Spatiotemporal Gene Expression Research Articles

Computational Approaches to Study Gene Regulatory Networks.

The goal of the gene regulatory network (GRN) inference is to determine the interactions between genes given heterogeneous data capturing spatiotemporal gene expression. Since transcription underlines all cellular processes, the inference of GRN is the first step in deciphering the determinants of the dynamics of biological systems. Here, we first describe the generic steps of the inference approaches that rely on similarity measures and group the similarity measures based on the computational methodology used. For each group of similarity measures, we not only review the existing approaches but also describe specifically the detailed steps of the existing state-of-the-art algorithms.

Non-CpG methylation by DNMT3B facilitates REST binding and gene silencing in developing mouse hearts.

The dynamic interaction of DNA methylation and transcription factor binding in regulating spatiotemporal gene expression is essential for embryogenesis, but the underlying mechanisms remain understudied. In this study, using mouse models and integration of in vitro and in vivo genetic and epigenetic analyses, we show that the binding of REST (repressor element 1 (RE1) silencing transcription factor; also known as NRSF) to its cognate RE1 sequences is temporally regulated by non-CpG methylation. This process is dependent on DNA methyltransferase 3B (DNMT3B) and leads to suppression of adult cardiac genes in developing hearts. We demonstrate that DNMT3B preferentially mediates non-CpG methylation of REST-targeted genes in the developing heart. Downregulation of DNMT3B results in decreased non-CpG methylation of RE1 sequences, reduced REST occupancy, and consequently release of the transcription suppression during later cardiac development. Together, these findings reveal a critical gene silencing mechanism in developing mammalian hearts that is regulated by the dynamic interaction of DNMT3B-mediated non-CpG methylation and REST binding.

Regulation of T-Cell Activation and Differentiation

Enhancers regulate spatiotemporal gene expression and impart cell-specific transcriptional outputs that drive cell identity. Super-enhancers (SEs), also known as stretch-enhancers, are a subset of enhancers especially important for genes associated with cell identity and genetic risk of disease. CD4+ T cells are critical for host defence and autoimmunity. Here we analysed maps of mouse T-cell SEs as a non-biased means of identifying key regulatory nodes involved in cell specification. We found that cytokines and cytokine receptors were the dominant class of genes exhibiting SE architecture in T cells. Nonetheless, the locus encoding Bach2, a key negative regulator of effector differentiation, emerged as the most prominent T-cell SE, revealing a network in which SE-associated genes critical for T-cell biology are repressed by BACH2. Disease-associated single-nucleotide polymorphisms for immune-mediated disorders, including rheumatoid arthritis, were highly enriched for T-cell SEs versus typical enhancers or SEs in other cell lineages. Intriguingly, treatment of T cells with the Janus kinase (JAK) inhibitor tofacitinib disproportionately altered the expression of rheumatoid arthritis risk genes with SE structures. Together, these results indicate that genes with SE architecture in T cells encompass a variety of cytokines and cytokine receptors but are controlled by a 'guardian' transcription factor, itself endowed with an SE. Thus, enumeration of SEs allows the unbiased determination of key regulatory nodes in T cells, which are preferentially modulated by pharmacological intervention. DisclosuresNo relevant conflicts of interest to declare.

Regulation of downstream neuronal genes by proneural transcription factors during initial neurogenesis in the vertebrate brain.

BackgroundNeurons arise in very specific regions of the neural tube, controlled by components of the Notch signalling pathway, proneural genes, and other bHLH transcription factors. How these specific neuronal areas in the brain are generated during development is just beginning to be elucidated. Notably, the critical role of proneural genes during differentiation of the neuronal populations that give rise to the early axon scaffold in the developing brain is not understood. The regulation of their downstream effectors remains poorly defined.ResultsThis study provides the first overview of the spatiotemporal expression of proneural genes in the neuronal populations of the early axon scaffold in both chick and mouse. Overexpression studies and mutant mice have identified a number of specific neuronal genes that are targets of proneural transcription factors in these neuronal populations.ConclusionTogether, these results improve our understanding of the molecular mechanisms involved in differentiation of the first neuronal populations in the brain.Electronic supplementary materialThe online version of this article (doi:10.1186/s13064-016-0077-7) contains supplementary material, which is available to authorized users.

