Biology subjects

Maqbool, A.

Publications and source records attributed to Maqbool, A..

At least 19 recordsLinked to original sources

bioRxiv2026

A conserved NLR activation switch governs global transcriptional control of antibiotic biosynthesis in Streptomyces

Nucleotide-binding and oligomerization domain-like receptors (NLRs) are conserved molecular switches that regulate innate immunity across diverse domains of life. While best known for their roles in immunity, bacterial NLR-related proteins also govern non-immune processes. In Streptomyces, the global transcriptional regulator AfsR employs an NLR-type architecture to control antibiotic production. Here, we identify a conserved histidine-aspartic acid motif within the nucleotide-binding and oligomerization domain of AfsR that functions as a universal NLR activation switch. Site-directed mutagenesis of this motif decouples AfsR from native upstream signals, locking it into a constitutive, autoactive "ON" state. Using integrated transcriptomics, ChIP-seq, and in vitro DNA-binding assays, we demonstrate that autoactivation expands the regulatory reach of AfsR via enhanced promoter affinity, directly controlling its canonical target afsS and uncovering wblH as an additional member of the AfsR regulon. Untargeted metabolomics further shows that autoactive AfsR drives systemic reprogramming of specialized metabolism, increasing the production of known antibiotics and awakening silent biosynthetic gene clusters in Streptomyces coelicolor and Streptomyces peucetius. Furthermore, phylogenomic analysis reveals that Actinomycetes encode multiple AfsR-like regulators with undefined roles in biosynthetic gene cluster regulation, suggesting a substantial reservoir of unexplored regulatory diversity that could be harnessed to expand natural product discovery. Together, our work provides fundamental insights into the functional diversity of NLRs and a framework for rationally engineering these regulators to unlock untapped microbial chemical diversity.

microbiology↗

bioRxiv2026

Computational design of de novo integrated domains enables rational control of pathogen effector recognition in plant NLR immune receptors.

The rapid evolution of plant pathogens poses a persistent threat to global agricultural sustainability, often outpacing the discovery and deployment of natural disease resistance genes. While bioengineering of plant intracellular immune receptors (NLRs) offers a potential solution, developing bespoke immune recognition remains constrained by the laborious characterisation of natural receptors and plant-pathogen interactions. Here, we describe a programmable framework that leverages generative AI protein design tools, RFdiffusion and ProteinMPNN, to design de novo integrated domains (IDs) against diverse pathogen effectors. By integrating these bespoke binders into the modular rice blast Pik-1/Pik-2 NLR receptor chassis, we successfully engineer recognition of a non-cognate virulence factor (effector) from the Panama disease pathogen, Fusarium oxysporum f. sp. cubense Tropical Race 4. Functional assays in Nicotiana benthamiana demonstrate that these de novo domains facilitate specific effector perception and initiate immune signalling, while structural and biophysical analyses confirm that de novo integrated domains maintain high structural fidelity to the initial designs and associate with their targets via the predicted interaction interfaces. Additionally, our findings provide orthogonal evidence for the role of integrated domains in regulation of NLR signalling, demonstrating integration of de novo IDs can either trigger autoactivity or, in some cases, lead to effector-mediated repression of cell death. By decoupling immune perception from natural evolutionary history through deploying AI-designed sensory domains, this work establishes a design-lead framework for generation of programmable plant immune receptors, providing a new avenue for bioengineering crops against emerging pathogens.

plant biology↗

bioRxiv2025

Rewinding the tape: historical contingency and functional constraints have shaped the evolution of APikL virulence effectors in the blast fungus

Protein evolution is influenced by historical contingencies and functional constraints, but their combined impact on rapidly diversifying pathogen virulence effectors remains poorly understood. Here, we combined ancestral state reconstructions and functional assays to recapitulate the evolution of the MAX-fold effector protein APikL2 of the plant pathogenic blast fungus Magnaporthe (syn. Pyricularia) oryzae, focusing on the ancestral and functionally critical amino acid residue D66 (Asp, Codon: GAT). "Rewinding the tape" experiments based on ancestral sequence resurrection revealed that, out of the seven potential amino acid substitutions derived from single nucleotide polymorphisms, only the naturally occurring D66N (Asp to Asn, GAT to AAT) expanded the binding spectrum to host plant proteins of the heavy metal associated (HMA) family. In contrast, three of the nonsynonymous substitutions were deleterious resulting in loss of binding to HMA proteins. Additionally, we identified three cases of homoplasy in the APikL effector family, involving HMA-binding interfaces, indicating recurrent convergent evolution. Our findings suggest an experimental framework for predicting evolutionary outcomes of pathogen effector--host target interactions with implications for plant disease resistance breeding.

