Biology subjects

Liu, F.

Publications and source records attributed to Liu, F..

At least 235 records · Page 13Linked to original sources

bioRxiv2022

Pyrophosphoproteomics: extensive protein pyrophosphorylation revealed in human cell lines

Reversible protein phosphorylation is a central signaling mechanism in eukaryotic cells. While the identification of canonical phosphorylation sites using mass-spectrometry (MS) based proteomics has become routine, annotation of non-canonical phosphorylation has remained a challenge. Here, we report a tailored pyrophosphoproteomics workflow to detect and reliably assign protein pyrophosphorylation in two human cell lines, providing the first direct evidence of endogenous protein pyrophosphorylation. Detection of protein pyrophosphorylation was reproducible, specific and consistent with previous biochemical evidence relating the installation of the modification to inositol pyrophosphates (PP-InsPs). We manually validated 148 pyrophosphosites across 71 human proteins, the most heavily pyrophosphorylated of which were the nucleolar proteins NOLC1 and TCOF1. A predictive workflow based on the MS data set was established to recognize putative pyrophosphorylation sequences, and UBF1, a nucleolar protein incompatible with the proteomics method, was biochemically shown to undergo pyrophosphorylation. When the biosynthesis of PP-InsPs was perturbed in a model cell line, proteins expressed in this background exhibited lower levels of pyrophosphorylation. Disruption of PP-InsP biosynthesis also significantly reduced rDNA transcription, potentially by lowering pyrophosphorylation on regulatory proteins NOLC1, TCOF1, and UBF1. Overall, protein pyrophosphorylation emerges as an archetype of non-canonical phosphorylation, and should be considered in future phosphoproteomic analyses.

biochemistry↗

bioRxiv2022

A novel antidepressant acting via allosteric inhibition of GluN2D-incorporated NMDA receptors at GABAergic interneurons

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated calcium-permeable excitatory channels. They have attracted great interest as potential targets for the treatment of depression in recent years. NMDARs typically assemble as heterotetramers composed of two GluN1 and two alternative GluN2 (2A-2D) subunits, the latter of which endow various subtypes with diverse gating and pharmacological properties. To date, limited molecules with GluN2 specificity have been identified to show antidepressant effects. Here, we identify a compound termed YY-23 extracted from Rhizoma Anemarrhenae allosterically inhibited the channel activities of GluN2C- or GluN2D-incorporated NMDARs, an effect that was presumably influenced by the S2 segment in the ligand-binding domain of the GluN2 subunit. We found that prefrontal GluN2D-containing NMDARs were predominantly expressed at GABAergic interneurons rather than pyramidal neurons. Furthermore, we revealed that YY-23 suppressed the activity of GluN2D-containing NMDARs and GABAergic neurotransmission in the medial prefrontal cortex (mPFC). As a consequence, this GABAergic disinhibition facilitated the excitatory transmission. Behavioural experiments showed that YY-23 acted as a rapid antidepressant in both stress-naive and stressed animal models, which was abolished in Grin2d-knockout mice. Together, our findings suggest that GluN2D-incorporated NMDARs on GABAergic interneurons might be promising therapeutic targets for the treatment of depression.

