Fédération de Recherche Agrobiosciences, Interactions et Biodiversité

Two plasma devices at atmospheric pressure (air dielectric barrier discharge and helium plasma jet) have been used to study the early germination of Arabidopsis thaliana seeds during the first days. Then, plasma activated waters are used during the later stage of plant development and growth until 42 days. The effects on both testa and endospserm ruptures during the germination stage are significant in the case of air plasma due to its higher energy and efficiency of producing reactive oxygen species than the case of helium plasma. The latter has shown distinct effects only for testa rupture. Analysis of germination stimulations are based on specific stainings for reactive oxygen species production, peroxidase activity and also membrane permeability tests. Furthermore, scanning electron microscopy (SEM) has shown a smoother seed surface for air plasma treated seeds that can explain the plasma induced-germination. During the growth stage, plants were watered using 4 kinds of water (tap and deionized waters activated or not by the low temperature plasma jet). With regards to other water kinds, the characterization of the tap water has shown a larger conductivity, acidity and concentration of reactive nitrogen and oxygen species. Only the tap water activated by the plasma jet has shown a significant effect on the plant growth. This effect could be correlated to reactive nitrogen species such as nitrite/nitrate species present in plasma activated tap water.

MicroRNAs (miRNAs) are transcribed as long primary transcripts (pri-miRNAs) by RNA polymerase II. Plant pri-miRNAs encode regulatory peptides called miPEPs, which specifically enhance the transcription of the pri-miRNA from which they originate. However, paradoxically, whereas miPEPs have been identified in different plant species, they are poorly conserved, raising the question of the mechanisms underlying their specificity. To address this point, we identify and re-annotate multiple Arabidopsis thaliana pri-miRNAs in order to identify ORF encoding miPEPs. The study of several identified miPEPs in different species show that non-conserved miPEPs are only active in their plant of origin, whereas conserved ones are active in different species. Finally, we find that miPEP activity relies on the presence of its own miORF, explaining both the lack of selection pressure on miPEP sequence and the ability for non-conserved peptides to play a similar role, i.e., to activate the expression of their corresponding miRNA.

Streptomycetes are soil-dwelling, filamentous actinobacteria and represent a prominent bacterial clade inside the plant root microbiota. The ability of streptomycetes to produce a broad spectrum of antifungal metabolites suggests that these bacteria could be used to manage plant diseases. Here, we describe the identification of a soil Streptomyces strain named AgN23 which strongly activates a large array of defense responses when applied on Arabidopsis thaliana leaves. AgN23 increased the biosynthesis of salicylic acid, leading to the development of salicylic acid induction deficient 2 (SID2)-dependent necrotic lesions. Size exclusion fractionation of plant elicitors secreted by AgN23 showed that these signals are tethered into high molecular weight complexes. AgN23 mycelium was able to colonize the leaf surface, leading to plant resistance against Alternaria brassicicola infection in wild-type Arabidopsis plants. AgN23-induced resistance was found partially compromised in salicylate, jasmonate, and ethylene mutants. Our data show that Streptomyces soil bacteria can develop at the surface of plant leaves to induce defense responses and protection against foliar fungal pathogens, extending their potential use to manage plant diseases.

In order to investigate the effects of low temperature plasmas on germination of Arabidopsis thaliana seeds, a dielectric barrier discharge device generating the plasma in ambient air was used. To highlight the different plasma effects on the seed surface, saline and osmotic stresses were considered in the case of reference Col-0 seeds and two further seed coat mutants gl2 and gpat5 to better analyse the seed surface changes and their consequences on germination. The GL2 gene encode a transcription factor controlling the balance between the biosynthesis of fatty acids in the embryo and the production of mucilage and flavonoid pigments in the seed coat. The GPAT5 gene encode for an acyltransferase necessary for the accumulation of suberin in the seed coat which is essential for the embryo protection. The testa and endosperm ruptures are identified to note the germination stage. An increasing of germination rate, possibly due to the modification of mantle layers structure, is observed in most of cases, even in presence of saline or osmotic stress, after plasma treatment. Furthermore, we demonstrated that the germination rate of the gl2 mutant seeds is increased by at most 47% after plasma treatment, contrariwise, the germination of gpat5 mutant being initially lower is inhibited by the same plasma treatment. The scanning electron microscopy pictures and confocal microscopy fluorescence both showed changes of the exterior aspects of the seeds after plasma treatment. Considering these results, we assumed that lipid compounds can be found on the surface. To validate this hypothesis, permeability tests were performed, and it was clearly shown that a permeability decrease is induced by the low temperature plasma treatment.