evolutionary biology↗

bioRxiv2025

Targeting Tenascin-C-Toll-like Receptor 4 signalling with Adhiron-derived small molecules - a viable strategy for reducing fibrosis in Systemic Sclerosis

Tissue fibrosis is a hallmark of systemic sclerosis (SSc) and results from the excessive production and deposition of collagen and other extracellular matrix proteins by resident fibroblasts. This excessive connective tissue accumulation leads to tissue disruption and subsequent dysfunction in the skin, lungs and other internal organs. Recent studies highlight a role for the matricellular protein Tenascin C in SSc, whereby its stimulation of Toll-like receptor 4 triggers self-sustaining fibroblast activation and ensuing fibrosis. We have utilised Adhiron guided ligand discovery to generate small molecules that target the fibrinogen-like globe domain of Tenascin C, a region involved in Toll-like receptor 4 activation and have demonstrated a reduction in the profibrotic phenotype of human dermal fibroblasts. These studies may facilitate the development of effective targeted therapy for fibrosis in SSc and other fibrotic diseases. Moreover, it highlights the utility of Adhiron guided ligand discovery to generate small molecule inhibitors to selectively modulate proteins. HighlightsO_LIAdhiron screening was undertaken to identify binders to the FBG domain of Tenascin C and the crystal structure of the FBG-Adhiron binding was resolved. C_LIO_LIAdhiron guided ligand discovery was used to generate small molecule inhibitors that selectively target the FBG domain of Tenascin C. C_LIO_LISPR was used to show binding of these molecules to the FBG domain of Tenascin C C_LIO_LIThe small molecule inhibitors generated attenuated TGF{beta}-mediated fibrosis in dermal fibroblasts C_LI

physiology↗

bioRxiv2025

A bacterial CARD-NLR immune system controls the release of gene transfer agents

Bacteria have evolved a wide array of immune systems to detect and defend against external threats including mobile genetic elements (MGEs) such as bacteriophages, plasmids, and transposons. MGEs are often selfish, exploiting their bacterial hosts to propagate, however they can also provide adaptive advantages through horizontal gene transfer. Gene transfer agents (GTAs), which are non-infectious domesticated prophages, represent a unique class of beneficial MGEs that facilitate bacterial gene transfer. Despite their domestication, GTAs retain phage-like features, including the requirement for host cell lysis to release particles, that may inadvertently trigger host immunity. How GTAs might avoid, subvert, or possibly adopt host immune systems to complete their life stages is poorly understood. Here, we identify a tripartite system, LypABC, that is essential for GTA-mediated cell lysis in Caulobacter crescentus. LypABC resembles caspase recruitment domain-nucleotide-binding leucine-rich repeat (CARD-NLR) anti-phage defence systems that mediate abortive infection wherein infected cells die to prevent phage proliferation, thereby protecting the overall bacterial population. LypABC-deficient cells produce host DNA-packed GTA particles and eventually die but cannot lyse to release GTA particles. Moreover, overproduction of LypABC is highly toxic to both GTA-producing and non-producing cells, highlighting the need for strict regulation. We find that such regulation is achieved transcriptionally by a repressor, RogB, which binds the promoters of lypABC and of essential GTA activator genes, thus coupling GTA activation and host cell lysis. While traditionally considered antagonistic towards MGEs, our findings here suggest that immunity components are versatile and can be adapted to support MGEs.

microbiology↗

bioRxiv2024

The conserved aphid saliva chemosensory protein effector Mp10 targets plant AMSH deubiquitinases at cellular membranes to suppress pattern-triggered immunity