neuroscience↗

bioRxiv2022

Searching across-cohort relatives via encrypted genotype regression

Explicitly sharing individual level data in genomics studies has many merits comparing to sharing summary statistics, including more strict QCs, common statistical analyses, relative identification and improved statistical power in GWAS, but it is hampered by privacy or ethical constraints. In this study, we developed encG-reg, a regression approach that can detect relatives of various degrees based on encrypted genomic data, which is immune of ethical constraints. The encryption properties of encG-reg are based on the random matrix theory by masking the original genotypic matrix without sacrificing precision of individual-level genotype data. We established a connection between the dimension of a random matrix, which masked genotype matrices, and the required precision of a study for encrypted genotype data. encG-reg has false positive and false negative rates equivalent to sharing original individual level data, and is computationally efficient when searching relatives. We split the UK Biobank into their respective centers, and then encrypted the genotype data. We observed that the relatives estimated using encG-reg was equivalently accurate with the estimation by KING, which is a widely used software but requires original genotype data. In a more complex application, we launched a finely devised multi-center collaboration across 5 research institutes in China, covering 9 cohorts of 54,092 GWAS samples. encG-reg again identified true relatives existing across the cohorts with even different ethnic backgrounds and genotypic qualities. Our study clearly demonstrates that encrypted genomic data can be used for data sharing without loss of information or data sharing barrier. Author SummaryEstimating pairwise genetic relatedness within a single cohort is straightforward. However, in practice, related samples are often distributed across different cohorts, making it challenging to estimate inter-cohort relatedness. In this study, we propose a method called encrypted genotype regression (encG-reg), which provides an unbiased estimation of inter-cohort relatedness using encrypted genotypes. The genotype matrix of each cohort is masked by a random matrix, which acts similarly to a private key in a cryptographic scheme. This masking process produces encrypted genotypes, which are a projection of the original genotype matrix. We derive the expectation and particularly the sampling variance for encG-reg, the latter involves eighth-order moments calculation. encG-reg allows us to accurately identify relatedness across cohorts, even for large-scale biobank data. To demonstrate the efficacy of encG-reg, we verified it in a multi-ethnicity UK Biobank dataset comprising 485,158 samples. For this case, we successfully tracked down to the 1st-degree relatedness (such as full sibs and parent-offspring). Furthermore, we used encG-reg in a collaboration involving 9 Chinese cohorts, encompassing a total of 54,092 samples from 5 genomic centers. It is worth noting that if the number of effective markers is sufficient encG-reg has the potential to detect even more distant degrees of relatedness beyond what we demonstrated.

genetics↗

bioRxiv2022

Bidirectional changes in postmitotic H3K27me3 distributions underlie cerebellar granule neuron maturation dynamics

The functional maturation of neurons is a prolonged process that extends well beyond mitotic exit and is mediated by the chromatin-dependent orchestration of gene transcription programs. We find that the postnatal maturation of cerebellar granule neurons (CGNs) requires dynamic changes in the genomic distribution of histone H3 lysine 27 trimethylation (H3K27me3), demonstrating a function for this chromatin modification beyond its role in cell fate specification. The developmental loss of H3K27me3 at promoters of genes that turn on as CGNs mature is facilitated by the lysine demethylase, and ASD-risk gene, Kdm6b. Interestingly, inhibition of the H3K27 methyltransferase EZH2 in newborn CGNs not only blocks the repression of progenitor genes but also impairs the induction of mature CGN genes, showing the importance of bidirectional H3K27me3 regulation across the genome. These data demonstrate that H3K27me3 turnover in developing postmitotic neurons regulates the temporal coordination of gene expression programs that underlie functional neuronal maturation.

molecular biology↗

bioRxiv2022

Heterologous boost with mRNA vaccines against SARS-CoV-2 Delta/Omicron variants following an inactivated whole-virus vaccine

The coronavirus SARS-CoV-2 has mutated quickly and caused significant global damage. This study characterizes two mRNA vaccines ZSVG-02 (Delta) and ZSVG-02-O (Omicron BA.1), and associating heterologous prime-boost strategy following the prime of a most widely administrated inactivated whole-virus vaccine (BBIBP-CorV). The ZSVG-02-O induces neutralizing antibodies that effectively cross-react with Omicron subvariants following an order of BA.1>BA.2>BA.4/5. In naive animals, ZSVG-02 or ZSVG-02-O induce humoral responses skewed to the vaccines targeting strains, but cellular immune responses cross-react to all variants of concern (VOCs) tested. Following heterologous prime-boost regimes, animals present comparable neutralizing antibody levels and superior protection across all VOCs. Single-boost only generated ancestral and omicron dual-responsive antibodies, probably by "recall" and "reshape" the prime immunity. New Omicron-specific antibody populations, however, appeared only following the second boost with ZSVG-02-O. Overall, our results support a heterologous boost with ZSVG-02-O, providing the best protection against current VOCs in inactivated virus vaccine- primed populations.

molecular biology↗

bioRxiv2022

Neuronal C-Reactive Protein Mediates Neuropathic Pain by Activating Nociceptive FcγRI-Coupled Signaling

