Cell specialization and coordination in Arabidopsis leaves upon pathogenic attack revealed by scRNA-seq.

Plant defense responses involve several biological processes that allow plants to fight against pathogenic attacks. How these different processes are orchestrated within organs and depend on specific cell types is poorly known. Here, using single-cell RNA sequencing (scRNA-seq) technology on three independent biological replicates, we identified several cell populations representing the core transcriptional responses of wild-type Arabidopsis leaves inoculated with the bacterial pathogen Pseudomonas syringae DC3000. Among these populations, we retrieved major cell types of the leaves (mesophyll, guard, epidermal, companion, and vascular S cells) with which we could associate characteristic transcriptional reprogramming and regulators, thereby specifying different cell-type responses to the pathogen. Further analyses of transcriptional dynamics, on the basis of inference of cell trajectories, indicated that the different cell types, in addition to their characteristic defense responses, can also share similar modules of gene reprogramming, uncovering a ubiquitous antagonism between immune and susceptible processes. Moreover, it appears that the defense responses of vascular S cells, epidermal cells, and mesophyll cells can evolve along two separate paths, one converging toward an identical cell fate, characterized mostly by lignification and detoxification functions. As this divergence does not correspond to the differentiation between immune and susceptible cells, we speculate that this might reflect the discrimination between cell-autonomous and non-cell-autonomous responses. Altogether our data provide an upgraded framework to describe, explore, and explain the specialization and the coordination of plant cell responses upon pathogenic challenge.

MPK3 and MPK6 control salicylic acid signaling by up-regulating NLR receptors during pattern- and effector-triggered immunity.

Arabidopsis thaliana mitogen-activated protein kinases 3 and 6 (MPK3/6) are activated transiently during pathogen-associated molecular pattern-triggered immunity (PTI) and durably during effector-triggered immunity (ETI). The functional differences between these two kinds of activation kinetics and how they coordinate the two layers of plant immunity remain poorly understood. Here, by suppressor analyses, we demonstrate that ETI-mediating nucleotide-binding domain leucine-rich repeat receptors (NLRs) and the NLR signaling components NDR1 and EDS1 can promote the salicylic acid sector of defense downstream of MPK3 activity. Moreover, we provide evidence that both sustained and transient MPK3/6 activities positively control the expression of several NLR genes, including AT3G04220 and AT4G11170. We further show that NDR1 and EDS1 contribute to the up-regulation of these two NLRs in both an ETI and a PTI context. Remarkably, whereas in ETI MPK3/6 activities are dependent on NDR1 and EDS1, they are not in PTI, suggesting crucial differences in the two signaling pathways. Finally, we demonstrate that expression of the NLR AT3G04220 is sufficient to induce expression of defense genes from the salicylic acid branch. Overall, this study expands our knowledge of MPK3/6 functions during immunity and provides new insights into the intricate interplay of PTI and ETI.

In vivo identification of putative CPK5 substrates in Arabidopsis thaliana.

Calcium signaling mediates most developmental processes and stress responses in plants. Among plant calcium sensors, the calcium-dependent protein kinases display a unique structure harboring both calcium sensing and kinase responding activities. AtCPK5 is an essential member of this family in Arabidopsis that regulates immunity and abiotic stress tolerance. To understand the underlying molecular mechanisms, we implemented a biochemical approach to identify in vivo substrates of AtCPK5. We generated transgenic lines expressing a constitutively active form of AtCPK5 under the control of a dexamethasone-inducible promoter. Lines expressing a kinase-dead version were used as a negative control. By comparing the phosphoproteome of the kinase-active and kinase-dead lines upon dexamethasone treatment, we identified 5 phosphopeptides whose abundance increased specifically in the kinase-active lines. Importantly, we showed that all 5 proteins were phosphorylated in vitro by AtCPK5 in a calcium-dependent manner, suggesting that they are direct targets of AtCPK5. We also detected several interaction patterns between the kinase and the candidates in the cytosol, membranes or nucleus, consistent with the ubiquitous localization of AtCPK5. Finally, we further validated the two phosphosites S245 and S280 targeted by AtCPK5 in the E3 ubiquitin ligase ATL31. Altogether, those results open new perspectives to decipher AtCPK5 biological functions.

Sustained Incompatibility between MAPK Signaling and Pathogen Effectors.

