys. However, many of the diseases we observe in crops are a consequence of host jumps from wild to cultivated plant species. This is reminiscent of zoonotic diseases, where host jumps from wild animals to humans result in new disease outbreaks. Here, we report that the notoriously devastating blast fungus Magnaporthe (Syn. Pyricularia) spp. occurs on wild grasses in south-western Germany. This region, which is at the center of the European cereal belt, has so far been viewed as having unfavorable climatic conditions for the blast disease. The newly identified blast fungus isolates have the capacity to infect wheat and barley cultivars, highlighting the risk of the disease spreading to staple cereal crops. In addition, there is potential for sexual recombination in local populations, which may increase the evolutionary potential of the fungus and might facilitate host jumps to cereal crops. Our findings emphasize the urgent need for surveillance of plant diseases on both wild hosts and crops.

plant biology↗

bioRxiv2022

A pandemic clonal lineage of the wheat blast fungus

Wheat, the most important food crop, is threatened by a blast disease pandemic. Here, we show that a clonal lineage of the wheat blast fungus recently spread to Asia and Africa following two independent introductions from South America. Through a combination of genome analyses and laboratory experiments, we show that the decade-old blast pandemic lineage can be controlled by the Rmg8 disease resistance gene and is sensitive to strobilurin fungicides. However, we also highlight the potential of the pandemic clone to evolve fungicide-insensitive variants and sexually recombine with African lineages. This underscores the urgent need for genomic surveillance to track and mitigate the spread of wheat blast outside of South America, and to guide pre-emptive wheat breeding for blast resistance.

evolutionary biology↗

bioRxiv2021

An atypical NLR protein modulates the NRC immune receptor network

The NRC immune receptor network has evolved in asterid plants from a pair of linked genes into a genetically dispersed and phylogenetically structured network of sensor and helper NLR (nucleotide-binding domain and leucine-rich repeat-containing) proteins. In some species, such as the model plant Nicotiana benthamiana and other Solanaceae, the NRC network forms up to half of the NLRome, and NRCs are scattered throughout the genome in gene clusters of varying complexities. Here, we describe NRCX, an atypical, but essential member of the NRC family that lacks canonical features of these NLR helper proteins, such as a functional N-terminal MADA motif and the capacity to trigger autoimmunity. In contrast to other NRCs, systemic gene silencing of NRCX markedly impairs plant growth resulting in a dwarf phenotype. Remarkably, dwarfism of NRCX silenced plants is partially dependent on NRCX paralogs NRC2 and NRC3, but not NRC4. Despite its negative impact on plant growth when silenced systemically, transient RNA interference of NRCX in mature N. benthamiana leaves doesnt result in visible cell death phenotypes. However, alteration of NRCX expression modulates the hypersensitive response mediated by NRC2 and NRC3 in a manner consistent with a negative role for NRCX in the NRC network. We conclude that NRCX is an atypical member of the NRC network that has evolved to contribute to the homeostasis of this genetically unlinked NLR network.

plant biology↗

bioRxiv2021

The helper NLR immune protein NRC3 mediates the hypersensitive cell death caused by the cell-surface receptor Cf-4

Cell surface pattern recognition receptors (PRRs) activate immune responses that can include the hypersensitive cell death. However, the pathways that link PRRs to the cell death response are poorly understood. Here, we show that the cell surface receptor-like protein Cf-4 requires the intracellular nucleotide-binding domain leucine-rich repeat containing receptor (NLR) NRC3 to trigger a confluent cell death response upon detection of the fungal effector Avr4 in leaves of Nicotiana benthamiana. This NRC3 activity requires an intact N-terminal MADA motif, a conserved signature of coiled-coil (CC)-type plant NLRs that is required for resistosome-mediated immune responses. A chimeric protein with the N-terminal 1 helix of Arabidopsis ZAR1 swapped into NRC3 retains the capacity to mediate Cf-4 hypersensitive cell death. Pathogen effectors acting as suppressors of NRC3 can suppress Cf-4-triggered hypersensitive cell-death. Our findings link the NLR resistosome model to the hypersensitive cell death caused by a cell surface PRR.

plant biology↗

bioRxiv2021

Two NLR immune receptors acquired high-affinity binding to a fungal effector through convergent evolution of their integrated domain

A subset of plant NLR immune receptors carry unconventional integrated domains in addition to their canonical domain architecture. One example is rice Pik-1 that comprises an integrated heavy metal-associated (HMA) domain. Here, we reconstructed the evolutionary history of Pik-1 and its NLR partner, Pik-2, and tested hypotheses about adaptive evolution of the HMA domain. Phylogenetic analyses revealed that the HMA domain integrated into Pik-1 before Oryzinae speciation over 15 million years ago and has been under diversifying selection. Ancestral sequence reconstruction coupled with functional studies showed that two Pik-1 allelic variants independently evolved from a weakly binding ancestral state to high-affinity binding of the blast fungus effector AVR-PikD. We conclude that for most of its evolutionary history the Pik-1 HMA domain did not sense AVR-PikD, and that different Pik-1 receptors have recently evolved through distinct biochemical paths to produce similar phenotypic outcomes. These findings highlight the dynamic nature of the evolutionary mechanisms underpinning NLR adaptation to plant pathogens.