BackgroundNeuropathic pain is difficult to treat in clinical practice, and the underlying mechanisms are insufficiently elucidated. Previous studies have demonstrated that Fc{gamma} receptor I (Fc{gamma}RI) is expressed in the neurons of the dorsal root ganglion (DRG) and may be involved in chronic pain. MethodsChronic constriction injury (CCI) was used to induce neuropathic pain in rats. Primary neuron-specific Fcgr1 conditional knockout (CKO) rats were established by crossing rats carrying a Fcgr1loxP+/+ with the PirtCRE+ line. Behavioral and molecular studies were conducted to evaluate the differences between wild-type and CKO rats after CCI. ResultsWe first revealed that CCI activated neuronal Fc{gamma}RI-related signaling in the DRG. CCI-induced neuropathic pain was alleviated in CKO rats. C-reactive protein (CRP) was increased in the DRG after nerve injury. Intraganglionic injection or overexpression of the recombinant CRP protein in the DRG evoked pain accompanied and activated neuronal Fc{gamma}RI. CRP-evoked pain was significantly reduced in CKO rats. Furthermore, microinjection of native IgG into the DRG alleviated neuropathic pain and the activation of neuronal Fc{gamma}RI-related signaling. ConclusionsOur results indicate that the activation of neuronal CRP/Fc{gamma}RI-related signaling plays an important role in the development of pain in CCI. Our findings may provide novel insights into the neuroimmune responses after peripheral nerve injury and might suggest potential therapeutic targets for neuropathic pain.

neuroscience↗

bioRxiv2022

CaMKII activity spreads by inter-holoenzyme phosphorylation

The dodecameric protein kinase CaMKII is expressed throughout the body. The alpha isoform is responsible for synaptic plasticity and participates in memory through its phosphorylation of synaptic proteins. Its elaborate subunit organization and propensity for autophosphorylation allow it to preserve neuronal plasticity across space and time. The prevailing hypothesis for the spread of CaMKII activity, involving shuffling of subunits between activated and naive holoenzymes, is broadly termed subunit exchange. In contrast to the expectations of previous work, we found little evidence for subunit exchange upon activation, and no effect of restraining subunits to their parent holoenzymes. Rather, mass photometry, crosslinking mass spectrometry, single molecule TIRF microscopy and biochemical assays identify inter-holoenzyme phosphorylation (IHP) as the mechanism for spreading phosphorylation. The transient, activity-dependent formation of groups of holoenzymes is well suited to the speed of neuronal activity. Our results place fundamental limits on the activation mechanism of this kinase.

biophysics↗

bioRxiv2022

Ena/VASP proteins at the crossroads of actin nucleation pathways in dendritic cell migration

As sentinels of our immune system dendritic cells (DCs) rely on efficient cell migration for patrolling peripheral tissues and delivering sampled antigens to secondary lymphoid organs for the activation of T-cells. Dynamic actin polymerization is key to their macropinocytic and migratory properties. Both major actin nucleation machineries, formins and the Arp2/3 complex, are critical for different aspects of DC functionality, by driving the generation of linear respectively branched actin filaments. However, the importance of a third group of actin nucleators, the Ena/VASP family, has not been addressed yet. Here, we show that the two family members Evl and VASP are expressed in murine DCs and that their loss negatively affects DC macropinocytosis, spreading, and migration. Our interactome analysis reveals Ena/VASP proteins to be ideally positioned for orchestrating the different actin nucleation pathways by binding to the formin mDia1 as well as to the WAVE regulatory complex, a stimulator of Arp2/3. In fact, Evl/VASP deficient murine DCs are more vulnerable to inhibition of Arp2/3 demonstrating that Ena/VASP proteins contribute to the robustness and efficiency of DC migration.

cell biology↗

bioRxiv2022

Human iPSC-derived neuron of 16p11.2 deletion reveals haplotype-specific expression of MAPK3 and its contribution to variable NDD phenotypes

Recurrent proximal 16p11.2 deletion (16p11.2del) is risk factor of diverse neurodevelopmental disorders (NDDs) with variable penetrance. Although previous human induced pluripotent stem cell (hiPSC) models of 16p11.2del confirmed disrupted neuron development, it is not known which gene(s) at this interval are mainly responsible for the abnormal cellular phenotypes and how the NDD penetrance is regulated. After haplotype phasing of 16p11.2 region, we generated hiPSCs for two 16p11.2del families with distinct residual haplotypes and variable NDD phenotypes. We also differentiated the hiPSCs to cortical neural cells and demonstrated MAPK3 as a driver signal of 16p11.2 region contributing to the dysfunctions in multiple pathways related to neuron development, which leads to altered morphological or electrophysiological properties in neuron cells. Furthermore, residual haplotype-specific MAPK3 expression was identified in 16p11.2del neuron cells, associating MAPK3 down-expression with the minor allele of the residual haplotype. Ten SNPs of the residual haplotype are mapped as enhancer SNPs (enSNPs) of MAPK3, eight enSNPs were functionally validated by luciferase assays, implying enSNPs contribute to residual haplotype-specific MAPK3 expression via cis-regulation. Finally, the analyses of three different patient cohorts showed that the residual haplotype of 16p11.2del is associated with variable NDD phenotypes.