In plants, Mitogen-Activated Protein Kinases (MAPKs) are important signaling components involved in developemental processes as well as in responses to biotic and abiotic stresses. In this review, we focus on the roles of MAPKs in Effector-Triggered Immunity (ETI), a specific layer of plant defense responses dependent on the recognition of pathogen effector proteins. Having inspected the literature, we synthesize the current state of knowledge concerning this topic. First, we describe how pathogen effectors can manipulate MAPK signaling to promote virulence, and how in parallel plants have developed mechanisms to protect themselves against these interferences. Then, we discuss the striking finding that the recognition of pathogen effectors can provoke a sustained activation of the MAPKs MPK3/6, extensively analyzing its implications in terms of regulation and functions. In line with this, we also address the question of how a durable activation of MAPKs might affect the scope of their substrates, and thereby mediate the emergence of possibly new ETI-specific responses. By highlighting the sometimes conflicting or missing data, our intention is to spur further research in order to both consolidate and expand our understanding of MAPK signaling in immunity.

Microstructure Optimization of Mg-Alloys by the ECAP Process Including Numerical Simulation, SPD Treatments, Characterization, and Hydrogen Sorption Properties.

Both numerical simulation and hardness measurements were used to determine the mechanical and microstructural behavior of AZ31 bulk samples when submitted to the Equal Channel Angular Pressing (ECAP) technique. Billets of this representative of Mg-rich alloys were submitted to different numbers of passes for various ECAP modes (anisotropic A, isotropic BC). The strain distribution, the grain size refinement, and the micro-hardness were used as indicators to quantify the effectiveness of the different processing routes. Structural characterizations at different scales were achieved using Scanning Electron Microscopy (SEM), micro-analysis, metallography, Small Angle Neutron Scattering SANS, X-Ray Diffraction (XRD), and texture determination. The grain and crystallite size distribution and orientation as well as defect impacts were determined. Anelastic Spectroscopy (AS) on mechanically deformed samples have shown that the temperature of ECAP differentiate the fragile to ductile regime. MgH2 consolidated powders were checked for using AS to detect potential hydrogen motions and interaction with host metal atoms. After further optimization, the different mechanically-treated samples were submitted to hydrogenation/dehydrogenation (H/D) cycles, which shows that, for a few passes, the BC mode is better than the A one, as supported by theoretical and experimental microstructure analyses. Accordingly, the hydrogen uptake and (H/D) reactions were correlated with the optimized microstructure peculiarities and interpreted in terms of Johnson-Avrami- Mehl-Kolmogorov (JAMK) and Jander models, successively.

Lifestyle of the biotroph Agrobacterium tumefaciens in the ecological niche constructed on its host plant.

Agrobacterium tumefaciens constructs an ecological niche in its host plant by transferring the T-DNA from its Ti plasmid into the host genome and by diverting the host metabolism. We combined transcriptomics and genetics for understanding the A. tumefaciens lifestyle when it colonizes Arabidopsis thaliana tumors. Transcriptomics highlighted: a transition from a motile to sessile behavior that mobilizes some master regulators (Hfq, CtrA, DivK and PleD); a remodeling of some cell surface components (O-antigen, succinoglucan, curdlan, att genes, putative fasciclin) and functions associated with plant defense (Ef-Tu and flagellin pathogen-associated molecular pattern-response and glycerol-3-phosphate and nitric oxide signaling); and an exploitation of a wide variety of host resources, including opines, amino acids, sugars, organic acids, phosphate, phosphorylated compounds, and iron. In addition, construction of transgenic A. thaliana lines expressing a lactonase enzyme showed that Ti plasmid transfer could escape host-mediated quorum-quenching. Finally, construction of knock-out mutants in A. tumefaciens showed that expression of some At plasmid genes seemed more costly than the selective advantage they would have conferred in tumor colonization. We provide the first overview of A. tumefaciens lifestyle in a plant tumor and reveal novel signaling and trophic interplays for investigating host-pathogen interactions.

Structural basis for high specificity of octopine binding in the plant pathogen Agrobacterium tumefaciens.

