Biology subjects

Thomas, H. R.

Publications and source records attributed to Thomas, H. R..

10 recordsLinked to original sources

bioRxiv2026

Salicylic acid mediates graft incompatibility by repressing auxin-dependent vascular reconnection in tomato

Graft incompatibility limits the combination of scions and rootstocks, yet the signals that block vascular reconnection remain poorly understood. Using incompatible tomato-pepper (Solanum lycopersicum-Capsicum annuum) grafts, we found that salicylic acid (SA) overaccumulates at the graft junction, predominantly in the rootstock, while incompatible scions exhibit enhanced pattern-triggered immunity. SA-deficient nahG-expressing tomato failed to rescue incompatibility and instead showed severely impaired self-graft healing, indicating that successful tissue reunion requires an optimal, rather than minimal, SA response. Defense-associated genes remained activated in failed grafts regardless of SA accumulation, placing SA downstream of incompatibility determination. Exogenous SA application phenocopied incompatibility, blocking xylem reconnection, inducing cell death, and suppressing auxin signaling. Incompatible grafts similarly displayed reduced auxin accumulation and response at the graft junction. Exogenous auxin partially rescued xylem reconnection in incompatible grafts but failed to activate cambial regulator genes or fully restore compatibility. We propose that graft compatibility depends on SA-auxin homeostasis, where moderate SA supports wound-associated defense and regeneration, whereas excessive SA suppresses the auxin-dependent program required for xylem differentiation. These findings identify SA as a dose-dependent regulator of graft healing in tomato and link excessive immune activation to failed vascular regeneration.

plant biology↗

bioRxiv2026

Somatic-to-germline transmission of horizontally acquired extrachromosomal circular DNA in Brassica graft chimeras

Whether somatically acquired traits can contribute to heritable variation has remained an open question in plant biology for over a century. Here, using graft-induced periclinal chimeras between Brassica juncea and Brassica oleracea, we identified extrachromosomal circular DNA (eccDNA) as mobile genetic elements capable of crossing histological boundaries and entering the germline. We demonstrated that grafting could promote the horizontal transfer of eccDNA between somatic cell layers, enabling its stable maintenance in recipient tissues and permitting transmission through sexual reproduction across multiple generations. We found that transmitted eccDNA is non-randomly distributed across the genome, preferentially originating from gene-dense regions, and enriched for features associated with molecular persistence, such as inverted repeats, hairpin-forming sequences, and autonomously replicating sequence (ARS) consensus motifs. These genetic regions conferred replication competence, as evidenced by autonomous propagation in an ARS-less heterologous yeast system. Sexual progeny carrying graft-acquired eccDNA exhibited reproducible and lineage-dependent alterations in leaf morphology and drought tolerance, accompanied by coordinated transcriptional reprogramming. Notably, a subset of inherited eccDNA remained transcriptionally active in progeny, producing transcripts absent from self-grafted controls. Our findings establish eccDNA as heritable extrachromosomal elements that link graft-mediated somatic genetic transfer with stable germline transmission, thereby expanding the molecular scope of heritable variation in plants and providing a conceptual framework for graft-based trait transmission.

plant biology↗

bioRxiv2025

Primary metabolism underpins the execution of immune responses in different tissues of the same plant

Cell-surface perception of microbes triggers a range of rapid immune responses in plants that include the induction of cell isolation by plasmodesmal closure, the production of reactive oxygen species, and changes to gene expression. Here, we identify that some of these immune response are differentially executed in leaves of different ages in the same plant. We observed that when compared to mature leaves, young, expanding leaves do not close their plasmodesmata, have a reduced transcriptional response to immune elicitors, and are more susceptible to a bacterial pathogen. Disconnecting leaf age from physiology, we determined that both plasmodesmal closure and the magnitude of transcriptional responses are dependent on whether the leaf is a carbon sink or a carbon source. To probe the relevance of differential regulation of plasmodesmata in sink and source tissues, we forced plasmodesmal closure in young sink leaves and found this perturbed the normal outputs of growth and defence. Thus, we propose that sink leaves do not close their plasmodesmata during immune reponses to prioritise carbon use for growth over defence, and consequently that primary metabolism underpins different immune response profiles in different leaves of the same plant.