Chemosensory proteins (CSPs) are a conserved family present in insects and other arthropods, recognized for their critical roles in both intra- and interspecies communication. However, the functional mechanisms of these proteins remain largely unexplored. In our previous research, we identified a CSP in aphid saliva, Mp10, from the peach-potato aphid Myzus persicae, which functions as an effector protein modulating host plant immunity. Mp10 suppresses pattern recognition receptor (PRR)-triggered immunity (PTI), the first layer of plant defence, while also inducing effector-triggered immunity (ETI). In this study, we elucidate the molecular mechanisms by which Mp10 suppresses PTI. Our findings reveal that Mp10 interacts with AMSH deubiquitinase enzymes in plants, as shown by yeast two-hybrid, co-immunoprecipitation (co-IP), and FRET-FLIM assays, with these interactions predominantly localized to intracellular membranes. Mp10 was found to modulate the dynamics of membrane-bound PRR receptor kinases in plant cells. Co-IP and mass spectrometry analyses demonstrated that Mp10 and AMSH2 associate with a range of PRR kinases, PRR-associated kinases, and proteins involved in the intracellular trafficking of membrane proteins. Mp10 reduces the accumulation of these kinases at the cell surface by promoting their internalization to internal membranes, thereby dampening PTI. Supporting this, a dominant-negative catalytically inactive variant of AMSH2 also inhibits PTI. Interestingly, Mp10 orthologues from other sap-feeding hemipteran insects exhibit similar immune-suppressive activities, and our findings show that their interaction with plant AMSH proteins is conserved, indicating this immune-suppression mechanism is evolutionarily ancient.

plant biology↗

bioRxiv2024

Proline-2'-deoxymugineic acid (PDMA) increases seed quality and yield by alleviating iron deficiency symptoms in soybean under calcareous-alkaline conditions

Iron (Fe) deficiency in crops, particularly in calcareous-alkaline soils, poses a major challenge due to Fe immobilization. While synthetic chelators like EDTA and EDDHA are commonly used to improve Fe availability, proline-2-deoxymugineic acid (PDMA) has emerged as a promising alternative, enhancing Fe nutrition in crops such as rice and cucumber. This study aimed to assess the effectiveness of PDMA on soybean growth and yield under calcareous-alkaline conditions. A pot trial demonstrated that PDMA improves Fe uptake, translocation, and storage in soybeans, leading to increased chlorophyll content, and enhanced root and shoot growth. Even at low dosages, PDMA significantly improved plant development, with the highest dosage (30 M) resulting in notable increases in Fe, Zn, Mn, and Mg concentrations in roots, leaves, and seeds, surpassing the effects of the synthetic chelator Fe-EDDHA in several parameters. Additionally, 30 M PDMA substantially boosted soybean yield, increasing pod and seed number, and 100-seed weight. It also improved seed quality by increasing protein and oil content. These findings suggest that PDMA offers a sustainable, effective alternative to traditional Fe chelators, providing a viable solution for addressing Fe deficiency and enhancing crop biofortification in challenging soil conditions.

plant biology↗

bioRxiv2024

Assembly of a segrosome by a CTP-independent ParB-like protein

The ATP- and CTP-dependent ParA-ParB-parS segrosome is a macromolecular complex that segregates chromosomes/plasmids in most bacterial species. CTP binding and hydrolysis enable ParB to slide on DNA and to bridge and condense DNA, thereby dictating the size and dynamics of the tripartite ParABS complex. Several other evolutionarily distinct systems can also segregate DNA, although the full diversity of bacterial DNA partition systems is not yet known. Here, we identify a CTP-independent ParABS system that maintains a conjugative plasmid SCP2 in the filamentous bacterium Streptomyces coelicolor. We demonstrate that an SCP2 ParB-like protein, ParT, loads onto DNA at an 18-bp parS site and diffuses away to the adjacent DNA despite lacking an apparent CTPase domain and detectable NTPase activity. We further show that parS DNA stimulates ParT transition from loading to a diffusing state to accumulate on DNA, and ParT activates the ATPase activity of its cognate partner protein ParA. We also identify numerous structural homologs of ParT, suggesting that CTP-independent diffusion on DNA might be widespread in bacteria despite being previously unappreciated. Overall, our findings uncover a CTP-independent DNA translocation as an alternative and unanticipated mechanism for the assembly of a bacterial DNA segregation complex and suggest that CTP binding and hydrolysis is not a fundamental feature of ParABS-like systems.

microbiology↗

bioRxiv2023

Activation of plant immunity through conversion of a helper NLR homodimer into a resistosome

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins can engage in complex interactions to detect pathogens and execute a robust immune response via downstream helper NLRs. However, the biochemical mechanisms of helper NLR activation by upstream sensor NLRs remain poorly understood. Here, we show that the coiled-coil helper NLR NRC2 accumulates in vivo as a homodimer that converts into a higher order oligomer upon activation by its upstream virus disease resistance protein Rx. The Cryo-EM structure of NRC2 in its resting state revealed intermolecular interactions that mediate homodimer formation. These dimerization interfaces have diverged between paralogous NRC proteins to insulate critical network nodes and enable redundant immune pathways. Our results expand the molecular mechanisms of NLR activation pointing to transition from homodimers to higher-order oligomeric resistosomes.