genetics↗

bioRxiv2022

A kinase-independent function of cyclin-dependent kinase 6 promotes outer radial glia expansion and neocortical folding

The neocortex, the center for higher brain function, first emerged in mammals and has become massively expanded and folded in humans, constituting almost half the volume of the human brain. Primary microcephaly, a developmental disorder in which the brain is smaller than normal at birth, mainly results from the number of neurons in the neocortex being reduced because of defects in neural progenitor cells (NPCs). Outer radial glia (oRGs), NPCs that are abundant in gyrencephalic species but rare in lisencephalic species, are thought to play key roles in the expansion and folding of the neocortex. However, how oRGs expand, whether they are necessary for neocortical folding, and whether defects in oRGs cause microcephaly remain important questions in the study of brain development, evolution, and disease. Here, we show that oRG expansion in mice, ferrets, and human cerebral organoids requires cyclin-dependent kinase 6 (CDK6), the mutation of which causes primary microcephaly via an unknown mechanism. In a mouse model in which increased Hedgehog signaling expands oRGs and intermediate progenitor cells and induces neocortical folding, CDK6 loss selectively decreased oRGs and abolished neocortical folding. Remarkably, this function of CDK6 in oRG expansion did not require its kinase activity, was not shared by the highly similar CDK4 and CDK2, and was disrupted by the mutation causing microcephaly. Therefore, our results indicate that CDK6 is conserved to promote oRG expansion; that oRGs are necessary for neocortical folding; and that defects in oRG expansion may cause primary microcephaly. Significance StatementPrimary microcephaly, a disorder in which the brain is smaller than normal at birth, disproportionately affects the neocortex. Although outer radial glia (oRGs) expansion is hypothesized to be important in neocortical expansion and folding, it remains unknown whether oRGs are necessary for neocortical folding and whether defective oRGs cause microcephaly. Moreover, how oRGs expand is not well understood. A mutation in CDK6 causes microcephaly via an unknown mechanism. Here, we show that CDK6 promotes oRG expansion and neocortical folding. This function of CDK6 does not require its kinase activity but is disrupted by a mutation that causes microcephaly. Our findings show that CDK6 is conserved to expand oRGs and provide evidence that oRG defects disrupt neocortical growth and folding.

neuroscience↗

bioRxiv2022

Genome-wide analysis of dendritic cell differentiation

Dendritic cells (DCs) are immune sentinel cells that comprise antigen-presenting conventional DCs (cDCs) and cytokine-producing plasmacytoid DCs (pDCs). Cytokine Flt3 ligand (Flt3L) supports the proliferation of hematopoietic progenitors, and is also necessary and sufficient for DC differentiation. Here we characterized the spontaneous differentiation of a Flt3L-dependent murine progenitor cell line into pDCs and "myeloid" cDCs (cDC2s), and interrogated it using a genome-wide CRISPR/Cas9 dropout screen. The screen revealed multiple regulators of DC differentiation including the glycosylphosphatidylinositol transamidase complex, the Nieman-Pick type C cholesterol transporter and arginine methyltransferase Carm1; the role of Carm1 in pDC and cDC2 differentiation was confirmed by conditional targeting in vivo. We also found that negative regulators of mTOR signaling, including the subunits of TSC and GATOR1 complexes, restricted progenitor growth but enabled DC differentiation. The results provide a comprehensive forward genetic analysis of DC differentiation, and help explain how the opposing processes of proliferation and differentiation could be driven by the same cytokine.

immunology↗

bioRxiv2022

Cross-link assisted spatial proteomics to map sub-organelle proteomes and membrane protein topology