BACKGROUND: Oomycetes are a group of filamentous eukaryotic microorganisms that have colonized all terrestrial and oceanic ecosystems, and they include prominent plant pathogens. The Aphanomyces genus is unique in its ability to infect both plant and animal species, and as such exemplifies oomycete versatility in adapting to different hosts and environments. Dissecting the underpinnings of oomycete diversity provides insights into their specificity and pathogenic mechanisms. RESULTS: By carrying out genomic analyses of the plant pathogen A. euteiches and the crustacean pathogen A. astaci, we show that host specialization is correlated with specialized secretomes that are adapted to the deconstruction of the plant cell wall in A. euteiches and protein degradation in A. astaci. The A. euteiches genome is characterized by a large repertoire of small secreted protein (SSP)-encoding genes that are highly induced during plant infection, and are not detected in other oomycetes. Functional analysis revealed an SSP from A. euteiches containing a predicted nuclear-localization signal which shuttles to the plant nucleus and increases plant susceptibility to infection. CONCLUSION: Collectively, our results show that Aphanomyces host adaptation is associated with evolution of specialized secretomes and identify SSPs as a new class of putative oomycete effectors.

Adipose tissue, as the main energy storage organ and through its endocrine activity, is interconnected with all physiological functions. It plays a fundamental role in energy homeostasis and in the development of metabolic disorders. Up to now, this tissue has been analysed as a pool of different cell types with very little attention paid to the organization and putative partitioning of cells. Considering the absence of a complete picture of the intimate architecture of this large soft tissue, we developed a method that combines tissue clearing, acquisition of autofluorescence or lectin signals by confocal microscopy, segmentation procedures based on contrast enhancement, and a new semi-automatic image analysis process, allowing accurate and quantitative characterization of the whole 3D fat pad organization. This approach revealed the unexpected anatomic complexity of the murine subcutaneous fat pad. Although the classical picture of adipose tissue corresponds to a superposition of simple and small ellipsoidal lobules of adipose cells separated by mesenchymal spans, our results show that segmented lobules display complex 3D poly-lobular shapes. Despite differences in shape and size, the number of these poly-lobular subunits is similar from one fat pad to another. Finally, investigation of the relationships of these subunits between each other revealed a never-described organization in two clusters with distinct molecular signatures and specific vascular and sympathetic nerve densities correlating with different browning abilities. This innovative procedure reveals that subcutaneous adipose tissue exhibits a subtle functional heterogeneity with partitioned areas, and opens new perspectives towards understanding its functioning and plasticity.

Intestinal organoids are self-organized 3-dimensional (3D) structures formed by a single layer of polarized epithelial cells. This innovative in vitro model is highly relevant to study physiology of the intestinal epithelium and its role in nutrition and barrier function. However, this model has never been developed in rabbits, while it would have potential applications for biomedical and veterinary research. Here, we cultured rabbit caecum organoids with either pharmacological inhibitors (2Ki medium) or L-WRN cells conditioned medium (L-WRN CM) to reconstitute the intestinal stem cell niche in vitro. Large spherical organoids were obtained with the 2Ki medium and this morphology was associated with a high level of proliferation and stem cells markers gene expression. In contrast, organoids cultured with L-WRN CM had a smaller diameter; a greater cell height and part of them were not spherical. When the L-WRN CM was used at low concentration (5%) for two days, the gene expression of stem cells and proliferation markers were very low, while absorptive and secretory cells markers and antimicrobial peptides were elevated. Epithelial cells within organoids were polarized in 3D cultures with 2Ki medium or L-WRN CM (apical side towards the lumen). We cultured dissociated organoid cells in 2D monolayers, which allowed accessibility to the apical compartment. Under these conditions, actin stress fibers were observed with the 2Ki medium, while perijonctionnal localization of actin was observed with the L-WRN CM suggesting, in 2D cultures as well, a higher differentiation level in the presence of L-WRN CM. In conclusion, rabbit caecum organoids cultured with the 2Ki medium were more proliferative and less differentiated than organoids cultured with L-WRN CM. We propose that organoids cultured with the 2Ki medium could be used to rapidly generate in vitro a large number of rabbit intestinal epithelial stem cells while organoids cultured with the L-WRN CM used at low concentration represent a suitable model to study differentiated rabbit epithelium.

