Biology subjects

Coaker, G.

Publications and source records attributed to Coaker, G..

17 recordsLinked to original sources

bioRxiv2026

Experimental Context Shapes PRR-Mediated Immune Output Sensitivity in Arabidopsis

Pattern recognition receptors (PRRs) mediate plant immune responses by detecting extracellular immunogenic patterns, including microbe-associated molecular patterns (MAMPs). PRR signaling is commonly assessed using assays such as reactive oxygen species (ROS) bursts, cytosolic calcium influx, mitogen-activated protein kinase (MAPK) activation, and seedling growth inhibition (SGI), which are performed in distinct experimental systems, including seedlings grown on artificial media and soil-grown rosettes. Here, we systematically compare receptor kinase immune outputs triggered by the bacterial MAMPs elf18 and flg22 in Arabidopsis thaliana seedlings and rosettes across a range of concentrations. Rosettes exhibited greater sensitivity than seedlings in ROS assays, whereas cytosolic calcium responses measured using the Aeqcyt/pMAQ2 reporter were stronger in seedlings, correlating with reduced reporter transcript accumulation in rosette tissue. MAPK activation was consistently stronger in rosettes, whereas SGI assays revealed higher sensitivity to elf18 than flg22 in seedlings despite flg22 inducing stronger early signaling outputs. Together, these results demonstrate that canonical PRR-mediated immune outputs are differentially sensitive to experimental context and should not be interpreted as interchangeable measures of immune activation. These findings highlight the importance of considering experimental conditions when comparing immune responses across assays and developmental stages.

plant biology↗

bioRxiv2026

Spatial organization of plant defense at the infection front

Plants lack specialized immune cells and instead rely on coordinated cellular responses to restrict pathogen invasion while preserving tissue integrity. How plant immunity spatially organizes these responses remains unclear. Using live-cell reporters in Arabidopsis infected with Pseudomonas syringae, we show that effector-triggered immunity, superimposed on pattern-triggered immunity, establishes a sustained yet spatially confined immune architecture at the infection front. Defense activation persists for days but remains restricted to a narrow ring of cells surrounding viable bacterial microcolonies. Over time, immune activity spreads to adjacent layers, forming a coordinated, multilayered defense zone. This zonation extends beyond transcriptional activation to polarized callose deposition at pathogen-facing cell walls, reinforcing a localized containment boundary that limits pathogen spread. Consistent with previous single-reporter studies, simultaneous visualization of salicylic acid (SA) and jasmonic acid (JA) biosynthesis and response markers reveals a radial hormone gradient, with SA-enriched cells proximal to bacterial colonies and JA-enriched cells in surrounding regions. Individual cells predominantly activate one pathway, indicating that SA-JA antagonism is resolved through spatial compartmentalization across neighboring cells. Together, these findings establish immune zonation as a strategy for robust pathogen containment while minimizing collateral tissue damage. Significance statementPlants lack specialized immune cells and must coordinate defense responses across tissues to contain infection. Although plant immune responses are known to be locally activated, how they are organized across cells to form a stable containment boundary has remained unclear. Using live imaging of fluorescent bacterial microcolonies, we show that plant immunity forms a spatially organized defense zone surrounding viable infection sites. Within this zone, distinct defense activities are arranged in a radial gradient, with salicylic acid-associated responses enriched in cells near the pathogen and jasmonic acid-associated responses in surrounding cells. Rather than being resolved solely within individual cells, antagonistic hormone pathways are separated across neighboring cells. This spatial organization reveals immune zonation as a strategy for confining pathogen spread while preserving tissue integrity.

plant biology↗

bioRxiv2025

The spectrum of diversity of nucleotide-binding leucine-rich repeat (NLR) genes in citrus and its relatives