plant biology↗

bioRxiv2025

Engineering precision zebrafish alleles of human disease

Animal models of human diseases are an essential component of understanding disease pathogenesis and serve as preclinical models for therapeutic evaluation. Recently human patient genome sequencing has defined unique patient variants that result in disease states with different phenotypes than those observed with null alleles. The UAB Center for Precision Animal Modeling (CPAM) serves to analyze patient variant pathogenicity and disease mechanisms through the generation of animal models. We have optimized a zebrafish gene editing platform to successfully generate 11 patient variants (first round: NF1 R1276Q, NF1 G484R, VMA21 G55V, SPOP D144N, SGO1 K23E, Pex10 H310D, and FKRP C318Y; second round: NF1 R681*, NF1 M992del, P53 R175H, and PKD2 L656W) and 1 research allele (p53 K120R). We used CRISPR/Cas9 guide directed cleavage along with single-stranded oligodeoxynucleotide (ssODNs) repair templates to generate these models. We evaluated multiple oligo orientations and sizes, but did not find a unified consensus orientation or size that significantly impacted efficiency, emphasizing the need to empirically evaluate multiple variations for the best homology directed repair (HDR) rate. We determined PCR amplicon Next Generation Sequencing (NGS) evaluation of HDR efficiency at the F0 embryo level is best for determining the ideal guide and oligo combination. Further NGS evaluation of DNA from progeny from F0s (germline derived), not F0 biopsy DNA, is essential to identify germline transmitting founders. Surprisingly we find that most founders exhibit a jackpot effect in the germ line but not in the somatic tissue. We found NGS superior to using ICE (Inference of CRISPR Edits) for determining HDR frequency. When applicable, allelic-specific PCR or allelic specific restriction digestion can be used to genotype mutation carrying F1 generation animals, however we demonstrated that false positives occur. Further, we successfully used high resolution melting curve analysis (HRMA) to differentiate and identify F1 animals with patient variants.

genetics↗

bioRxiv2024

Disrupted development of sensory systems and the cerebellum in a zebrafish ebf3a mutant

Mutations in the transcription factor EBF3 results in a neurodevelopmental disorder, and studies in animal models indicate that it has a critical role in neuronal differentiation. The molecular pathways and neuron types disrupted by its loss, however, have not been thoroughly investigated. Nor have the outcomes of these changes on behavior and brain activity. Here, we generated and characterized a zebrafish ebf3a loss-of-function mutant. We discovered morphological and neural phenotypes, including an overall smaller brain size, particularly in the hypothalamus, cerebellum, and hindbrain. Brain function was also compromised, with activity strongly increased in the cerebellum and abnormal behavior at baseline and in response to visual and acoustic stimuli. From RNA-sequencing of developing larvae, notable changes included significant downregulation of genes that mark olfactory sensory neurons, the lateral line, and cerebellar Purkinje neurons. This study sets the stage for determining which downstream pathways underlie the emergence of the observed phenotypes and establishes multiple strong phenotypes that could form the basis of a drug screen.

neuroscience↗

bioRxiv2024

Re-analysis of mobile mRNA datasets highlights challenges in the detection of mobile transcripts from short-read RNA-Seq data

Short-read RNA-Seq analyses of grafted plants have led to the proposal that large numbers of mRNAs move over long distances between plant tissues, acting as potential signals. The detection of transported transcripts by RNA-Seq is both experimentally and computationally challenging, requiring successful grafting, delicate harvesting, rigorous contamination controls and data processing approaches that can identify rare events in inherently noisy data. Here, we perform a meta-analysis of existing datasets and examine the associated bioinformatic pipelines. Our analysis reveals that technological noise, biological variation and incomplete genome assemblies give rise to features in the data that can distort the interpretation. Taking these considerations into account, we find that a substantial number of transcripts that are currently annotated as mobile are left without support from the available RNA-Seq data. Whilst several annotated mobile mRNAs have been validated, we cannot exclude that others may be false positives. The identified issues may also impact other RNA-Seq studies, in particular those using single nucleotide polymorphisms (SNPs) to detect variants.

plant biology↗

bioRxiv2024

Plasmodesmal closure elicits stress responses

Plant cells are connected to their neighbors via plasmodesmata facilitating the exchange of nutrients and signaling molecules. During immune responses, plasmodesmata close, but how this contributes towards a full immune response is unknown. To investigate this, we developed two transgenic lines with which we could induce plasmodesmal closure independently of immune elicitors, using the over-active CALLOSE SYNTHASE3 allele icals3m and the C-terminus of PDLP1 to drive callose deposition at plasmodesmata. Induction of plasmodesmal closure increased the expression of stress responsive genes, salicylic acid accumulation and resistance to Pseudomonas syringae DC3000. More homogeneous plasmodesmal closure using icals3m also led to the accumulation of starch and sugars, decreased leaf growth, as well as hypersusceptibility to Botrytis cinerea. Based on the profile of responses, we conclude that plasmodesmal closure itself activates stress signaling, raising questions of what signals mediate this response and whether these responses occur in all circumstances when plasmodesmata close.

plant biology↗

bioRxiv2024

Graft incompatibility between pepper and tomato can be attributed to genetic incompatibility between diverged immune systems