Yin-yang actions of histone methylation regulatory complexes in the brain.

Dysregulation of histone methylation has emerged as a major driver of neurodevelopmental disorders including intellectual disabilities and autism spectrum disorders. Histone methyl writer and eraser enzymes generally act within multisubunit complexes rather than in isolation. However, it remains largely elusive how such complexes cooperate to achieve the precise spatiotemporal gene expression in the developing brain. Histone H3K4 methylation (H3K4me) is a chromatin signature associated with active gene-regulatory elements. We review a body of literature that supports a model in which the RAI1-containing H3K4me writer complex counterbalances the LSD1-containing H3K4me eraser complex to ensure normal brain development. This model predicts H3K4me as the nexus of previously unrelated neurodevelopmental disorders.

Divergence of ectodermal and mesodermal gene regulatory network linkages in early development of sea urchins

Developmental gene regulatory networks (GRNs) are assemblages of gene regulatory interactions that direct ontogeny of animal body plans. Studies of GRNs operating in the early development of euechinoid sea urchins have revealed that little appreciable change has occurred since their divergence ∼90 million years ago (mya). These observations suggest that strong conservation of GRN architecture was maintained in early development of the sea urchin lineage. Testing whether this holds for all sea urchins necessitates comparative analyses of echinoid taxa that diverged deeper in geological time. Recent studies highlighted extensive divergence of skeletogenic mesoderm specification in the sister clade of euechinoids, the cidaroids, suggesting that comparative analyses of cidaroid GRN architecture may confer a greater understanding of the evolutionary dynamics of developmental GRNs. Here I report spatiotemporal patterning of 55 regulatory genes and perturbation analyses of key regulatory genes involved in euechinoid oral-aboral patterning of nonskeletogenic mesodermal and ectodermal domains in early development of the cidaroid Eucidaris tribuloides These results indicate that developmental GRNs directing mesodermal and ectodermal specification have undergone marked alterations since the divergence of cidaroids and euechinoids. Notably, statistical and clustering analyses of echinoid temporal gene expression datasets indicate that regulation of mesodermal genes has diverged more markedly than regulation of ectodermal genes. Although research on indirect-developing euechinoid sea urchins suggests strong conservation of GRN circuitry during early embryogenesis, this study indicates that since the divergence of cidaroids and euechinoids, developmental GRNs have undergone significant, cell type-biased alterations.

Spatiotemporal expression and transcriptional regulation of heme oxygenase and biliverdin reductase genes in zebrafish (Danio rerio) suggest novel roles during early developmental periods of heightened oxidative stress

Heme oxygenase 1 (HMOX1) degrades heme into biliverdin, which is subsequently converted to bilirubin by biliverdin reductase (BVRa or BVRb) in a manner analogous to the classic anti-oxidant glutathione-recycling pathway. To gain a better understanding of the potential antioxidant roles the BVR enzymes may play during development, the spatiotemporal expression and transcriptional regulation of zebrafish hmox1a, bvra and bvrb were characterized under basal conditions and in response to pro-oxidant exposure. All three genes displayed spatiotemporal expression patterns consistent with classic hematopoietic progenitors during development. Transient knockdown of Nrf2a did not attenuate the ability to detect bvra or bvrb by ISH, or alter spatial expression patterns in response to cadmium exposure. While hmox1a:mCherry fluorescence was documented within the intermediate cell mass, a transient location of primitive erythrocyte differentiation, expression was not fully attenuated in Nrf2a morphants, but real-time RT-PCR demonstrated a significant reduction in hmox1a expression. Furthermore, Gata-1 knockdown did not attenuate hmox1a:mCherry fluorescence. However, while there was a complete loss of detection of bvrb expression by ISH at 24hpf, bvra expression was greatly attenuated but still detectable in Gata-1 morphants. In contrast, 96 hpf Gata-1 morphants displayed increased bvra and bvrb expression within hematopoietic tissues. Finally, temporal expression patterns of enzymes involved in the generation and maintenance of NADPH were consistent with known changes in the cellular redox state during early zebrafish development. Together, these data suggest that Gata-1 and Nrf2a play differential roles in regulating the heme degradation enzymes during an early developmental period of heightened cellular stress.