Agrobacterium pathogens of octopine- and nopaline-types force host plants to produce either octopine or nopaline compounds, which they use as nutrients. Two Agrobacterium ABC-transporters and their cognate periplasmic binding proteins (PBPs) OccJ and NocT import octopine and nopaline/octopine, respectively. Here, we show that both octopine transport and degradation confer a selective advantage to octopine-type A. tumefaciens when it colonizes plants. We report the X-ray structures of the unliganded PBP OccJ and its complex with octopine as well as a structural comparison with NocT and the related PBP LAO from Salmonella enterica, which binds amino acids (lysine, arginine and ornithine). We investigated the specificity of OccJ, NocT and LAO using several ligands such as amino acids, octopine, nopaline and octopine analogues. OccJ displays a high selectivity and nanomolar range affinity for octopine. Altogether, the structural and affinity data allowed to define an octopine binding signature in PBPs and to construct a OccJ mutant impaired in octopine binding, a selective octopine-binding NocT and a non-selective octopine-binding LAO by changing one single residue in these PBPs. We proposed the PBP OccJ as a major trait in the ecological specialization of octopine-type Agrobacterium pathogens when they colonize and exploit the plant host.

Constitutive activity of the Arabidopsis MAP Kinase 3 confers resistance to Pseudomonas syringae and drives robust immune responses.

Mitogen Activated Protein Kinases (MAPKs) are known to be important mediators of plant responses to biotic and abiotic stresses. In a recent report, we enlarged the understanding of the Arabidopsis thaliana MPK3 functions showing that the expression of a constitutively active (CA) form of the protein led to auto-immune phenotypes. CA-MPK3 plants are dwarf and display defense responses that are characterized by the accumulation of salicylic acid and phytoalexins as well as by the upregulation of several defense genes. Consistently with these data, we present here results demonstrating that, compared with wild type controls, CA-MPK3 plants are more resistant to the hemibiotrophic pathogen Pseudomonas syringae DC3000. Based on our previous work, we also discuss the mechanisms of robust plant immunity controlled by sustained MPK3 activity, focusing especially on the roles of disease resistance proteins.

Constitutively Active Arabidopsis MAP Kinase 3 Triggers Defense Responses Involving Salicylic Acid and SUMM2 Resistance Protein.

Mitogen-activated protein kinases (MAPKs) are important regulators of plant immunity. Most of the knowledge about the function of these pathways is derived from loss-of-function approaches. Using a gain-of-function approach, we investigated the responses controlled by a constitutively active (CA) MPK3 in Arabidopsis thalianaCA-MPK3 plants are dwarfed and display a massive derepression of defense genes associated with spontaneous cell death as well as the accumulation of reactive oxygen species, phytoalexins, and the stress-related hormones ethylene and salicylic acid (SA). Remarkably CA-MPK3/sid2 and CA-MPK3/ein2-50 lines, which are impaired in SA synthesis and ethylene signaling, respectively, retain most of the CA-MPK3-associated phenotypes, indicating that the constitutive activity of MPK3 can bypass SA and ethylene signaling to activate defense responses. A comparative analysis of the molecular phenotypes of CA-MPK3 and mpk4 autoimmunity suggested convergence between the MPK3- and MPK4-guarding modules. In support of this model, CA-MPK3 crosses with summ1 and summ2, two known suppressors of mpk4, resulted in a partial reversion of the CA-MPK3 phenotypes. Overall, our data unravel a novel mechanism by which the MAPK signaling network contributes to a robust defense-response system.

Quorum-quenching limits quorum-sensing exploitation by signal-negative invaders.

Some bacteria produce and perceive quorum-sensing (QS) signals that coordinate several behaviours, including the costly processes that are exoenzyme production and plasmid transfer. In the case of plasmid transfer, the emergence of QS signal-altered invaders and their policing are poorly documented. In Agrobacterium tumefaciens, the virulence Ti-plasmid encodes both synthesis and sensing of QS-signals, which promote its transfer from a donor to a recipient cell. Here, we reported that QS-altered A. tumefaciens mutants arose during experimental evolution. All showed improved growth compared to their ancestor. Genome sequencing revealed that, though some had lost the Ti-plasmid, most were defective for QS-signal synthesis and Ti-plasmid conjugation (traR mutations) and one exhibited a QS-signal exploitation behaviour, using signal produced by other cells to enhance its own Ti-plasmid transfer. We explored mechanisms that can limit this QS-hijacking. We showed that the A. tumefaciens capacity to inactivate QS-signals by expressing QS-degrading enzyme could attenuate dissemination of the QS signal-negative Ti-plasmids. This work shows that enzymatic QS-disruption whether encoded by the QS-producing Ti-plasmid itself, by a companion plasmid in the same donor cells, or by one in the recipient cells, in all cases can serve as a mechanism for controlling QS exploitation by QS signal-negative mutants.

Fitness costs restrict niche expansion by generalist niche-constructing pathogens.