Genomic clusters of immune genes, including those encoding nucleotide-binding leucine-rich repeat (NLR) proteins, are a model for exploring the dynamics of genomic regions in flux. Rapid sequence evolution of immune genes, including NLRs, and variation in their gene content, may enable long-lived plants, which lack adaptive immune systems, to keep pace with the fast evolution of pathogens. To explore the patterns and processes shaping the evolution of NLR gene content in a genus of long-lived tree species, we unified the annotation of NLR genes across 11 accessions (or 15 haplotypes) from the genus Citrus and its relatives, including three new diploid genome assemblies. A majority of NLRs were arranged in genomic clusters composed of paralogous genes, typically from a single gene family. Even larger clusters, with 10 or more NLRs, were limited to genes derived from one or few gene families. These patterns suggested that genomic clustering of NLRs arose through local expansion of phylogenetically related NLRs, but the mechanistic processes driving these patterns are not clear. Local gene duplication can be mediated by multiple processes, including transposon-mediated gene capture and subsequent proliferation, and non-allelic repair of double stranded breaks, including unequal recombination. Examples of retrotransposon-mediated duplication of NLRs were identified, but these were not sufficient to explain massive regional expansions. Signatures of unequal recombination are challenging to identify. Focusing on recent lineage-specific sequence duplications, at least one case of unequal recombination was identified, supporting a role for unequal recombination in shaping genomic variation in these regions.

evolutionary biology↗

bioRxiv2025

mamp-ml: A deep learning approach to epitope immunogenicity in plants

Eukaryotes detect biomolecules through surface-localized receptors, key signaling components. A subset of receptors survey for pathogens, induce immunity, and restrict pathogen growth. Comparative genomics of both hosts and pathogens has unveiled vast sequence variation in receptors and potential ligands, creating an experimental bottleneck. We have developed mamp-ml, a machine learning framework for predicting plant receptor-ligand interactions. We leveraged existing functional data from over two decades of foundational research, together with the large protein language model ESM-2, to build a pipeline and model that predicts immunogenic outcomes using a combination of receptor-ligand features. Our model achieves 73% prediction accuracy on a held-out test set, even when an experimental structure is lacking. Our approach enables high-throughput screening of LRR receptor-ligand combinations and provides a computational framework for engineering plant immune systems.

immunology↗

bioRxiv2024

Tomato roots exhibit distinct, development-specific responses to bacterial-derived peptides

O_LIPlants possess cell-surface recognition receptors that detect molecular patterns from microbial invaders and initiate an immune response. Understanding the conservation of pattern-triggered immunity within different plant organs and across species is crucial to its sustainable and effective use in plant disease management but is currently unclear. C_LIO_LIWe examined the activation and immune response patterns of three pattern recognition receptors (PRRs: SlFLS2, SlFLS3, and SlCORE) in different developmental regions of roots and in leaves of multiple accessions of domesticated and wild tomato (Solanum lycopersicum and S. pimpinellifolium) using biochemical and genetic assays. C_LIO_LIRoots from different tomato accessions differed in the amplitude and dynamics of their immune response, but all exhibited developmental-specific PTI responses in which the root early differentiation zone was the most sensitive to molecular patterns. PRR signaling pathways also showed distinct but occasionally overlapping responses downstream of each immune receptor in tomato roots. C_LIO_LIThese results reveal that each PRR initiates a unique PTI pathway and suggest that the specificity and complexity of tomato root immunity are tightly linked to the developmental stage, emphasizing the importance of spatial and temporal regulation in PTI. C_LI

plant biology↗

bioRxiv2024

Unlocking expanded flagellin perception through rational receptor engineering

The surface-localized receptor kinase FLS2 detects the flg22 epitope from bacterial flagella. FLS2 is conserved across land plants, but bacterial pathogens exhibit polymorphic flg22 epitopes. Most FLS2 homologs possess narrow perception ranges, but four with expanded perception ranges have been identified. Using diversity analyses, AlphaFold-Multimer modeling, and amino acid properties, key residues enabling expanded recognition were mapped to FLS2s concave surface, interacting with the co-receptor and polymorphic flg22 residues. Synthetic biology enabled engineering of expanded recognition from QvFLS2 (Quercus variabilis) and FLS2XL (Vitis riparia) into homologs with canonical perception. Evolutionary analyses across three plant orders showed residues under positive selection aligning with those binding the co-receptor and flg22s C-terminus, suggesting more alleles with expanded perception exist. Our experimental data enabled the identification of specific receptor amino acid properties and AlphaFold3 metrics that facilitate predicting FLS2-flg22 recognition. This study provides a framework for rational receptor engineering to enhance pathogen restriction.