plant biology↗

bioRxiv2023

Bimodular Architecture of Bacterial Effector SAP05 Drives Ubiquitin-Independent Targeted Protein Degradation

In eukaryotes, targeted protein degradation (TPD) typically depends on a series of interactions among ubiquitin ligases that transfer ubiquitin molecules to substrates leading to degradation by the 26S proteasome. We previously discovered that the bacterial effector protein SAP05 mediates ubiquitin-independent TPD. SAP05 forms a ternary complex via interactions with the von Willebrand Factor Type A (vWA) domain of the proteasomal ubiquitin receptor Rpn10 and the Zinc-finger (ZnF) domains of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) and GATA BINDING FACTOR (GATA) transcription factors (TFs). This leads to direct TPD of the TFs by the 26S proteasome. Here, we report the crystal structures of the SAP05-vWA complex at 2.17 [A] resolution and of the SAP05-ZnF(SPL5) complex at 2.20 [A] resolution. Structural analyses revealed that SAP05 displays a unique bimodular architecture with two distinct non-overlapping surfaces, a loop surface with three protruding loops that form electrostatic interactions with ZnF, and a sheet surface featuring two {beta}-sheets, loops and -helices that establish polar interactions with vWA. SAP05 binding to ZnF TFs involves single amino acids responsible for multiple contacts, while SAP05 binding to vWA is more stable due to the necessity of multiple mutations to break the interaction. In addition, positioning of the SAP05 complex on the 26S proteasome points to a mechanism of protein degradation. Collectively, our findings demonstrate how a small bacterial bimodular protein can bypass the canonical UPS cellular proteolysis pathway, enabling ubiquitin-independent TPD in eukaryotic cells. This knowledge holds significant potential for the creation of novel TPD technologies.

biochemistry↗

bioRxiv2021

Regressive evolution of an effector following a host jump in the Irish Potato Famine Pathogen Lineage

In order to infect a new host species, the pathogen must evolve to enhance infection and transmission in the novel environment. Although we often think of evolution as a process of accumulation, it is also a process of loss. Here, we document an example of regressive evolution in the Irish potato famine pathogen (Phytophthora infestans) lineage, providing evidence that a key sequence motif in the effector PexRD54 has degenerated following a host jump. We began by looking at PexRD54 and PexRD54-like sequences from across Phytophthora species. We found that PexRD54 emerged in the common ancestor of Phytophthora clade 1b and 1c species, and further sequence analysis showed that a key functional motif, the C-terminal ATG8-interacting motif (AIM), was also acquired at this point in the lineage. A closer analysis showed that the P. mirabilis PexRD54 (PmPexRD54) AIM appeared unusual, the otherwise-conserved central residue mutated from a glutamate to a lysine. We aimed to determine whether this PmPexRD54 AIM polymorphism represented an adaptation to the Mirabilis jalapa host environment. We began by characterizing the M. jalapa ATG8 family, finding that they have a unique evolutionary history compared to previously characterized ATG8s. Then, using co-immunoprecipitation and isothermal titration calorimetry assays, we showed that both full-length PmPexRD54 and the PmPexRD54 AIM peptide bind very weakly to the M. jalapa ATG8s. Through a combination of binding assays and structural modelling, we showed that the identity of the residue at the position of the PmPexRD54 AIM polymorphism can underpin high-affinity binding to plant ATG8s. Finally, we conclude that the functionality of the PexRD54 AIM was lost in the P. mirabilis lineage, perhaps owing to as-yet-unknown pressure on this effector in the new host environment. Author SummaryPathogens evolve in concert with their hosts. When a pathogen begins to infect a new host species, known as a "host jump," the pathogen must evolve to enhance infection and transmission. These evolutionary processes can involve both the gain and loss of genes, as well as dynamic changes in protein function. Here, we describe an example of a pathogen protein that lost a key functional domain following a host jump, a salient example of "regressive evolution." Specifically, we show that an effector protein from the plant pathogen Phytopthora mirabilis, a host-specific lineage closely related to the Irish potato famine pathogen Phytopthora infestans, has a derived amino acid polymorphism that results in a loss of interaction with certain host machinery.

plant biology↗

bioRxiv2021

Dynamic accumulation of a helper NLR at the plant-pathogen interface underpins pathogen recognition