A gene-centered C.elegans protein-DNA interaction network provides a framework for functional predictions.

Transcription factors (TFs) play a central role in controlling spatiotemporal gene expression and the response to environmental cues. A comprehensive understanding of gene regulation requires integrating physical protein–DNA interactions (PDIs) with TF regulatory activity, expression patterns, and phenotypic data. Although great progress has been made in mapping PDIs using chromatin immunoprecipitation, these studies have only characterized ~10% of TFs in any metazoan species. The nematode C. elegans has been widely used to study gene regulation due to its compact genome with short regulatory sequences. Here, we delineated the largest gene‐centered metazoan PDI network to date by examining interactions between 90% of C. elegans TFs and 15% of gene promoters. We used this network as a backbone to predict TF binding sites for 77 TFs, two‐thirds of which are novel, as well as integrate gene expression, protein–protein interaction, and phenotypic data to predict regulatory and biological functions for multiple genes and TFs.

Transcriptional Regulation of Arabidopsis Polycomb Repressive Complex 2 Coordinates Cell-Type Proliferation and Differentiation.

Spatiotemporal regulation of transcription is fine-tuned at multiple levels, including chromatin compaction. Polycomb Repressive Complex 2 (PRC2) catalyzes the trimethylation of Histone 3 at lysine 27 (H3K27me3), which is the hallmark of a repressive chromatin state. Multiple PRC2 complexes have been reported in Arabidopsis thaliana to control the expression of genes involved in developmental transitions and maintenance of organ identity. Here, we show that PRC2 member genes display complex spatiotemporal gene expression patterns and function in root meristem and vascular cell proliferation and specification. Furthermore, PRC2 gene expression patterns correspond with vascular and nonvascular tissue-specific H3K27me3-marked genes. This tissue-specific repression via H3K27me3 regulates the balance between cell proliferation and differentiation. Using enhanced yeast one-hybrid analysis, upstream regulators of the PRC2 member genes are identified, and genetic analysis demonstrates that transcriptional regulation of some PRC2 genes plays an important role in determining PRC2 spatiotemporal activity within a developing organ.

DamX Controls Reversible Cell Morphology Switching in Uropathogenic Escherichia coli.

ABSTRACTThe ability to change cell morphology is an advantageous characteristic adopted by multiple pathogenic bacteria in order to evade host immune detection and assault during infection. Uropathogenic Escherichia coli (UPEC) exhibits such cellular dynamics and has been shown to transition through a series of distinct morphological phenotypes during a urinary tract infection. Here, we report the first systematic spatio-temporal gene expression analysis of the UPEC transition through these phenotypes by using a flow chamber-based in vitro infection model that simulates conditions in the bladder. This analysis revealed a novel association between the cell division gene damX and reversible UPEC filamentation. We demonstrate a lack of reversible bacterial filamentation in a damX deletion mutant in vitro and absence of a filamentous response by this mutant in a murine model of cystitis. While deletion of damX abrogated UPEC filamentation and secondary surface colonization in tissue culture and in mouse infections, transient overexpression of damX resulted in reversible UPEC filamentation. In this study, we identify a hitherto-unknown damX-mediated mechanism underlying UPEC morphotypical switching. Murine infection studies showed that DamX is essential for establishment of a robust urinary tract infection, thus emphasizing its role as a mediator of virulence. Our study demonstrates the value of an in vitro methodology, in which uroepithelium infection is closely simulated, when undertaking targeted investigations that are challenging to perform in animal infection models.

H3K27me3 Does Not Orchestrate the Expression of Lineage-Specific Markers in hESC-Derived Hepatocytes InVitro.

SummaryAlthough pluripotent stem cells can be differentiated into the hepatocyte lineages, such cells retain an immature phenotype. As the chromatin state of regulatory regions controls spatiotemporal gene expression during development, we evaluated changes in epigenetic histone marks in lineage-specific genes throughout in vitro hepatocyte differentiation from human embryonic stem cells (hESCs). Active acetylation and methylation marks at promoters and enhancers correlated with progressive changes in gene expression. However, repression-associated H3K27me3 marks at these control regions showed an inverse correlation with gene repression during transition from hepatic endoderm to a hepatocyte-like state. Inhibitor of Enhancer of Zeste Homolog 2 (EZH2) reduced H3K27me3 decoration but did not improve hepatocyte maturation. Thus, H3K27me3 at regulatory regions does not regulate transcription and appears dispensable for hepatocyte lineage differentiation of hESCs in vitro.