plant biology↗

bioRxiv2020

A vector system for fast-forward in vivo studies of the ZAR1 resistosome in the model plant Nicotiana benthamiana

Nicotiana benthamiana has emerged as a complementary experimental system to Arabidopsis. It enables fast-forward in vivo analyses primarily through transient gene expression and is particularly popular in the study of plant immunity. Recently, our understanding of NLR plant immune receptors has greatly advanced following the discovery of Arabidopsis ZAR1 resistosome. Here, we describe a novel vector system of 52 plasmids that enables functional studies of the ZAR1 resistosome in N. benthamiana. We showed that ZAR1 stands out among the coiled coil class of NLRs for being highly conserved across distantly related dicot plant species and confirmed NbZAR1 as the N. benthamiana ortholog of Arabidopsis ZAR1. NbZAR1 triggers autoimmune cell death in N. benthamiana and this activity is dependent on a functional N-terminal 1 helix. C-terminally tagged NbZAR1 remains functional in N. benthamiana thus enabling cell biology and biochemical studies in this plant system. We conclude that the NbZAR1 open source plasmids form an additional experimental system to Arabidopsis for in planta resistosome studies.

plant biology↗

bioRxiv2020

Genomic rearrangements generate hypervariable mini-chromosomes in host-specific lineages of the blast fungus

Supernumerary mini-chromosomes-a unique type of genomic structural variation-have been implicated in the emergence of virulence traits in plant pathogenic fungi. However, the mechanisms that facilitate the emergence and maintenance of mini-chromosomes across fungi remain poorly understood. In the blast fungus Magnaporthe oryzae, mini-chromosomes have been first described in the early 1990s but, until very recently, have been overlooked in genomic studies. Here we investigated structural variation in four isolates of the blast fungus M. oryzae from different grass hosts and analyzed the sequences of mini-chromosomes in the rice, foxtail millet and goosegrass isolates. The mini-chromosomes of these isolates turned out to be highly diverse with distinct sequence composition. They are enriched in repetitive elements and have lower gene density than core-chromosomes. We identified several virulence-related genes in the mini-chromosome of the rice isolate, including the polyketide synthase Ace1 and the effector gene AVR-Pik. Macrosynteny analyses around these loci revealed structural rearrangements, including inter-chromosomal translocations between core- and mini-chromosomes. Our findings provide evidence that mini-chromosomes independently emerge from structural rearrangements of core-chromosomes and might contribute to adaptive evolution of the blast fungus. Author summaryThe genomes of plant pathogens often exhibit an architecture that facilitates high rates of dynamic rearrangements and genetic diversification in virulence associated regions. These regions, which tend to be gene sparse and repeat rich, are thought to serve as a cradle for adaptive evolution. Supernumerary chromosomes, i.e. chromosomes that are only present in some but not all individuals of a species, are a special type of structural variation that have been observed in plants, animals, and fungi. Here we identified and studied supernumerary mini-chromosomes in the blast fungus Magnaporthe oryzae, a pathogen that causes some of the most destructive plant diseases. We found that rice, foxtail millet and goosegrass isolates of this pathogen contain mini-chromosomes with distinct sequence composition. All mini-chromosomes are rich in repetitive genetic elements and have lower gene densities than core-chromosomes. Further, we identified virulence-related genes on the mini-chromosome of the rice isolate. We observed large-scale genomic rearrangements around these loci, indicative of a role of mini-chromosomes in facilitating genome dynamics. Taken together, our results indicate that mini-chromosomes facilitate genome rearrangements and possibly adaptive evolution of the blast fungus.

genomics↗

bioRxiv2019

An N-terminal motif in NLR immune receptors is functionally conserved across distantly related plant species

The molecular codes underpinning the functions of plant NLR immune receptors are poorly understood. We used in vitro Mu transposition to generate a random truncation library and identify the minimal functional region of NLRs. We applied this method to NRC4--a helper NLR that functions with multiple sensor NLRs within a Solanaceae receptor network. This revealed that the NRC4 N-terminal 29 amino acids are sufficient to induce hypersensitive cell death. This region is defined by the consensus MADAxVSFxVxKLxxLLxxEx (MADA motif) that is conserved at the N-termini of NRC family proteins and ~20% of coiled-coil (CC)-type plant NLRs. The MADA motif matches the N-terminal 1 helix of Arabidopsis NLR protein ZAR1, which undergoes a conformational switch during resistosome activation. Immunoassays revealed that the MADA motif is functionally conserved across NLRs from distantly related plant species. NRC-dependent sensor NLRs lack MADA sequences indicating that this motif has degenerated in sensor NLRs over evolutionary time.

plant biology↗