Biology subjects

Walley, J.

Publications and source records attributed to Walley, J..

11 recordsLinked to original sources

bioRxiv2026

A co-proteomic view of metabolite-specific interactions in the Botrytis cinerea-Arabidopsis pathosystem

To successfully infect their myriad hosts, generalist plant pathogens must tolerate a vast arsenal of plant specialized defense metabolites. To understand how host-specific metabolites influence plant-generalist pathogen interactions, we conducted a co-proteomic analysis of both Arabidopsis thaliana and Botrytis cinerea proteomes from the same samples during early infection. The Arabidopsis proteomic responses to Botrytis center around induction and suppression of defense metabolite pathways, particularly camalexin and glucosinolates. Several Botrytis proteins involved in key virulence pathways were induced within 32-48 hours, including potential defense metabolite detoxification proteins. Co-proteomic analysis using a panel of Arabidopsis genotypes with differing glucosinolate profiles revealed that disruptions to the glucosinolate pathway had broad changes on the Arabidopsis proteome, and that Botrytis induces specific proteins in response to presence/absence of Arabidopsis defense metabolites. Among the proteins that were induced quickly on infection and linked to the presence of glucosinolates, we validated a novel isothiocyanate hydrolase in Botrytis, BcSaxA, that catabolizes isothiocyanates in vitro. Gene expression data further indicated BcSaxA is expressed only in dicot hosts containing isothiocyanates. Our study describes a highly dynamic host proteome during infection with Botrytis and elucidates metabolite-specific infection strategies for a generalist pathogen.

plant biology↗

bioRxiv2025

Dynamic ASK1 proximity networks uncover SCF-dependent and noncanonical roles in ABA and drought adaptation

Plants rely on rapid proteome remodeling to withstand fluctuating environmental conditions, yet how the ubiquitin system dynamically coordinates these multilayered responses remains unclear. Here we define the in vivo proximity interactome of ARABIDOPSIS SKP1-LIKE 1 (ASK1), the core adaptor of SCF ubiquitin ligases, under acute abscisic acid (ABA) signaling and prolonged drought. TurboID-based proximity labeling coupled with quantitative proteomics revealed that ASK1 assembles highly condition-specific protein networks, distinguishing canonical SCF modules from broader noncanonical associations with transcriptional, chromatin, translational, vesicle-trafficking, and proteostasis machinery. Acute ABA exposure rapidly recruits F-box proteins and ABA-responsive transcription factors while engaging ribosomal and chromatin modules, whereas drought drives ASK1 into expanded proteostasis and stress-signaling assemblies, including chaperone-cochaperone systems, transcriptional repressors, and autophagy-endomembrane components. Global proteomics shows that ASK1 overexpression enhances accumulation of drought-protective and ABA-responsive proteins while repressing immune and ROS-scavenging pathways, indicating a shift in resource allocation. Together, these results describe ASK1 as a multifunctional proteostasis and signaling hub that integrates SCF-dependent and SCF-independent pathways to coordinate transcriptional, translational, and proteolytic reprogramming during plant adaptation to stress.

plant biology↗

bioRxiv2024

Transcription factor binding site divergence across maize inbred lines drives transcriptional and phenotypic variation

Regulatory elements are important constituents of plant genomes that have shaped ancient and modern crops. Their identification, function, and diversity in crop genomes however are poorly characterized, thus limiting our ability to harness their power for further agricultural advances using induced or natural variation. Here, we use DNA affinity purification-sequencing (DAP-seq) to map transcription factor (TF) binding events for 200 maize TFs belonging to 30 distinct families and heterodimer pairs in two distinct inbred lines historically used for maize hybrid plant production, providing empirical binding site annotation for 5.3% of the maize genome. TF binding site comparison in B73 and Mo17 inbreds reveals widespread differences, driven largely by structural variation, that correlate with gene expression changes. TF binding site presence-absence variation helps clarify complex QTL such as vgt1, an important determinant of maize flowering time, and DICE, a distal enhancer involved in herbivore resistance. Modification of TF binding regions via CRISPR-Cas9 mediated editing alters target gene expression and phenotype. Our functional catalog of maize TF binding events enables collective and comparative TF binding analysis, and highlights its value for agricultural improvement.