RNA-binding Protein Musashi Homologue 1 Regulates Kidney Fibrosis by Translational Inhibition of p21 and Numb mRNA

RNA-binding proteins (RBPs) are recognized as key posttranscriptional regulators that not only modulate the spatiotemporal expression of genes during organism development but also regulate disease pathogenesis. Very limited information exists on the potential role of RBPs in modulating kidney fibrosis, which is a major hallmark of chronic kidney disease. Here, we report a novel mechanism in kidney fibrosis involving a RBP, Musashi homologue 1 (Msi1), which is expressed in tubular epithelial cells. Using two mechanistically distinct mouse models of kidney fibrosis, we show that Msi1 protein levels are significantly down-regulated in the kidneys following fibrosis. We found that Msi1 functions by negatively regulating the translation of its target mRNAs, p21 and Numb, whose protein levels are markedly increased in kidney fibrosis. Also, Msi1 overexpression and knockdown in kidney epithelial cells cause p21- and Numb-mediated cell cycle arrest. Furthermore, we observed that Numb looses its characteristic membrane localization in fibrotic kidneys and therefore is likely unable to inhibit Notch resulting in tubular cell death. Oleic acid is a known inhibitor of Msi1 and injecting oleic acid followed by unilateral ureteral obstruction surgery in mice resulted in enhanced fibrosis compared with the control group, indicating that inhibiting Msi1 activity renders the mice more susceptible to fibrosis. Given that deregulated fatty acid metabolism plays a key role in kidney fibrosis, these results demonstrate a novel connection between fatty acid and Msi1, an RNA-binding protein, in kidney fibrosis.

Spatiotemporal SERT expression in cortical map development

The cerebral cortex is organized into morphologically distinct areas that provide biological frameworks underlying perception, cognition, and behavior. Profiling mouse and human cortical transcriptomes have revealed temporal-specific differential gene expression modules in distinct neocortical areas during cortical map establishment. However, the biological roles of spatiotemporal gene expression in cortical patterning and how cortical topographic gene expression is regulated are largely unknown. Here, we characterize temporal- and spatial-defined expression of serotonin (5-HT) transporter (SERT) in glutamatergic neurons during sensory map development in mice. SERT is transiently expressed in glutamatergic thalamic neurons projecting to sensory cortices and in pyramidal neurons in the prefrontal cortex (PFC) and hippocampus (HPC) during the period that lays down the basic functional neural circuits. We previously identified that knockout of SERT in the thalamic neurons blocks 5-HT uptake by their thalamocortical axons, resulting in excessive 5-HT signaling that impairs sensory map architecture. In contrast, here we show that selective SERT knockout in the PFC and HPC neurons does not perturb sensory map patterning. These data suggest that transient SERT expression in specific glutamatergic neurons provides area-specific instructions for cortical map patterning. Hence, genetic and pharmacological manipulations of this SERT function could illuminate the fundamental genetic programming of cortex-specific maps and biological roles of temporal-specific cortical topographic gene expression in normal development and mental disorders.

CTCF-mediated Chromatin Loop for the Posterior Hoxc Gene Expression in MEF Cells.