The specific functions of cellular organelles and sub-compartments depend on their protein content, which can be characterized by spatial proteomics approaches. However, many spatial proteomics methods are limited in their ability to resolve organellar sub-compartments, profile multiple sub-compartments in parallel, and/or characterize membrane-associated proteomes. Here, we develop a cross-link assisted spatial proteomics (CLASP) strategy that addresses these shortcomings. Using human mitochondria as a model system, we show that CLASP can elucidate spatial proteomes of all mitochondrial sub-compartments and provide topological insight into the mitochondrial membrane proteome in a single experiment. Biochemical and imaging-based follow-up studies demonstrate that CLASP allows discovering mitochondria-associated proteins and revising previous protein sub-compartment localization and membrane topology data. This study extends the scope of cross-linking mass spectrometry beyond protein structure and interaction analysis towards spatial proteomics, establishes a method for concomitant profiling of sub-organelle and membrane proteomes, and provides a resource for mitochondrial spatial biology.

systems biology↗

bioRxiv2022

An endolysosome membrane transformation process for engulfment of autophagosomes independently of ESCRT

Endolysosome, usually regarded as the cellular recycling bin, digests materials from multiple origins. The materials from different origins are delivered to endolysosome through the vesicle fusion, kiss-and-run mechanisms, CMA or microautophagy. However, it remains unknown whether endolysosome can receive cargo via other ways. Here, we reported another endolysosomal process for receiving materials. In this, endolysosome receives materials by extending two arms which embraces autophagosomes and engulfs the autophagosomes ultimately, but not via the conventional fusion of autophagosomes with endolysosomes. We named this process as LEA (lysosome eats autophagosome) and the endolysosome arms as LF (endolysosome filopodia) provisionally. The endolysosomes with engulfed autophagosomes (LEA endolysosomes) get more autophagosomes via the fusion with other LEA endolysosomes. The engulfed autophagosomes are labelled by F-actin on their membranes and have ER protein Sec61{beta} and peroxisome protein Pex14 inside their lumens, but mitochondria are excluded outside endolysosome. Our discovery of LEA and LF reveal an unidentified endolysosome transformation process which is responsible for receiving cargoes.

cell biology↗

bioRxiv2022

Spatial, Quantitative and Functional Deconstruction of Virus and Host Protein Interactions Inside Intact Cytomegalovirus Particles

Herpesviruses assemble large enveloped particles that are difficult to characterize structurally due to their size, fragility and complex proteome with partially amorphous nature. Here we use cross-linking mass spectrometry and quantitative proteomics to derive a spatially resolved interactome map of intact human cytomegalovirus virions. This enabled the de novo allocation of 32 viral proteins into four spatially resolved virion layers, each organized by a dominant viral scaffold protein. The viral protein UL32 engages with all layers in an N-to-C-terminal radial orientation bridging nucleocapsid to viral membrane. In addition, we observed the layer-specific recruitment of 82 host proteins, a subset of which are constitutively and selectively incorporated via specific host-virus interactions. We uncover how the recruitment of PP1 phosphatase and 14-3-3 proteins by UL32 affects early and late steps during viral biogenesis. Collectively, this study provides global structural insights into the native configuration of virus and host protein interactions inside herpesvirus particles.

microbiology↗

bioRxiv2022

C-Jun N-terminal Kinase Promotes Stress Granule Assembly and Neurodegeneration in C9orf72-mediated ALS and FTD

Stress granules (SGs), RNA/protein condensates assembled in cells under stress, are believed to play a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, how SG assembly is regulated and related to pathomechanism is incompletely understood. Here, we show that ER stress activates JNK via IRE1 in fly and cellular models of C9orf72-mediated ALS/FTD (c9ALS/FTD), the most common genetic form of ALS/FTD. Furthermore, activated JNK promotes SG assembly induced by poly(GR) and poly(PR), two toxic proteins implicated in c9ALS/FTD, by promoting the transcription of G3BP1, a key SG protein. Consistent with these findings, JNK or IRE1 inhibition reduced SG formation, G3BP1 mRNA and protein levels, and neurotoxicity in cells overexpressing poly(GR) and poly(PR) or neurons derived from c9ALS/FTD patient induced pluripotent stem cells (iPSCs). Our findings connect ER stress, JNK, and SG assembly in a unified pathway contributing to c9ALS/FTD neurodegeneration.