plant biology↗

bioRxiv2024

Gene editing of the E3 ligase PIRE1 fine-tunes ROS production for enhanced bacterial disease resistance in tomato

Reactive oxygen species (ROS) accumulation is required for effective plant defense. Accumulation of the Arabidopsis NADPH oxidase RBOHD is regulated by phosphorylation of a conserved C-terminal residue (T912) leading to ubiquitination by the RING E3 ligase PIRE. Arabidopsis PIRE knockouts exhibit enhanced ROS production and resistance to the foliar pathogen Pseudomonas syringae. Here, we identified 170 PIRE homologs, which emerged in Tracheophytes and expanded in Angiosperms. We investigated the role of Solanum lycopersicum (tomato) PIRE homologs in regulating ROS production, RBOH stability, and disease resistance. Mutational analyses of residues corresponding to T912 in the tomato RBOHD ortholog, SlRBOHB, affected protein accumulation and ROS production in a PIRE-dependent manner. Using CRISPR-cas9, we generated mutants in two S. lycopersicum PIRE homologs (SlPIRE). SlPIRE1 edited lines (Slpire1) in the tomato cultivar M82 displayed enhanced ROS production upon treatment with flg22, an immunogenic epitope of flagellin. Furthermore, Slpire1 exhibited decreased disease symptoms and bacterial accumulation when inoculated with foliar bacterial pathogens Pseudomonas syringae and Xanthomonas campestris. However, Slpire1 exhibited similar levels of colonization as wild type upon inoculation with diverse soilborne pathogens. These results indicate that phosphorylation and ubiquitination crosstalk regulate RBOHs in multiple plant species, and PIRE is a promising target for foliar disease control. This study also highlights the pathogen-specific role of PIRE, indicating its potential for targeted manipulation to enhance foliar disease resistance without affecting root-associated interactions, positioning PIRE as a promising target for improving overall plant health.

plant biology↗

bioRxiv2024

Direct binding of a fungal effector by the wheat RWT4 tandem kinase activates defense

Plants have intricate innate immune receptors that detect pathogens. Research has intensely focused on two receptor classes recognizing external and internal threats. Recent research has identified a class of disease resistance proteins called tandem kinase proteins (TKPs). We investigated RWT4, a wheat TKP that confers resistance to the devastating fungal pathogen Magnaporthe oryzae. We established a rice protoplast system, revealing RWT4 specifically recognizes the AvrPWT4 effector, leading to the transcription of defense genes and inducing cell death. RWT4 possesses both kinase and pseudokinase domains, with its kinase activity essential for defense. RWT4 directly interacts with and transphosphorylates AvrPWT4. Biolayer interferometry revealed both RWT4 kinase and pseudokinase regions bind the effector. Sequence similarity and structural modeling revealed an integrated partial kinase duplication in RWT4s kinase region as critical for effector interaction and defense activation. Collectively, these findings demonstrate that TKPs can directly bind a recognized effector, leading to downstream defense activation.

plant biology↗

bioRxiv2023

Evolutionary dynamics of proteinaceous MAMPs reveals intrabacterial antagonism of plant immune perception