We investigated the molecular and ecological mechanisms involved in niche expansion, or generalism, versus specialization in sympatric plant pathogens. Nopaline-type and octopine-type Agrobacterium tumefaciens engineer distinct niches in their plant hosts that provide different nutrients: nopaline or octopine, respectively. Previous studies revealed that nopaline-type pathogens may expand their niche to also assimilate octopine in the presence of nopaline, but consequences of this phenomenon on pathogen dynamics in planta were not known. Here, we provided molecular insight into how the transport protein NocT can bind octopine as well as nopaline, contributing to niche expansion. We further showed that despite the ability for niche expansion, nopaline-type pathogens had no competitive advantage over octopine-type pathogens in co-infected plants. We also demonstrated that a single nucleotide polymorphism in the nocR gene was sufficient to allow octopine assimilation by nopaline-type strains even in absence of nopaline. The evolved nocR bacteria had higher fitness than their ancestor in octopine-rich transgenic plants but lower fitness in tumors induced by octopine-type pathogens. Overall, this work elucidates the specialization of A. tumefaciens to particular opine niches and explains why generalists do not always spread despite the advantage associated with broader nutritional niches.

Plant GABA:proline ratio modulates dissemination of the virulence Ti plasmid within the Agrobacterium tumefaciens hosted population.

Accumulation of amino acids is a common plant response to several biotic and abiotic stresses, even if the roles of these accumulations remain often poorly understood. In a recent study we measured the levels of different amino acids in tumors of Arabidopsis thaliana induced by the phytopathogen Agrobacterium tumefaciens and correlated these data with changes of gene expressions in both organisms. This led to the demonstration that the non-protein amino acid GABA plays an important role for the adaptation of the bacteria to the plant tumor environment, and especially in the control of the virulent Ti plasmid dissemination. Here we present a model that describes how different GABA:proline ratios in the A. thaliana host may have different impacts on the conjugation of A. tumefaciens Ti plasmid, and advance the view that the amino acid metabolism of plant hosts could be critical for the propagation of the virulence genes in A. tumefaciens populations.

The plant GABA signaling downregulates horizontal transfer of the Agrobacterium tumefaciens virulence plasmid.

In the tumor-inducing (Ti) Agrobacterium tumefaciens, quorum sensing activates the horizontal transfer of the virulent Ti plasmid. In pure culture, this process can be impaired by the A. tumefaciens BlcC lactonase, whose expression is induced by gamma-aminobutyrate (GABA). It was therefore hypothesized that host GABA content might modulate quorum sensing and virulence gene dissemination during A. tumefaciens infection. We examined GABA metabolism and transport in Arabidopsis thaliana tumors combining transcriptomic, metabolomic and histological approaches. In addition, using genetically modified plants and bacteria, we evaluated the impact of plant host GABA content on Ti plasmid dissemination. The results showed that GABA and free proline, which acts as an antagonist of GABA uptake in A. tumefaciens, accumulated in wild-type tumors relative to uninfected plant tissues. Moreover, comparisons of tumors induced on Col-0 and her1 plants showed that the increase in the plant GABA : proline ratio was associated with both the upregulated expression of the blcC gene and the decreased dissemination of Ti plasmid in tumor-colonizing A. tumefaciens populations. This work demonstrates experimentally that the variation in the GABA content in plant tumors can interfere with the dissemination of A. tumefaciens Ti plasmids, and therefore highlights plant GABA content as an important trait in the struggle against pathogenic bacteria.

A Pyranose-2-Phosphate Motif Is Responsible for Both Antibiotic Import and Quorum-Sensing Regulation in Agrobacterium tumefaciens.

Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs.

Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host.

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (K(D) of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens.

Functions and regulation of quorum-sensing in Agrobacterium tumefaciens.

In Agrobacterium tumefaciens, horizontal transfer and vegetative replication of oncogenic Ti plasmids involve a cell-to-cell communication process called quorum-sensing (QS). The determinants of the QS-system belong to the LuxR/LuxI class. The LuxI-like protein TraI synthesizes N-acyl-homoserine lactone molecules which act as diffusible QS-signals. Beyond a threshold concentration, these molecules bind and activate the LuxR-like transcriptional regulator TraR, thereby initiating the QS-regulatory pathway. For the last 20 years, A. tumefaciens has stood as a prominent model in the understanding of the LuxR/LuxI type of QS systems. A number of studies also unveiled features which are unique to A. tumefaciens QS, some of them being directly related to the phytopathogenic lifestyle of the bacteria. In this review, we will present the current knowledge of QS in A. tumefaciens at both the genetic and molecular levels. We will also describe how interactions with plant host modulate the QS pathway of A. tumefaciens, and discuss what could be the advantages for the agrobacteria to use such a tightly regulated QS-system to disseminate the Ti plasmids.