Plants employ sensor-helper pairs of NLR immune receptors to recognize pathogen effectors and activate immune responses. Yet the subcellular localization of NLRs pre- and post-activation during pathogen infection remains poorly known. Here we show that NRC4, from the NRC solanaceous helper NLR family, undergoes dynamic changes in subcellular localization by shuttling to and from the plant-pathogen haustorium interface established during infection by the Irish potato famine pathogen Phytophthora infestans. Specifically, prior to activation, NRC4 accumulates at the extra-haustorial membrane (EHM), presumably to mediate response to perihaustorial effectors, that are recognized by NRC4-dependent sensor NLRs. However not all NLRs accumulate at the EHM, as the closely related helper NRC2, and the distantly related ZAR1, did not accumulate at the EHM. NRC4 required an intact N-terminal coiled coil domain to accumulate at the EHM, whereas the functionally conserved MADA motif implicated in cell death activation and membrane insertion was dispensable for this process. Strikingly, a constitutively autoactive NRC4 mutant did not accumulate at the EHM and showed punctate distribution that mainly associated with the plasma membrane, suggesting that post-activation, NRC4 probably undergoes a conformation switch to form clusters that do not preferentially associate with the EHM. When NRC4 is activated by a sensor NLR during infection however, NRC4 formed puncta mainly at the EHM and to a lesser extent at the plasma membrane. We conclude that following activation at the EHM, NRC4 may spread to other cellular membranes from its primary site of activation to trigger immune responses. Significance statementPlant NLR immune receptors function as intracellular sensors of pathogen virulence factors known as effectors. In resting state, NLRs localize to subcellular sites where the effectors they sense operate. However, the extent to which NLRs alter their subcellular distribution during infection remains elusive. We describe dynamic changes in spatiotemporal localization of an NLR protein in infected plant cells. Specifically, the NLR protein accumulates at the newly synthesized plant-pathogen interface membrane, where the corresponding effectors are deployed. Following immune recognition, the activated receptor re-organizes to form punctate structures that target the cell periphery. We propose that NLRs are not necessarily stationary immune receptors, but instead may spread to other cellular membranes from the primary site of activation to boost immune responses.

plant biology↗

bioRxiv2021

Parasite co-opts a ubiquitin receptor to induce a plethora of developmental changes

Obligate parasites can induce complex and substantial phenotypic changes in their hosts in ways that favour their transmission to other trophic levels. However, mechanisms underlying these changes remain largely unknown. Here, we demonstrate how SAP05 protein effectors from insect-vectored plant pathogenic phytoplasmas take control of several plant developmental processes to simultaneously prolong host lifespan and induce witchs broom-like proliferations of leaf and sterile shoots, organs colonized by phytoplasmas and vectors. SAP05 acts by mediating the concurrent degradation of SPL and GATA developmental regulators via a process that uniquely relies on hijacking the plant ubiquitin receptor RPN10 independently of substrate lysine ubiquitination. RPN10 is highly conserved among eukaryotes, but SAP05 does not bind insect vector RPN10. A two-amino-acid substitution within plant RPN10 generates a functional variant that is resistant to SAP05 activities. Therefore, one effector protein enables obligate parasitic phytoplasmas to induce a plethora of developmental phenotypes in their hosts.

molecular biology↗

bioRxiv2021

Plant pathogens convergently evolved to counteract redundant nodes of an NLR immune receptor network

In plants, NLR (nucleotide-binding domain and leucine-rich repeat-containing) proteins can form receptor networks to confer hypersensitive cell death and innate immunity. One class of NLRs, known as NRCs (NLR required for cell death), are central nodes in a complex network that protects against multiple pathogens and comprises up to half of the NLRome of solanaceous plants. Given the prevalence of this NLR network, we hypothesized that pathogens convergently evolved to secrete effectors that target NRC activities. To test this, we screened a library of 167 bacterial, oomycete, nematode and aphid effectors for their capacity to suppress the cell death response triggered by the NRC-dependent disease resistance proteins Prf and Rpi-blb2. Among five of the identified suppressors, one cyst nematode protein and one oomycete protein suppress the activity of autoimmune mutants of NRC2 and NRC3, but not NRC4, indicating that they specifically counteract a subset of NRC proteins independently of their sensor NLR partners. Whereas the cyst nematode effector SPRYSEC15 binds the nucleotide-binding domain of NRC2 and NRC3, the oomycete effector AVRcap1b suppresses the response of these NRCs via the membrane trafficking-associated protein NbTOL9a (Target of Myb 1-like protein 9a). We conclude that plant pathogens have evolved to counteract central nodes of the NRC immune receptor network through different mechanisms. Coevolution with pathogen effectors may have driven NRC diversification into functionally redundant nodes in a massively expanded NLR network.

plant biology↗

bioRxiv2020

Multiple variants of the blast fungus effector AVR-Pik bind the HMA domain of the rice protein OsHIPP19 with high affinity.