Plants and animals detect biomolecules termed Microbe-Associated Molecular Patterns (MAMPs) and induce immunity. Agricultural production is severely impacted by pathogens which can be controlled by transferring immune receptors. However, most studies use a single MAMP epitope and the impact of diverse multi-copy MAMPs on immune induction is unknown. Here we characterized the epitope landscape from five proteinaceous MAMPs across 4,228 plant-associated bacterial genomes. Despite the diversity sampled, natural variation was constrained and experimentally testable. Immune perception in both Arabidopsis and tomato depended on both epitope sequence and copy number variation. For example, Elongation Factor Tu is predominantly single copy and 92% of its epitopes are immunogenic. Conversely, 99.9% of bacterial genomes contain multiple Cold Shock Proteins and 46% carry a non-immunogenic form. We uncovered a new mechanism for immune evasion, intrabacterial antagonism, where a non-immunogenic Cold Shock Protein blocks perception of immunogenic forms encoded in the same genome. These data will lay the foundation for immune receptor deployment and engineering based on natural variation. Significance StatementPlants recognize pathogens as non-self using innate immune receptors. Receptors on the cell surface can recognize amino acid epitopes present in pathogen proteins. Despite many papers investigating receptor signaling, the vast majority use a single epitope. Here, we analyzed the natural variation across five different epitopes and experimentally characterized their perception in plants. We highlight the importance of analyzing all epitope copies within a pathogen genome. Through genetic and biochemical analyses, we revealed a mechanism for immune evasion, intrabacterial antagonism, where a non-immunogenic epitope blocks perception of immunogenic forms encoded in a single genome. These data can directly inform disease control strategies by enabling prediction of receptor utility and deployment for current and emerging pathogens.

microbiology↗

bioRxiv2022

The oomycete MAMP, arachidonic acid, and an Ascophyllum nodosum-derived plant biostimulant induce defense metabolome remodeling in tomato

Arachidonic acid (AA) is an oomycete-derived MAMP capable of eliciting robust defense responses and inducing resistance in plants. Similarly, extract (ANE) from the brown seaweed Ascophylum nodosum, a plant biostimulant that contains AA, can also prime plants for defense against pathogen challenge. A previous parallel study comparing the transcriptomes of AA and ANE root-treated tomato demonstrated significant overlap in transcriptional profiles, a shared induced resistance phenotype, and changes in accumulation of various defense-related phytohormones. In this work, untargeted metabolomic analysis via liquid chromatography-mass spectrometry was conducted to investigate the local and systemic metabolome-wide remodeling events elicited by AA- and ANE-root treatment in tomato. Our study demonstrated AA and ANEs capacity to locally and systemically alter the metabolome of tomato with enrichment of chemical classes and accumulation of metabolites associated with defense-related secondary metabolism. AA and ANE root-treated plants showed enrichment of fatty acyl-glycosides and strong modulation of hydroxycinnamic acids and derivatives. Identification of specific metabolites whose accumulation was affected by AA and ANE treatment revealed shared metabolic changes related to ligno-suberin biosynthesis and the synthesis of phenolic compounds. This study highlights the extensive local and systemic metabolic changes in tomato induced by treatment with a fatty acid MAMP and a seaweed-derived plant biostimulant with implications for induced resistance and crop improvement.

plant biology↗

bioRxiv2022

Single-cell profiling of complex plant responses to Pseudomonas syringae infection

Plant response to pathogen infection varies within a leaf, yet this heterogeneity is not well resolved. We exposed Arabidopsis to Pseudomonas syringae or mock treatment and profiled >11,000 individual cells using single-cell RNA sequencing. Integrative analysis of cell populations from both treatments identified distinct pathogen responsive cell clusters exhibiting transcriptional responses ranging from immunity to susceptibility. Pseudotime analyses through pathogen infection revealed a continuum of disease progression from an immune to susceptible state. Confocal imaging of promoter reporter lines for transcripts enriched in immune cell clusters expressed surrounding substomatal cavities colonized or in close proximity to bacterial colonies, suggesting cells within immune clusters represent sites of early pathogen invasion. Susceptibility clusters exhibited more general localization and were highly induced at later stages of infection. Overall, our work uncovers cellular heterogeneity within an infected leaf and provides unique insight into plant differential response to infection at a single-cell level.

plant biology↗

bioRxiv2022

Overlapping local and systemic defense induced by an oomycete fatty acid MAMP and brown seaweed extract in tomato