plant biology↗

bioRxiv2023

Improved Super-Resolution Ribosome Profiling Revealed Prevalent Translation of Upstream ORFs and Small ORFs in Arabidopsis

A crucial step in functional genomics is identifying actively translated open reading frames (ORFs) that link biological functions. The challenge lies in identifying short ORFs, as they are greatly impacted by data quality and depth. Here, we improved the coverage of super-resolution Ribo-seq in Arabidopsis, revealing uncharacterized translation events in nucleus-, chloroplast-, and mitochondria-encoded genes. We identified 7,751 unconventional translation events, including 6,996 upstream ORFs (uORFs) and 209 downstream ORFs on annotated protein-coding genes, as well as 546 ncORFs on presumed non-coding RNAs. Proteomics data confirmed the production of stable proteins from some of the unannotated translation events. We present evidence of active translation on primary transcripts of tasiRNAs (TAS1-4) and microRNAs (pri-miR163, pri-miR169), and periodic ribosome stalling supporting co-translational decay. Additionally, we developed a method for identifying extremely short uORFs, including 370 minimum uORF (AUG-stop), and 2,984 tiny uORFs (2-10 aa), as well as 681 uORFs that overlap with each other. Remarkably, these short uORFs exhibit strong translational repression as longer uORFs. We also systematically discovered 594 uORFs regulated by alternative splicing, suggesting widespread isoform-specific translational control. Finally, these prevalent uORFs are associated with numerous important pathways. In summary, our improved Arabidopsis translational landscape provides valuable resources to study gene expression regulation.

genomics↗

bioRxiv2023

A soybean rust effector protease suppresses host immunity and cleaves a 3-deoxy-7-phosphoheptulonate synthase

The devastating soybean rust (SBR) pathogen, Phakopsora pachyrhizi, encodes many secreted proteins, but only two have been functionally characterized for their roles in rust virulence. Here, we demonstrate that transient expression of P. pachyrhizi effector candidate 15 (PpEC15), an aspartic protease, leads to enhanced bacterial growth in planta, suppression of callose deposition, reduced expression of plant defense-related marker genes and suppression of pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species (ROS). Stable expression of PpEC15 in soybean suppresses PAMP-induced ROS production and enhances bacterial growth, indicating that, collectively, PpEC15 suppresses host and non-host innate immune responses. Yeast-two-hybrid and proximity labeling identified putative PpEC15 interacting partners including a peptide-chain release factor (PCRF), a NAC83 (NAM, ATAF, and CUC) transcription factor, and a DAHP (3-deoxy-7-phosphoheptulonate) synthase. We further show that PpEC15 can cleave DAHP but does not cleave PCRF or NAC83. Virus-induced gene silencing of NAC83, PCRF and DAHP altered PAMP-induced ROS production and salicylic acid production, indicating that these proteins may be involved in immune signaling. Collectively, our data show that PpEC15 is conserved across P. pachyrhizi isolates and other economically important rust species and is involved in the suppression of plant basal defense responses. Understanding the role of PpEC15 in P. pachyrhizi virulence will provide a foundation for designing targeted intervention strategies to generate rust-resistant crops.

plant biology↗

bioRxiv2023

Transcription Factor Dynamics in Cross-Regulation of Plant Hormone Signaling Pathways

Cross-regulation between hormone signaling pathways is indispensable for plant growth and development. However, the molecular mechanisms by which multiple hormones interact and co-ordinate activity need to be understood. Here, we generated a cross-regulation network explaining how hormone signals are integrated from multiple pathways in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. To do so we comprehensively characterized transcription factor activity during plant hormone responses and reconstructed dynamic transcriptional regulatory models for six hormones; abscisic acid, brassinosteroid, ethylene, jasmonic acid, salicylic acid and strigolactone/karrikin. These models incorporated target data for hundreds of transcription factors and thousands of protein-protein interactions. Each hormone recruited different combinations of transcription factors, a subset of which were shared between hormones. Hub target genes existed within hormone transcriptional networks, exhibiting transcription factor activity themselves. In addition, a group of MITOGEN-ACTIVATED PROTEIN KINASES (MPKs) were identified as potential key points of cross-regulation between multiple hormones. Accordingly, the loss of function of one of these (MPK6) disrupted the global proteome, phosphoproteome and transcriptome during hormone responses. Lastly, we determined that all hormones drive substantial alternative splicing that has distinct effects on the transcriptome compared with differential gene expression, acting in early hormone responses. These results provide a comprehensive understanding of the common features of plant transcriptional regulatory pathways and how cross-regulation between hormones acts upon gene expression.

