Biology subjects

Stacey, G.

Publications and source records attributed to Stacey, G..

13 recordsLinked to original sources

bioRxiv2026

The chitin receptor-interacting protein LIK1 regulates extracellular ATP signaling via interaction with P2K1 in Arabidopsis thaliana

Previously, the chitin receptor-interacting protein kinase LIK1 (LysM receptor kinase 1/CERK1-interacting kinase) was shown to play an important role in regulating chitin signaling and plant defense. A limited proteolysis proteomics study revealed several LIK1-derived peptides that showed differential abundance between ATP-treated and mock-treated Arabidopsis samples, suggesting a possible involvement of LIK1 in extracellular ATP (eATP) signaling. To explore this possibility, LIK1 mutants were obtained and examined for their response to ATP. The results showed that mutations in LIK1 significantly reduced the expression of eATP-responsive genes. In addition, LIK1 was found to interact with the eATP receptor P2K1 and to be phosphorylated by it. The LIK1 protein was localized to the plasma membrane and its gene expression appeared to be ubiquitous. Collectively, these findings indicate that LIK1 not only contributes to chitin signaling but also participates in eATP signaling, highlighting its potential role as a shared component in multiple signaling pathways to regulate plant responses to diverse internal and external cues.

plant biology↗

bioRxiv2025

Global analysis of membrane protein S-acylation in the model plant Arabidopsis thaliana

Protein S-acylation is the addition of fatty acids to the cysteine residues in a protein, catalyzed by protein S-acyltransferases (PATs). Despite extensive research on protein S-acylation in animals, our understanding of this process in plants remains limited. In this study, we sought to characterize the S-acylproteome of membrane proteins in Arabidopsis and identify potential substrates for two important plant immunity-related PATs (PAT5 and PAT9). To achieve this, S-acylated membrane proteins were first enriched via our optimized acyl-biotinyl exchange strategies at both the protein-level and peptide-level. The enriched samples were then analyzed by label-free quantitative liquid chromatography-mass spectrometry. The results from the two enrichment methods demonstrated that they were complementary in identifying S-acylated proteins and S-acylation sites. Using these methods, over 2500 S-acylation sites in more than 2000 putative S-acylated proteins were identified. Proteins involved in vesicle trafficking, plant phosphorylation, immune responses, and signal transduction pathways were significantly enriched. Additionally, certain amino acid patterns surrounding the S-acylation sites were identified. Comparisons of the S-acylproteomes between the wild type and the PAT5 and PAT9 mutants revealed over 100 potential substrates for both S-acyltransferases. The high quality of our data was supported by the significant overlap with the previously reported data and successful experimental verification of selected candidate proteins. Overall, our study revealed a well-represented S-acylproteome for Arabidopsis (especially its membrane proteins) and identified potential substrates for PAT5 and PAT9. These findings will facilitate the functional characterization of S-acylated proteins in plants.

plant biology↗

bioRxiv2025

Phloem-Specific Translational Regulation of Soybean Nodulation: Insights from a Phloem-Targeted TRAP-Seq Approach

Soybean (Glycine max) root nodulation is a symbiotic process that requires complex molecular and cellular coordination. The phloem plays a crucial role not only in nutrient transport but also in long-distance signaling that regulates nodulation. However, the molecular mechanisms underlying phloem-specific regulation during nodulation remain poorly characterized. Here, we developed a phloem-specific Translating Ribosome Affinity Purification sequencing (TRAP-seq) system to investigate the translational dynamics of phloem-associated genes during nodulation. Using a phloem-specific promoter (Glyma.01G040700) combined with the GAL4-UAS amplification system, we successfully captured the translatome of soybean root phloem at early (72 hours post-inoculation, hpi) and late (21 days post-inoculation, dpi) nodulation stages. Differential expression analysis revealed dynamic translational reprogramming, with 2,636 differentially expressed genes (DEGs) at 72 hpi and 8,422 DEGs at 21 dpi. Gene ontology and pathway enrichment analyses showed stage-specific regulatory shifts, including early activation of ethylene and defense pathways and late-stage enhancement of nutrient transport and vascular development. Transcription factor analysis identified GmbHLH121 as a key phloem-specific regulator of nodulation. Functional validation using RNAi knockdown and overexpression experiments demonstrated that GmbHLH121 negatively regulates nodule formation, likely acting downstream of or independently from early nodulation signaling pathways. Additionally, we uncovered dynamic regulation of cell wall-modifying enzymes (PME and PMEI) in the phloem, implicating their role in modulating plasmodesmata permeability and facilitating symplastic connectivity during nodulation. Our findings highlight the critical role of phloem-mediated translational regulation in coordinating root nodulation, emphasizing the phloem as an active regulatory hub for long-distance signaling and symbiotic efficiency.