neuroscience↗

bioRxiv2022

Mutually exclusive spectral biclustering and its applications in cancer subtyping

Many soft biclustering algorithms have been developed and applied to various biological and biomedical data analyses. However, until now, few mutually exclusive (hard) biclustering algorithms have been proposed although they can be extremely useful for identify disease or molecular subtypes based on genomic or transcriptomic data. We considered the biclustering problem of expression matrices as a bipartite graph partitioning problem and developed a novel biclustering algorithm, MESBC, based on Dhillons spectral method to detect mutually exclusive biclusters. MESBC simultaneously detects relevant features (genes) and corresponding subgroups, and therefore automatically uses the signature features for each subtype to perform the clustering, improving the clustering performance. MESBC could accurately detect the pre-specified biclusters in simulations, and the identified biclusters were highly consistent with the true labels. Particularly, in setting with high noise, MESBC outperformed existing NMF and Dhillons method and provided markedly better accuracy. Analysis of two TCGA datasets (LUAD and BRAC cohorts) revealed that MESBC provided similar or more accurate prognostication (i.e., smaller p value) for overall survival in patients with breast and lung cancer, respectively, compared to the existing, gold-standard subtypes for breast (PAM50) and lung cancer (integrative clustering). In the TCGA lung cancer patients, MESBC detected two clinically relevant, rare subtypes that other biclustering or integrative clustering algorithms could not detect. These findings validated our hypothesis that MESBC could improve molecular subtyping in cancer patients and potentially facilitate better individual patient management, risk stratification, patient selection, therapeutic assignments, as well as better understanding gene signatures and molecular pathways for development of novel therapeutic agents.

bioinformatics↗

bioRxiv2022

Extended Brain Sources Estimation via Unrolled Optimization Neural Network

Electroencephalography (EEG)/Magnetoencephalography (MEG) source imaging aims to seek an estimation of underlying activated brain sources to explain the observed EEG/MEG recording. Due to the ill-posed nature of inverse problem, solving EEG/MEG Source Imaging (ESI) requires design of regularization or prior terms to guarantee a unique solution. Traditionally, the design of regularization terms is based on preliminary assumptions on the spatio-temporal structure in the source space. In this paper, we propose a novel paradigm to solve the ESI problem by using Unrolled Optimization Neural Network (UONN) (1) to improve the efficiency compared to traditional iterative algorithms; (2) to establish a data-driven way to model the source solution structure instead of using hand-crafted regularizations; (3) to learn the hyperparameter automatically in a data-driven manner. The proposed framework is based on unfolding of the iterative optimization algorithm with neural network modules. The proposed new learning framework is the first one that use the unrolled optimization neural network to solve the ESI problem. The newly designed framework can effectively learn the source extents pattern and achieved significantly improved performance compared to benchmark algorithms.

bioengineering↗

bioRxiv2022

Engineering of Sr33 and Sr50 plant immune receptors to alter recognition specificity and autoactivity

Plants possess cytoplasmic immune receptors called nucleotide-binding leucine-rich repeat receptors (NLRs) that recognize the presence of a pathogen through a range of mechanisms: direct binding of effectors or indirect recognition of effector actions. The direct binding of effectors has been shown to be mediated through the NLRs leucine-rich repeat (LRR) domain. Accurate prediction of amino acids involved in these direct interactions can greatly enhance understanding of effector recognition and inform efforts to engineer new resistance. In this study, we utilized two homologous NLR resistance genes from wheat, Sr33 and Sr50, that recognize distinct effectors by directly binding to them through their LRR domain. While the effector recognized by Sr50 is known and described as AvrSr50, the effector recognized by Sr33 remains unknown. Through a combination of phylogenetics, allele diversity analysis in the LRR and structural modeling, we identified the amino acids in Sr50 likely to physically interact with its effector. Mutation of these sites helped identify 12 amino acids we hypothesized to be sufficient to mediate effector binding in Sr50. Changing these 12 corresponding amino acids in Sr33, we showed AvrSr50-dependent initiation of cell death in wheat protoplasts and Nicotiana benthamiana. Furthermore, we were able to pinpoint and change amino acid residues that govern autoactivity of Sr50 in the wheat protoplast cell death assay. These findings are a major advance towards the successful engineering of new effector recognition specificities in direct binder NLRs.

plant biology↗