Eicosapolyenoic fatty acids are integral components of oomycete pathogens that can act as microbe-associated molecular patterns (MAMPs) to induce disease resistance in plants. Defense inducing eicosapolyenoic fatty acids include arachidonic (AA) and eicosapentaenoic acids, and are strong elicitors in solanaceous plants with bioactivity in other plant families. Similarly, extracts of the brown seaweed, Ascophyllum nodosum, used in sustainable agriculture as a biostimulant of plant growth, may also induce disease resistance. A. nodosum, similar to other macroalgae, is rich in eicosapolyenoic fatty acids, which comprise as much as 25% of total fatty acid composition. We investigated the response of roots and leaves from AA or a commercial A. nodosum extract (ANE) on root-treated tomatoes via RNA sequencing, phytohormone profiling, and disease assays. AA and ANE significantly altered transcriptional profiles relative to control plants, inducing numerous defense-related genes with both substantial overlap as well as differences in gene expression patterns. Root treatment with AA and, to a lesser extent, ANE also altered both salicylic acid and jasmonic acid levels while inducing local and systemic resistance to oomycete and bacterial pathogen challenge. Thus, our study highlights overlap in both local and systemic defense induced by AA and ANE, with potential for inducing broad-spectrum resistance against pathogens.

plant biology↗

bioRxiv2021

Effectors from a Bacterial Vector-Borne Pathogen Exhibit Diverse Subcellular Localization, Expression Profiles and Manipulation of Plant Defense

Climate change is predicted to increase the prevalence of vector borne disease due to expansion of insect populations. Candidatus Liberibacter solanacearum (CLso) is a phloem-limited pathogen associated with multiple economically important diseases in Solanaceous crops. Little is known about the strategies and pathogenicity factors CLso uses to colonize vector and host. We determined the CLso effector repertoire by predicting SEC secreted proteins across four different CLso haplotypes, investigated effector localization in planta, and profiled effector expression in vector and host. The localization of CLso effectors in Nicotiana revealed diverse eukaryotic subcellular targets. The majority of tested effectors were unable to suppress plant immune responses, indicating they possess unique activities. Expression profiling in tomato and the psyllid Bactericera cockerelli indicated CLso differentially interacts with its host and vector and can switch effector expression in response to the environment. This study reveals CLso effectors possess complex expression patterns, target diverse host organelles and the majority are unable to suppress host immune responses. A mechanistic understanding of Lso effector function will reveal novel targets and provide insight into phloem biology.

plant biology↗

bioRxiv2021

A genetic toolkit for investigating Clavibacter: markerless deletion, permissive site identification and an integrative plasmid

The development of knockout mutants and expression variants are critical for understanding genotype-phenotype relationships. However, advancements of these techniques in Gram-positive actinobacteria have stagnated over the last decade. Actinobacteria in the Clavibacter genus are composed of diverse crop pathogens which cause a variety of wilt and cankering diseases. Here, we present a suite of tools for genetic manipulation in the tomato pathogen C. michiganensis including a markerless deletion system, an integrative plasmid, and an R package for identification of permissive sites for plasmid integration. The vector pSelAct-KO is a recombination based, markerless knockout system that uses dual selection to engineer seamless deletions of a region of interest, providing opportunities for repeated higher-order genetic knockouts. The efficacy of pSelAct-KO was demonstrated in C. michiganensis and confirmed using whole genome sequencing. We developed permissR, an R package to identify permissive sites for chromosomal integration, which can be used in conjunction with pSelAct-Express, a non-replicating integrative plasmid that enables recombination into a permissive genomic location. Expression of eGFP by pSelAct-Express was verified in two candidate permissive regions predicted by permissR in C. michiganensis. These molecular tools are essential advancements for investigating Gram-positive actinobacteria, particularly for important pathogens in the Clavibacter genus.