plant biology↗

bioRxiv2024

Assembly, comparative analysis, and utilization of a single haplotype reference genome for soybean

Cultivar Williams 82 has served as the reference genome for the soybean research community since 2008, but is known to have areas of genomic heterogeneity among different sub-lines. This work provides an updated assembly (version Wm82.a6) derived from a specific sub-line known as Wm82-ISU-01 (seeds available under USDA accession PI 704477). The genome was assembled using Pacific BioSciences HiFi reads and integrated into chromosomes using HiC. The 20 soybean chromosomes assembled into a genome of 1.01Gb, consisting of 36 contigs. The genome annotation identified 48,387 gene models, named in accordance with previous assembly versions Wm82.a2 and Wm82.a4. Comparisons of Wm82.a6 with other near-gapless assemblies of Williams 82 reveal large regions of genomic heterogeneity, including regions of differential introgression from the genotype Kingwa within approximately 30 Mb and 25 Mb segments on chromosomes 03 and 07, respectively. Additionally, our analysis revealed a previously unknown large ([~]20 Mb) heterogeneous region in the pericentromeric region of chromosome 12, where Wm82.a6 matches the Williams haplotype while the other two near-gapless assemblies do not match the haplotype of either parent of Williams 82. In addition to the Wm82.a6 assembly, we also assembled the genome of soybean line Fiskeby III, a rich resource for abiotic stress resistance genes. A genome comparison of Wm82.a6 with Fiskeby III revealed the nucleotide and structural polymorphisms between the two genomes within a QTL region for iron deficiency chlorosis resistance. The Wm82.a6 and Fiskeby III genomes described here will enhance comparative and functional genomics capacities and applications in the soybean community.

plant biology↗

bioRxiv2024

The Rhizobial effector NopT targets Nod factor receptors to regulate symbiosis in Lotus japonicus

It is well-documented that type-III effectors are required by Gram-negative pathogens to directly target different host cellular pathways to promote bacterial infection. However, in the context of legume-rhizobium symbiosis, the role of rhizobial effectors in regulating plant symbiotic pathways remains largely unexplored. Here, we show that NopT, a YopT-type cysteine protease of Sinorhizobium fredii NGR234 directly targets the plants symbiotic signaling pathway by associating with two Nod factor receptors (NFR1 and NFR5 of Lotus japonicus). NopT inhibits cell death triggered by co-expression of NFR1/NFR5 in Nicotiana benthamiana. Full-length NopT physically interacts with NFR1 and NFR5. NopT proteolytically cleaves NFR5 both in vitro and in vivo, but can be inactivated by NFR1 as a result of phosphorylation. NopT plays an essential role in mediating rhizobial infection in L. japonicus. Autocleaved NopT retains the ability to cleave NFR5 but no longer interacts with NFR1. Interestingly, genomes of certain Sinorhizobium species only harbor nopT genes encoding truncated proteins without the autocleavage site. These results reveal an intricate interplay between rhizobia and legumes, in which a rhizobial effector protease targets NFR5 to suppress symbiotic signaling. NFR1 appears to counteract this process by phosphorylating the effector. This discovery highlights the role of a bacterial effector in regulating a signaling pathway in plants and opens up the perspective of developing kinase-interacting proteases to fine-tune cellular signaling processes in general. One-sentence summaryThe rhizobial effector NopT and two Nod factor receptors of Lotus japonicus, NFR1 and NFR5, mutually interact to precisely regulate rhizobial symbiosis.

plant biology↗

bioRxiv2023

Soybean RIN4 represents a mechanistic link between plant immune and symbiotic signaling