O_LIGraft compatibility is the capacity of two plants to form cohesive vascular connections. Tomato and pepper are incompatible graft partners; however, the underlying cause of graft rejection between these two species remains unknown. C_LIO_LIWe diagnosed graft incompatibility between tomato and diverse pepper varieties based on weakened biophysical stability, decreased growth, and persistent cell death using trypan blue and TUNEL assays. Transcriptomic analysis of cell death in the junction was performed using RNA-sequencing, and molecular signatures for incompatible graft response were characterized based on meta-transcriptomic comparisons with other biotic processes. C_LIO_LIWe show that tomato is broadly incompatible with diverse pepper cultivars. These incompatible graft partners activate prolonged transcriptional changes that are highly enriched for defense processes. Amongst these processes was broad NLR upregulation and hypersensitive response. Using transcriptomic datasets for a variety of biotic stress treatments, we identified a significant overlap in the genetic profile of incompatible grafting and plant parasitism. In addition, we found over 1000 genes that are uniquely upregulated in incompatible grafts. C_LIO_LIBased on NLR overactivity, DNA damage, and prolonged cell death we have determined that tomato and pepper graft incompatibility is likely caused by a form of genetic incompatibility, which triggers a hyperimmune-response. C_LI

plant biology↗

bioRxiv2021

Evolutionarily conserved genetic interactions between nphp-4 and bbs-5 mutations exacerbate ciliopathy phenotypes

Primary cilia are sensory and signaling hubs with a protein composition that is distinct from the rest of the cell due to the barrier function of the transition zone (TZ) at the base of the cilium. Protein transport across the TZ is mediated in part by the BBSome, and mutations disrupting TZ and BBSome proteins cause human ciliopathy syndromes. Ciliopathies have phenotypic variability even among patients with identical genetic variants, suggesting a role for modifier loci. To identify potential ciliopathy modifiers, we performed a mutagenesis screen on nphp-4 mutant C. elegans and uncovered a novel allele of bbs-5. Nphp-4;bbs-5 double mutant worms have phenotypes not observed in either individual mutant strain. To test whether this genetic interaction is conserved, we also analyzed zebrafish and mice mutants. While Nphp4 mutant zebrafish appeared overtly normal, Bbs5 mutants exhibited scoliosis. When combined, Nphp4;Bbs5 double mutant zebrafish did not exhibit synergistic effects, but the lack of a phenotype in Nphp4 mutants makes interpreting these data difficult. In contrast, viable Nphp4;Bbs5 double mutant mice were not obtained and there were fewer mice than expected carrying three mutant alleles. Additionally, postnatal loss of Bbs5 in mice using a conditional allele compromised survival when combined with a Nphp4 allele. As cilia are formed in the double mutant mice, the exacerbated phenotype is likely a consequence of disrupted ciliary signaling. Collectively, these data support an evolutionarily conserved genetic interaction between Bbs5 and Nphp4 alleles that may contribute to the variability in ciliopathy phenotypes.

genetics↗

bioRxiv2020

Atypical Mediofrontal Theta Oscillations Underlying Cognitive Control in Kindergarteners with Autism Spectrum Disorder

BackgroundChildren with autism spectrum disorder (ASD) often exhibit deficits in cognitive control. Neuroimaging approaches have implicated disruptions to medio-frontal cortex (MFC) structure and function. However, prior work has not directly tested whether young children with ASD exhibit disruptions to task-related theta oscillations thought to arise from the MFC. MethodsForty-three children with ASD and 24 age- and gender-matched typically developing (TD) peers performed a child-friendly Go/No-go task while 64-channel electroencephalography (EEG) was recorded at kindergarten-entry. Time-frequency approaches were employed to assess the magnitude of mediofrontal theta oscillations immediately following error (vs. correct) responses ("early theta"), as well as later emerging theta oscillations ("late theta"). We tested whether error-related mediofrontal theta oscillations differed as a function of diagnosis (ASD/typical) and timing (early/late theta). Additionally, links to social and academic outcomes were tested. ResultsOverall, children showed increased theta power following error vs. correct responses. Compared to TD children, children with ASD exhibited a selective reduction in error-related mediofrontal theta power during the late theta time window. There were no significant group differences for early theta power. Moreover, reduced error-related theta power during the late, but not early, time window significantly predicted poorer academic and social skills. ConclusionsKindergarteners with ASD demonstrated a selective reduction in error-related mediofrontal theta power during a relatively late time window, which is consistent with impairments in specific cognitive processes that recruit top-down control. Targeting these particular cognitive control processes via intervention prior to school-entry may promote more successful functional outcomes for children with ASD.

neuroscience↗