microbiology↗

bioRxiv2020

ER bodies are induced by Pseudomonas syringae and negatively regulate immunity

ER bodies are endoplasmic reticulum-derived organelles present in plants belonging to the Brassicales order. In Arabidopsis thaliana, ER bodies are ubiquitous in cotyledons and roots, and present only in certain cell types in rosette leaves. However, both wounding and jasmonic acid treatment induce the formation of ER bodies in leaves. Formation of this structure is dependent on the transcription factor NAI1. The main components of the ER bodies are {beta}-glucosidases (BGLUs), enzymes that hydrolyze specialized compounds. In Arabidopsis, PYK10 (BGLU23) and BGLU18 are the most abundant ER body proteins. In this work, we found that ER bodies are downregulated as a consequence of the immune responses induced by bacterial flagellin perception. Arabidopsis mutants defective in ER body formation show enhanced responses upon flagellin perception and enhanced resistance to bacterial infections. Furthermore, the bacterial toxin coronatine induces the formation of de novo ER bodies in leaves and its virulence function is partially dependent on this structure. Finally, we show that performance of the polyphagous beet armyworm herbivore, Spodoptera exigua, increases in plants lacking ER bodies. Altogether, we provide new evidence for the role of the ER bodies in plant immune responses.

plant biology↗

bioRxiv2020

Citrus vascular proteomics highlights the role of peroxidases and serine proteases during Huanglongbing disease progression

Huanglongbing (HLB) is the most devastating and widespread citrus disease. All commercial citrus varieties are susceptible to the HLB-associated bacterium, Candidatus Liberibacter asiaticus (CLas), which resides in the phloem. The phloem is part of the plant vascular system and is involved in sugar transport. To investigate the plant response to CLas, we enriched for proteins surrounding the phloem in an HLB susceptible sweet orange variety, Washington navel (Citrus sinensis (L) Osbeck). Quantitative proteomics revealed global changes in the citrus proteome after CLas inoculation. Plant metabolism and translation were suppressed, while defense-related proteins such as peroxidases, proteases and protease inhibitors were induced in the vasculature. Transcript accumulation and enzymatic activity of plant peroxidases in CLas infected sweet orange varieties under greenhouse and field conditions were assessed. While peroxidase transcript accumulation was induced in CLas infected sweet orange varieties, peroxidase enzymatic activity varied. Specific serine proteases were upregulated in Washington navel in the presence of CLas based on quantitative proteomics. Subsequent activity-based protein profiling revealed increased activity of two serine proteases, and reduced activity of one protease in two C. sinensis sweet orange varieties under greenhouse and field conditions. The observations in the current study highlight global reprogramming of the citrus vascular proteome and differential regulation of enzyme classes in response to CLas infection. These results open an avenue for further investigation of diverse responses to HLB across different environmental conditions and citrus genotypes.

plant biology↗

bioRxiv2020

Three previously characterized resistances to yellow rust are encoded by a single locus Wtk1

The wild emmer wheat (Triticum turgidum ssp. dicoccoides; WEW) yellow (stripe) rust resistance genes Yr15, YrG303 and YrH52 were discovered in natural populations from different geographic locations. They all localize to chromosome 1B but were thought to be non-allelic based on differences in resistance response. We recently cloned Yr15 as a Wheat Tandem Kinase 1 (WTK1) and showed here that these three resistance loci co-segregate in fine-mapping populations and share identical full-length genomic sequence of functional Wtk1. Independent EMS mutagenized susceptible yrG303 and yrH52 lines carried single nucleotide mutations in Wtk1 that disrupted function. A comparison of the mutations for yr15, yrG303 and yrH52 mutants showed that while key conserved residues were intact, other conserved regions in critical kinase subdomains were frequently affected. Thus, we concluded that Yr15-, YrG303- and YrH52-mediated resistances to yellow rust are encoded by a single locus Wtk1. Introgression of Wtk1 into multiple genetic backgrounds resulted in variable phenotypic responses, confirming that Wtk1-mediated resistance is part of a complex immune response network. WEW natural populations subjected to natural selection and adaptation have potential to serve as a good source for evolutionary studies of different traits and multifaceted gene networks. HighlightWe demonstrate that Yr15, YrG303 and YrH52 resistances are encoded by the Wtk1 locus, but express variable resistance responses to yellow rust in a genetic background dependent manner.

genetics↗