Modulation of chromatin structure has been proposed as a molecular mechanism underlying the spatiotemporal collinear expression of Hox genes during development. CCCTC-binding factor (CTCF)-mediated chromatin organization is now recognized as a crucial epigenetic mechanism for transcriptional regulation. Thus, we examined whether CTCF-mediated chromosomal conformation is involved in Hoxc gene expression by comparing wild-type mouse embryonic fibroblast (MEF) cells expressing anterior Hoxc genes with Akt1 null MEFs expressing anterior as well as posterior Hoxc genes. We found that CTCF binding between Hoxc11 and -c12 is important for CTCF-mediated chromosomal loop formation and concomitant posterior Hoxc gene expression. Hypomethylation at this site increased CTCF binding and recapitulated the chromosomal conformation and posterior Hoxc gene expression patterns observed in Akt1 null MEFs. From this work we found that CTCF at the C12|11 does not function as a barrier/boundary, instead let the posterior Hoxc genes switch their interaction from inactive centromeric to active telomeric genomic niche, and concomitant posterior Hoxc gene expression. Although it is not clear whether CTCF affects Hoxc gene expression solely through its looping activity, CTCF-mediated chromatin structural modulation could be an another tier of Hox gene regulation during development. © 2016 IUBMB Life, 68(6):436-444, 2016.

The RecA-Dependent SOS Response Is Active and Required for Processing of DNA Damage during Bacillus subtilis Sporulation.

The expression of and role played by RecA in protecting sporulating cells of Bacillus subtilis from DNA damage has been determined. Results showed that the DNA-alkylating agent Mitomycin-C (M-C) activated expression of a PrecA-gfpmut3a fusion in both sporulating cells’ mother cell and forespore compartments. The expression levels of a recA-lacZ fusion were significantly lower in sporulating than in growing cells. However, M-C induced levels of ß-galactosidase from a recA-lacZ fusion ~6- and 3-fold in the mother cell and forespore compartments of B. subtilis sporangia, respectively. Disruption of recA slowed sporulation and sensitized sporulating cells to M-C and UV-C radiation, and the M-C and UV-C sensitivity of sporangia lacking the transcriptional repair-coupling factor Mfd was significantly increased by loss of RecA. We postulate that when DNA damage is encountered during sporulation, RecA activates the SOS response thus providing sporangia with the repair machinery to process DNA lesions that may compromise the spatio-temporal expression of genes that are essential for efficient spore formation.

Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons.

The rat is a preferred model system over the mouse for neurological studies, and cell type-specific Cre expression in the rat enables precise ablation of gene function in neurons of interest, which is especially valuable for neurodegenerative disease modeling and optogenetics. Yet, few such Cre rats are available. Here we report the characterization of two Cre rats, tyrosine hydroxylase (TH)-Cre and dopamine active transporter (DAT or Slc6a3)-Cre, by using a combination of immunohistochemistry (IHC) and mRNA fluorescence in situ hybridization (FISH) as well as a fluorescent reporter for Cre activity. We detected Cre expression in expected neurons in both Cre lines. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats. In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins. The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson’s disease.

Epigenomic Landscape of Human Fetal Brain, Heart, and Liver

The epigenetic regulation of spatiotemporal gene expression is crucial for human development. Here, we present whole-genome chromatin immunoprecipitation followed by high throughput DNA sequencing (ChIP-seq) analyses of a wide variety of histone markers in the brain, heart, and liver of early human embryos shortly after their formation. We identified 40,181 active enhancers, with a large portion showing tissue-specific and developmental stage-specific patterns, pointing to their roles in controlling the ordered spatiotemporal expression of the developmental genes in early human embryos. Moreover, using sequential ChIP-seq, we showed that all three organs have hundreds to thousands of bivalent domains that are marked by both H3K4me3 and H3K27me3, probably to keep the progenitor cells in these organs ready for immediate differentiation into diverse cell types during subsequent developmental processes. Our work illustrates the potentially critical roles of tissue-specific and developmental stage-specific epigenomes in regulating the spatiotemporal expression of developmental genes during early human embryonic development.