The legume-rhizobium symbiosis represents a unique and beneficial interaction between legumes and nitrogen-fixing soil bacteria, called rhizobia. The initiation and development of this symbiosis is complex and begins with recognition of key molecular signals, produced by the plant and its symbiont, which determine symbiotic compatibility. Current data suggest that the invading symbiont initially triggers plant immune responses that are subsequently suppressed. Hence, there is growing evidence that features of plant immunity may be relevant to symbiotic establishment. RIN4 is a key immune regulator in plants, regulating basal immunity and it is also targeted by pathogen effector proteins that either confer susceptibility or resistance, depending on the presence of the appropriate resistance protein. Surprisingly, we found that RIN4 was rapidly phosphorylated upon rhizobial inoculation of soybean root hairs. RNAi silencing and mutant studies indicate that RIN4 expression is essential for effective nodulation of soybean. RIN4 phosphorylation occurs within a fifteen amino acid motif, which is highly conserved within the Fabales (legumes) and Rosales orders, which comprise species capable of nitrogen-fixing endosymbiosis with rhizobia. RIN4 proteins mutated in this conserved phosphorylation site failed to support efficient soybean nodulation. Phosphorylation of this site is mediated by the symbiotic receptor-like kinase, SymRK, a well-studied member of the symbiotic signaling pathway. The data implicate RIN4 phosphorylation as a key mediator of rhizobial compatibility, interconnecting symbiotic and immune signaling pathways. SignificanceThe nitrogen fixing legume-rhizobium symbiosis is a cornerstone of sustainable agriculture, with ongoing efforts to transfer this unique ability to non-leguminous crop plants. Plants are surrounded by a myriad of microbes in the soil, and, therefore, require constant surveillance in order to distinguish between a pathogen or symbiont. Plants monitor for specific molecular signals that indicate pathogen or symbiont presence. We show that RIN4, a key immune regulator, plays an essential role in promoting the development of the symbiotic nitrogen-fixing relationship between soybean and its compatible symbiont Bradyrhizobium japonicum. Therefore, RIN4 is likely a key player in mediating the appropriate response upon infection by friend or foe.

plant biology↗

bioRxiv2022

The mitogen-activated protein kinase kinase kinase, ILK5, regulates plant purinergic receptor-mediated, innate immunity

Mitogen-activated protein (MAP) kinase signaling cascades play important roles in the regulation of eukaryotic defense against various pathogens. Activation of the extracellular ATP (eATP) receptor P2K1 triggers MAP kinase 3 and 6 (MPK3/6) phosphorylation, which leads to elevated defense responses in Arabidopsis. However, the mechanism by which P2K1 activates the MAPK cascade is unclear. In this study, we identified Raf-like MAPKKK ILK5 as a downstream substrate of the P2K1 kinase. P2K1 phosphorylates ILK5 on serine 192. The interaction between P2K1 and ILK5 was confirmed both in vitro and in planta and their interaction was enhanced by ATP treatment. Similar to P2K1 expression, ILK5 expression levels were highly induced by treatment with ATP, flg22, Pseudomonas syringae pv. tomato DC3000, and various abiotic stresses, such as wounding. ILK5 interacts with both MKK4 and MKK5, but only MKK5 is phosphorylated by ILK5. Moreover, phosphorylation of MPK3/6 was significantly reduced upon ATP treatment in ilk5 mutant plants, relative to wild-type. The ilk5 mutant plants showed higher susceptibility to P. syringae pathogen infection relative to wild-type plants. Plants expressing only the mutant ILK5S192A protein, lacking kinase activity, did not activate the MAPK cascade upon ATP addition. Taken together, the results suggest that eATP activation of P2K1 results in transphosphorylation of the Raf-like MAPKKK ILK5, which subsequently triggers the MAPK cascade, culminating in activation of MAPK3 and 6 associated with an elevated innate immunity response. Significance statementPathogens invasion and subsequent wound stress activates extracellular ATP-mediated purinergic signaling cascades, a danger associated molecular pattern (DAMP) signal, which induces phosphorylation of MAPKs. Previous studies revealed that the P2K1 purinergic receptor increases MPK3/6 phosphorylation in response to eATP signaling cascades in Arabidopsis. However, the specific mechanism by which this occurs remains unknown. Here, we describe the isolation and characterization of Raf-like MAPKKK ILK5 (Integrin-linked Kinase 5) as a downstream substrate of P2K1 kinase activity. Initiation of an eATP-dependent signaling pathway by phosphorylation of ILK5 with subsequent activation of MKK5, leading to activation of MPK3/6 and downstream events is crucial to the plant innate immunity response.