Microbial plant pathogens secrete effector proteins which manipulate the host to promote infection. Effectors can be recognised by plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, initiating an immune response. The AVR-Pik effector from the rice blast fungus Magnaporthe oryzae is recognised by a pair of rice NLR receptors, Pik-1 and Pik-2. Pik-1 contains a non-canonical integrated heavy metal-associated (HMA) domain, which directly binds AVR-Pik to activate plant defences. Non-canonical integrated domains are widespread in plant NLRs and are thought to resemble the host target of the recognised effector. AVR-Pik interacts with specific rice HMA domain-containing proteins, namely heavy metal-associated isoprenylated plant proteins (HIPPs) and heavy metal-associated plant proteins (HPPs). Here, we define the biochemical and structural basis of the interaction between AVR-Pik and OsHIPP19, and compare the interaction with the HMA domain of Pik-1. Using analytical gel filtration and surface plasmon resonance, we show that multiple AVR-Pik variants, including the stealthy variants AVR-PikC and AVR-PikF which do not interact with any characterised Pik-1 alleles, bind to OsHIPP19 with nanomolar affinity. The crystal structure of OsHIPP19 in complex with AVR-PikF reveals differences at the interface that underpin high-affinity binding of OsHIPP19-HMA to a wider set of AVR-Pik variants than achieved by the integrated HMA domain of Pik-1. Our results provide a foundation for engineering the HMA domain of Pik-1 to extend binding to currently unrecognised AVR-Pik variants and expand disease resistance in rice to divergent pathogen strains.

plant biology↗

bioRxiv2020

Jurassic NLR: conserved and dynamic evolutionary features of the atypically ancient immune receptor ZAR1

In plants, NLR immune receptors generally exhibit hallmarks of rapid evolution even at the intraspecific level. We used iterative sequence similarity searches coupled with phylogenetic analyses to reconstruct the evolutionary history of ZAR1, an atypically conserved NLR that traces its origin to early flowering plant lineages [~]220 to 150 million years ago (Jurassic period). We discovered 120 ZAR1 orthologs in 88 species, including the monocot Colacasia esculenta, the magnoliid Cinnamomum micranthum and the majority of eudicots, notably the early diverging eudicot species Aquilegia coerulea. Ortholog sequence analyses revealed highly conserved features of ZAR1, including regions for pathogen effector recognition, intramolecular interactions and cell death activation. We functionally reconstructed the cell death activity of ZAR1 and its partner receptor-like cytoplasmic kinase (RLCK) from distantly related plant species, experimentally validating the hypothesis that ZAR1 has evolved to be a partner with RLCKs early in its evolution. In addition, ZAR1 acquired novel features, such as a C-terminal integration of a thioredoxin-like domain. ZAR1 duplicated into two paralog families, which underwent distinct evolutionary paths. We conclude that ZAR1 stands out among angiosperm NLRs for having experienced relatively limited gene duplication and expansion throughout its deep evolutionary history. Nonetheless, ZAR1 did also give rise to non-canonical NLR proteins with integrated domains and degenerated molecular features.

plant biology↗

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↗

bioRxiv2018

N-terminal β-strand underpins biochemical specialization of an ATG8 isoform

ATG8 is a highly-conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and numerous proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins remains unclear. Here, we describe a comprehensive protein-protein interaction resource, obtained using in planta immunoprecipitation followed by mass spectrometry, to define the potato ATG8 interactome. We discovered that ATG8 isoforms bind distinct sets of plant proteins with varying degrees of overlap. This prompted us to define the biochemical basis of ATG8 specialization by comparing two potato ATG8 isoforms using both in vivo protein interaction assays and in vitro quantitative binding affinity analyses. These experiments revealed that the N-terminal {beta}-strand--and, in particular, a single amino acid polymorphism--underpins binding specificity to the substrate PexRD54 by shaping the hydrophobic pocket that accommodates this proteins ATG8 interacting motif. Additional proteomics experiments indicated that the N-terminal {beta}-strand shapes the ATG8 interactor profiles, defining interaction specificity with about 80 plant proteins. Our findings are consistent with the view that ATG8 isoforms comprise a layer of specificity in the regulation of selective autophagy pathways in plants.

plant biology↗