plant biology↗

bioRxiv2022

Integration of multi-omics data reveals interplay between brassinosteroid and TORC signaling in Arabidopsis

Brassinosteroids (BR) and Target of Rapamycin Complex (TORC) are two major actors coordinating plant growth and stress responses. BRs function through a signaling pathway to extensively regulate gene expression and TORC is known to regulate translation and autophagy. Recent studies revealed that these two pathways crosstalk, but a system-wide view of their interplay is still missing. Thus, we quantified the level of 23,975 transcripts, 11,183 proteins, and 27,887 phosphorylation sites in wild-type Arabidopsis and in mutants with altered levels of either BRASSINOSTEROID INSENSITIVE 2 (BIN2) or REGULATORY ASSOCIATED PROTEIN OF TOR 1B (RAPTOR1B), two key players in BR and TORC signaling, respectively. We found that perturbation of BIN2 or RAPTOR1B levels affects a common set of gene-products involved in growth and stress responses. Furthermore, we used the multi-omic data to reconstruct an integrated signaling network. We screened 41 candidate genes identified from the reconstructed network and found that loss of function mutants of many of these proteins led to an altered BR response and/or modulated autophagy activity. Altogether, these results establish a predictive network that defines different layers of molecular interactions between BR-or TORC-regulated growth and autophagy.

plant biology↗

bioRxiv2022

Brassinosteroids modulate autophagy through phosphorylation of RAPTOR1B by the GSK3-like kinase BIN2 in Arabidopsis

Macroautophagy/autophagy is a conserved recycling process that maintains cellular homeostasis during environmental stress. Autophagy is negatively regulated by TARGET OF RAPAMYCIN (TOR), a nutrient-regulated protein kinase that in plants is activated by several phytohormones, leading to increased growth. However, the detailed molecular mechanisms by which TOR integrates autophagy and hormone-signaling are poorly understood. Here, we show that TOR modulates brassinosteroid (BR)-regulated plant growth and stress-response pathways. Active TOR was required for full BR-induced growth in Arabidopsis thaliana. Autophagy was constitutively up-regulated upon blocking BR biosynthesis or signaling, and down-regulated by increasing the activity of the BR pathway. BRASSINOSTEROID-INSENSITIVE 2 (BIN2) kinase, a GSK3-like kinase functioning as a negative regulator in BR signaling, directly phosphorylated Regulatory-Associated Protein of TOR 1B (RAPTOR1B), a substrate-recruiting subunit in the TOR complex, at a conserved serine residue within a typical BIN2 phosphorylation motif. Mutation of RAPTOR1B serine 916 to alanine, to block phosphorylation by BIN2, repressed autophagy and increased phosphorylation of the TOR substrate autophagy-related protein 13a (ATG13a). By contrast, this mutation had only a limited effect on growth. We present a model in which RAPTOR1B is phosphorylated and inhibited by BIN2 when BRs are absent, activating the autophagy pathway. When BRs signal and inhibit BIN2, RAPTOR1B is thus less inhibited by BIN2 phosphorylation. This leads to increased TOR activity and ATG13a phosphorylation, and decreased autophagy activity. Our studies define a new mechanism by which coordination between BR and TOR signaling pathways helps to maintain the balance between plant growth and stress responses.