plant biology↗

bioRxiv2022

Extracellular ATP plays an important role in systemic wound response activation

Mechanical wounding occurs in plants during biotic (e.g., herbivore or pathogen attack) or abiotic (e.g., wind damage or freezing) stresses and is associated with the activation of multiple signaling pathways. These initiate many wound responses at the wounded tissues, as well as trigger long-distance signaling pathways that activate wound responses in tissues that were not affected by the initial wounding event (termed systemic wound response). Among the different systemic signals activated by wounding are electric signals, calcium and reactive oxygen species (ROS) waves, and different plant hormones such as jasmonic acid. The release of glutamate from cells at the wounded tissues was recently proposed to trigger several different systemic signal transduction pathways via glutamate-like receptors (GLRs). However, the role of another important compound released from cells during wounding (i.e., extracellular ATP; eATP) in triggering systemic responses is not clear. Here we show that eATP that accumulates in wounded leaves and is sensed by the purinoreceptor kinase P2K is required for the activation of the ROS wave during wounding. Application of eATP to unwounded leaves triggered the ROS wave, and the activation of the ROS wave by wounding or eATP application was suppressed in mutants deficient in P2K (i.e., p2k1-3, p2k2, and p2k1-3p2k2). In addition, the expression of several systemic wound response transcripts was suppressed in mutants deficient in P2K during wounding. Our findings reveal that in addition to sensing glutamate via GLRs, eATP sensed by P2Ks is playing a key role in the triggering of systemic wound responses in plants. One sentence summaryExtracellular ATP plays an important role in triggering the ROS wave and systemic transcriptomics responses during wounding.

plant biology↗

bioRxiv2021

Large-scale miRNA-Target Data Analysis to Discover miRNA Co-regulation Network of Abiotic Stress Tolerance in Soybeans

Although growing evidence shows that microRNA (miRNA) regulates plant growth and development, miRNA regulatory networks in plants are not well understood. Current experimental studies cannot characterize miRNA regulatory networks on a large scale. This information gap provides a good opportunity to employ computational methods for global analysis and to generate useful models and hypotheses. To address this opportunity, we collected miRNA-target interactions (MTIs) and used MTIs from Arabidopsis thaliana and Medicago truncatula to predict homologous MTIs in soybeans, resulting in 80,235 soybean MTIs in total. A multi-level iterative bi-clustering method was developed to identify 483 soybean miRNA-target regulatory modules (MTRMs). Furthermore, we collected soybean miRNA expression data and corresponding gene expression data in response to abiotic stresses. By clustering these data, 37 MTRMs related to abiotic stresses were identified including stress-specific MTRMs and shared MTRMs. These MTRMs have gene ontology (GO) enrichment in resistance response, iron transport, positive growth regulation, etc. Our study predicts soybean miRNA-target regulatory modules with high confidence under different stresses, constructs miRNA-GO regulatory networks for MTRMs under different stresses and provides miRNA targeting hypotheses for experimental study. The method can be applied to other biological processes and other plants to elucidate miRNA co-regulation mechanisms.

plant biology↗

bioRxiv2020

Integrated Collection of Stem Cell Bank data, a data portal for standardized stem cell information

The last decade has witnessed an extremely rapid increase in the number of newly established stem cell lines. However, due to the lack of a standardized format, data exchange among stem cell line resources has been challenging, and no system can search all stem cell lines across resources worldwide. To solve this problem, we have developed Integrated Collection of Stem Cell Bank data (ICSCB) (http://icscb.stemcellinformatics.org/), the largest database search portal for stem cell line information, based on the standardized data items and terms of the MIACARM framework. Currently, ICSCB can retrieve >16,000 cell lines from four major data resources in Europe, Japan, and the United States. ICSCB is automatically updated to provide the latest cell line information, and its integrative search helps users collect cell line information for over 1,000 diseases including many rare diseases worldwide, which has been a formidable task, thereby distinguishing itself from other database search portals.

bioinformatics↗

bioRxiv2020

Genome-wide Association Studies Reveal Important Candidate Genes for the Bacillus pumilus TUAT-1-Arabidopsis thaliana Interaction