Concerted transfer of the virulence Ti plasmid and companion At plasmid in the Agrobacterium tumefaciens-induced plant tumour.

The plant pathogen Agrobacterium tumefaciens C58 harbours three independent type IV secretion (T4SS) machineries. T4SST-DNA promotes the transfer of the T-DNA to host plant cells, provoking tumour development and accumulation of opines such as nopaline and agrocinopines. T4SSpTi and T4SSpAt control the bacterial conjugation of the Ti and At plasmids respectively. Expression of T4SSpTi is controlled by the agrocinopine-responsive transcriptional repressor AccR. In this work, we compared the genome-wide transcriptional profile of the wild-type A. tumefaciens strain C58 with that of its accR KO-mutant to delineate the AccR regulon. In addition to the genes that encode agrocinopine catabolism and T4SSpTi , we found that AccR also regulated genes coding for nopaline catabolism and T4SSpAt . Further opine detection and conjugation assays confirmed the enhancement of nopaline consumption and At plasmid conjugation frequency in accR. Moreover, co-regulation of the T4SSpTi and T4SSpAt correlated with the co-transfer of the At and Ti plasmids both in vitro and in plant tumours. Finally, unlike T4SSpTi , T4SSpAt activation does not require quorum-sensing. Overall this study highlights the regulatory interplays between opines, At and Ti plasmids that contribute to a concerted dissemination of the two replicons in bacterial populations colonizing the plant tumour.

Plant γH2AX foci are required for proper DNA DSB repair responses and colocalize with E2F factors.

Cellular responses to DNA double-strand breaks (DSBs) are linked in mammals and yeasts to the phosphorylated histones H2AX (γH2AX) repair foci which are multiproteic nuclear complexes responsible for DSB sensing and signalling. However, neither the components of these foci nor their role are yet known in plants. In this paper, we describe the effects of γH2AX deficiency in Arabidopsis thaliana plants challenged with DSBs in terms of genotoxic sensitivity and E2F-mediated transcriptional responses. We further establish the existence, restrictive to the G1/S transition, of specific DSB-induced foci containing tobacco E2F transcription factors, in both A. thaliana roots and BY-2 tobacco cells. These E2F foci partially colocalize with γH2AX foci while their formation is ataxia telangiectasia mutated (ATM)-dependent, requires the E2F transactivation domain with its retinoblastoma-binding site and is optimal in the presence of functional H2AXs. Overall, our results unveil a new interplay between plant H2AX and E2F transcriptional activators during the DSB response.

Ribonucleotide reductase regulation in response to genotoxic stress in Arabidopsis.

Ribonucleotide reductase (RNR) is an essential enzyme that provides dNTPs for DNA replication and repair. Arabidopsis (Arabidopsis thaliana) encodes three AtRNR2-like catalytic subunit genes (AtTSO2, AtRNR2A, and AtRNR2B). However, it is currently unclear what role, if any, each gene contributes to the DNA damage response, and in particular how each gene is transcriptionally regulated in response to replication blocks and DNA damage. To address this, we investigated transcriptional changes of 17-d-old Arabidopsis plants (which are enriched in S-phase cells over younger seedlings) in response to the replication-blocking agent hydroxyurea (HU) and to the DNA double-strand break inducer bleomycin (BLM). Here we show that AtRNR2A and AtRNR2B are specifically induced by HU but not by BLM. Early AtRNR2A induction is decreased in an atr mutant, and this induction is likely required for the replicative stress checkpoint since rnr2a mutants are hypersensitive to HU, whereas AtRNR2B induction is abolished in the rad9-rad17 double mutant. In contrast, AtTSO2 transcription is only activated in response to double-strand breaks (BLM), and this activation is dependent upon AtE2Fa. Both TSO2 and E2Fa are likely required for the DNA damage response since tso2 and e2fa mutants are hypersensitive to BLM. Interestingly, TSO2 gene expression is increased in atr versus wild type, possibly due to higher ATM expression in atr. On the other hand, a transient ATR-dependent H4 up-regulation was observed in wild type in response to HU and BLM, perhaps linked to a transient S-phase arrest. Our results therefore suggest that individual RNR2-like catalytic subunit genes participate in unique aspects of the cellular response to DNA damage in Arabidopsis.