plant biology↗

bioRxiv2021

Phosphorylation dynamics in a flg22 induced, heterotrimeric G protein dependent signaling network in Arabidopsis thaliana reveals a candidate PP2A phosphatase involved in AtRGS1 trafficking

flg22 is a 22-amino peptide released from bacterial flagellin, a Microbe-Associated Molecular Pattern (MAMP) that is recognized by the plant cell as a signal indicating that bacteria are present. On its own, flg22 initiates a rapid increase in cytoplasmic calcium, extracellular reactive oxygen species, and activation of a Mitogen Activated Protein Kinase (MAPK) cascade, all of which are activated within 15 minutes after the cell perceives flg22. Here we show a massive change in protein abundance and phosphorylation state of the Arabidopsis root cell proteome within this 15-minute duration in wildtype and a mutant deficient in G-protein coupled signaling. Integration of phosphoproteome with protein-protein interactome data followed by network topology analyses discovered that many of the flg22-induced phosphoproteome changes fall on proteins that comprise the G protein interactome and on the most highly populated hubs of the immunity network. Approximately 95% of the phosphorylation changes in the G-protein interactome depend on a functional heterotrimeric G protein complex, some occur on proteins that interact directly with components of G-coupled signal transduction. One of these is ATB, a substrate-recognition sub-unit of the PP2A Ser/Thr phosphatase and an interactor to Arabidopsis thaliana REGULATOR OF G SIGNALING 1 protein (AtRGS1), a 7-transmembrane spanning modulator of the nucleotide-binding state of the core G protein complex. AtRGS1 is phosphorylated by BAK1, a component of the flg22 receptor, to initiate AtRGS1 endocytosis. A null mutation of ATB confers high basal endocytosis of AtRGS1, suggesting sustained phosphorylated status. Loss of ATB confers traits associated with loss of AtRGS1. Because the basal level of AtRGS1 is lower in the atb null mutant in a proteasome-dependent manner, we propose that phosphorylation-dependent endocytosis of AtRGS1 is part of a mechanism to degrade AtRGS1 which then sustains activation of the G protein complex. Thus, the role of ATB is now established as a central component of phosphorylation-dependent regulation of system dynamics in innate immunity.

systems biology↗

bioRxiv2021

Interplay between brassinosteroids and TORC signaling in Arabidopsis revealed by integrated multi-dimensional analysis

Brassinosteroids (BR) and Target of Rapamycin Complex (TORC) are two major processes coordinating plant growth and stress responses. BRs function through a signaling pathway to extensively regulate gene expression and TORC is known to regulate translation and autophagy. Recent studies revealed that these two pathways crosstalk, but a system-wide view of their interplay is still missing. Thus, we performed transcriptome, proteome, and phosphoproteome profiling of Arabidopsis mutants with altered levels of either BIN2 or RAPTOR1B, two key players in BR and TORC signaling, respectively. We found that perturbation of BIN2 or RAPTOR1B levels affects a common set of gene-products involved in growth and stress responses. Additionally, we performed Multiplexed Assay for Kinase Specificity (MAKS), which provided a system-wide view of direct BIN2 substrates. Furthermore, phosphoproteomic data was used to reconstruct a kinase-signaling network and to identify novel proteins dependent on BR and/or TORC signaling pathways. Loss of function mutants of many of these proteins led to an altered BR response and/or modulated autophagy activity. Altogether, these results provide genome-wide evidence for crosstalk between BR and TORC signaling and established a kinase signaling network that defines the molecular mechanisms of BR and TORC interactions in the regulation of plant growth/stress balance.

plant biology↗

bioRxiv2019

Integrated Multi-omic Framework of the Plant Response to Jasmonic Acid

Understanding the systems-level actions of transcriptional responses to hormones provides insight into how the genome is reprogrammed in response to environmental stimuli. Here, we investigate the signaling pathway of the hormone jasmonic acid (JA), which controls a plethora of critically important processes in plants and is orchestrated by the transcription factor MYC2 and its closest relatives in Arabidopsis thaliana. We generated an integrated framework of the response to JA that spans from the activity of master and secondary-regulatory transcription factors, through gene expression outputs and alternative splicing to protein abundance changes, protein phosphorylation and chromatin remodeling. We integrated time series transcriptome analysis with (phospho)proteomic data to reconstruct gene regulatory network models. These enable us to predict previously unknown points of crosstalk from JA to other signaling pathways and to identify new components of the JA regulatory mechanism, which we validated through targeted mutant analysis. These results provide a comprehensive understanding of how a plant hormone remodels cellular functions and plant behavior, the general principles of which provide a framework for analysis of cross-regulation between other hormone and stress signaling pathways.

plant biology↗