The plant growth promoting bacterium (PGPB) Bacillus pumilus TUAT-1 is an indole acetic acid producer that can increase plant growth. Inoculation with this strain has been shown to confer greater plant tolerance to drought and saline conditions. Although the ability of TUAT-1 to enhance plant growth is well documented, little is known about what mechanisms underlie the plant response to this bacterium. Applying genome-wide association study (GWAS), we evaluated the interaction between TUAT-1 and Arabidopsis thaliana, screening 288 plant ecotypes for root architecture traits comparing non-inoculated and inoculated plants. Most of the ecotypes were significantly affected by TUAT-1 inoculation (66.7%) for at least one of the root traits measured. For example, some ecotypes responded positively increasing root growth while others showed reduced growth upon inoculation. A total of 96 ecotypes (33.3%) did not respond significantly to PGPB inoculation. These results are consistent with the widely reported strain-genotype specificity shown in many plant-microbe interactions. The GWAS analysis revealed significant SNPs associated to specific root traits leading to identification of several genes putatively involved in enabling the Bacillus pumilus TUAT-1 and A. thaliana association and contributing to plant growth promotion. Our results show that root architecture features are genetic separable traits associated with plant growth in association with TUAT-1. Our findings validate previous reported genes involved in Bacillus spp.-plant interaction, growth promotion and highlight potential genes involved in plant microbe interaction. We suggest that plant-bacterial interaction and the plant growth promotion are quantitative and multigenic traits. This knowledge expands our understanding of the functional mechanisms driving plant growth promotion by PGPB.

plant biology↗

bioRxiv2019

S-Acylation of plant immune receptors mediates immune signaling in plasma membrane nanodomains

S-Acylation is a reversible protein post-translational modification mediated by Protein S-acyltransferases (PATs). Here, we demonstrate that three plant immune receptors P2K1 (DORN1), FLS2 and CERK1, representing three distinct families of receptor like-kinases, are S-acylated by Arabidopsis PAT5 and PAT9 on the plasma membrane (PM). Mutations in Atpat5 and Atpat9 resulted in an elevated plant immune response, increased phosphorylation and decreased degradation of FLS2, P2K1 and CERK1 during immune signaling. Mutation of key cysteine residues S-acylated in these receptors resulted in a similar phenotype as exhibited by Atpat5/Atpat9 mutant plants. The data indicate that S-acylation also controls localization of the receptors in distinct PM nanodomains and, thereby, controls the formation of specific protein-protein interactions. Our study reveals that S-acylation plays a critical role in mediating spatiotemporal dynamics of plant receptors within PM nanodomains, suggesting a key role for this modification in regulating the ability of plants in respond to external stimuli.

plant biology↗

bioRxiv2019

Profiling the E. coli Membrane Interactome Captured in Peptidisc Libraries

Protein-correlation-profiling (PCP), in combination with quantitative proteomics, has emerged as a high-throughput method for the rapid identification of dynamic protein complexes in native conditions. While PCP has been successfully applied to soluble proteomes, characterization of the membrane interactome has lagged, partly due to the necessary use of detergents to maintain protein solubility. Here, we apply the peptidisc, a one-size fits all membrane mimetic, for the capture of the Escherichia coli cell envelope proteome and its high-resolution fractionation in the absence of detergent. Analysis of the SILAC-labeled peptidisc library via PCP allows generation of over 4900 possible binary interactions out of >700,000 random associations. Using well-characterized membrane protein systems such as the SecY translocon, the Bam complex and the MetNI transporter, we find our dataset very useful for identifying transient and surprisingly novel protein interactions. For example, we discover a trans-periplasmic super-complex comprising subunits of the Bam and Sec translocons, including membrane-bound chaperones YfgM and PpiD. We identify RcsF and OmpA as bone fide interactors of BamA, and we show that interaction of MetQ with the ABC transporter MetNI depends on its N-terminal lipid anchor. We also discover NlpA as a novel interactor of MetNIQ. Most of these interactions are largely undetected by standard detergent-based purification. Together, the peptidisc workflow applied to the proteomic field is emerging as a promising novel approach to characterize membrane protein interactions under native expression conditions and without genetic manipulation.

biochemistry↗