Chamber Specific Gene Expression Landscape of the Zebrafish Heart

The organization of structure and function of cardiac chambers in vertebrates is defined by chamber-specific distinct gene expression. This peculiarity and uniqueness of the genetic signatures demonstrates functional resolution attributed to the different chambers of the heart. Altered expression of the cardiac chamber genes can lead to individual chamber related dysfunctions and disease patho-physiologies. Information on transcriptional repertoire of cardiac compartments is important to understand the spectrum of chamber specific anomalies. We have carried out a genome wide transcriptome profiling study of the three cardiac chambers in the zebrafish heart using RNA sequencing. We have captured the gene expression patterns of 13,396 protein coding genes in the three cardiac chambers—atrium, ventricle and bulbus arteriosus. Of these, 7,260 known protein coding genes are highly expressed (≥10 FPKM) in the zebrafish heart. Thus, this study represents nearly an all-inclusive information on the zebrafish cardiac transcriptome. In this study, a total of 96 differentially expressed genes across the three cardiac chambers in zebrafish were identified. The atrium, ventricle and bulbus arteriosus displayed 20, 32 and 44 uniquely expressing genes respectively. We validated the expression of predicted chamber-restricted genes using independent semi-quantitative and qualitative experimental techniques. In addition, we identified 23 putative novel protein coding genes that are specifically restricted to the ventricle and not in the atrium or bulbus arteriosus. In our knowledge, these 23 novel genes have either not been investigated in detail or are sparsely studied. The transcriptome identified in this study includes 68 differentially expressing zebrafish cardiac chamber genes that have a human ortholog. We also carried out spatiotemporal gene expression profiling of the 96 differentially expressed genes throughout the three cardiac chambers in 11 developmental stages and 6 tissue types of zebrafish. We hypothesize that clustering the differentially expressed genes with both known and unknown functions will deliver detailed insights on fundamental gene networks that are important for the development and specification of the cardiac chambers. It is also postulated that this transcriptome atlas will help utilize zebrafish in a better way as a model for studying cardiac development and to explore functional role of gene networks in cardiac disease pathogenesis.

A quantitative validated model reveals two phases of transcriptional regulation for the gap gene giant in Drosophila

Understanding eukaryotic transcriptional regulation and its role in development and pattern formation is one of the big challenges in biology today. Most attempts at tackling this problem either focus on the molecular details of transcription factor binding, or aim at genome-wide prediction of expression patterns from sequence through bioinformatics and mathematical modelling. Here we bridge the gap between these two complementary approaches by providing an integrative model of cis-regulatory elements governing the expression of the gap gene giant (gt) in the blastoderm embryo of Drosophila melanogaster. We use a reverse-engineering method, where mathematical models are fit to quantitative spatio-temporal reporter gene expression data to infer the regulatory mechanisms underlying gt expression in its anterior and posterior domains. These models are validated through prediction of gene expression in mutant backgrounds. A detailed analysis of our data and models reveals that gt is regulated by domain-specific CREs at early stages, while a late element drives expression in both the anterior and the posterior domains. Initial gt expression depends exclusively on inputs from maternal factors. Later, gap gene cross-repression and gt auto-activation become increasingly important. We show that auto-regulation creates a positive feedback, which mediates the transition from early to late stages of regulation. We confirm the existence and role of gt auto-activation through targeted mutagenesis of Gt transcription factor binding sites. In summary, our analysis provides a comprehensive picture of spatio-temporal gene regulation by different interacting enhancer elements for an important developmental regulator.

The Arabidopsis NADPH oxidases RbohD and RbohF display differential expression patterns and contributions during plant immunity.

Plant NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) that perform a wide range of functions. RbohD and RbohF, two of the 10 Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes including the response to pathogens. We hypothesized that the spatio-temporal control of RbohD and RbohF gene expression might be critical in determining their multiplicity of functions. Transgenic Arabidopsis plants with RbohD and RbohF promoter fusions to β-glucuronidase and Luciferase reporter genes were generated. Analysis of these plants revealed a differential expression pattern for RbohD and RbohF throughout plant development and during immune responses. RbohD and RbohF gene expression was differentially modulated by pathogen-associated molecular patterns. Histochemical stains and in vivo expression analysis showed a correlation between the level of RbohD and RbohF promoter activity, H2O2 accumulation and the amount of cell death in response to the pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Plectosphaerella cucumerina. A promoter-swap strategy revealed that the promoter region of RbohD was required to drive production of ROS by this gene in response to pathogens. Moreover, RbohD promoter was activated during Arabidopsis interaction with a non-virulent P. cucumerina isolate, and susceptibility tests with the double mutant rbohD rbohF uncovered a new function for these oxidases in basal resistance. Altogether, our results suggest that differential spatio-temporal expression of the Rboh genes contributes to fine-tune RBOH/NADPH oxidase-dependent ROS production and signaling in Arabidopsis immunity.