Nitrogen-fixing symbionts of legumes have appeared after the emergence of legumes on earth, approximately 70 to 130 million years ago. Since then, symbiotic proficiency has spread to distant genera of α- and β-proteobacteria, via horizontal transfer of essential symbiotic genes and subsequent recipient genome remodeling under plant selection pressure. To tentatively replay rhizobium evolution in laboratory conditions, we previously transferred the symbiotic plasmid of the Mimosa symbiont Cupriavidus taiwanensis in the plant pathogen Ralstonia solanacearum, and selected spontaneous nodulating variants of the chimeric Ralstonia sp. using Mimosa pudica as a trap. Here, we pursued the evolution experiment by submitting two of the rhizobial drafts to serial ex planta-in planta (M. pudica) passages that may mimic alternating of saprophytic and symbiotic lives of rhizobia. Phenotyping 16 cycle-evolved clones showed strong and parallel evolution of several symbiotic traits (i.e., nodulation competitiveness, intracellular infection, and bacteroid persistence). Simultaneously, plant defense reactions decreased within nodules, suggesting that the expression of symbiotic competence requires the capacity to limit plant immunity. Nitrogen fixation was not acquired in the frame of this evolutionarily short experiment, likely due to the still poor persistence of final clones within nodules compared with the reference rhizobium C. taiwanensis. Our results highlight the potential of experimental evolution in improving symbiotic proficiency and for the elucidation of relationship between symbiotic capacities and elicitation of immune responses.

MiPEPs are short natural peptides encoded by microRNAs in plants. Exogenous application of miPEPs increases the expression of their corresponding miRNA and, consequently, induces consistent phenotypical changes. Therefore, miPEPs carry huge potential in agronomy as gene regulators that do not require genome manipulation. However, to this end, it is necessary to know their mode of action, including where they act and how they enter the plants. Here, after analyzing the effect of Arabidopsis thaliana miPEP165a on root and aerial part development, we followed the internalization of fluorescent-labelled miPEP165a into roots and compared its uptake into endocytosis-altered mutants to that observed in wild-type plants treated or not with endocytosis inhibitors. The results show that entry of miPEP165a involves both a passive diffusion at the root apex and endocytosis-associated internalization in the differentiation and mature zones. Moreover, miPEP165a is unable to enter the central cylinder and does not migrate from the roots to the aerial part of the plant, suggesting that miPEPs have no systemic effect.

Background and Aims: The plant Hirtella physophora, the ant Allomerus decemarticulatus and a fungus, Trimmatostroma sp., form a tripartite association. The ants manipulate both the plant trichomes and the fungus to build galleries under the stems of their host plant used to capture prey. In addition to its structural role, the fungus also improves nutrient uptake by the host plant. But it still remains unclear whether the fungus plays an indirect or a direct role in transferring nutrients to the plant. This study aimed to trace the transfer of N from the fungus to the plant's stem tissue. Methods: Optical microscopy and transmission electron microscopy (TEM) were used to investigate the presence of fungal hyphae in the stem tissues. Then, a 15N-labelling experiment was combined with a nanoscale secondary-ion mass spectrometry (NanoSIMS 50) isotopic imaging approach to trace the movement of added 15N from the fungus to plant tissues. Key Results: The TEM images clearly showed hyphae inside the stem tissue in the cellular compartment. Also, fungal hyphae were seen perforating the wall of the parenchyma cell. The 15N provisioning of the fungus in the galleries resulted in significant enrichment of the 15N signature of the plant's leaves 1 d after the 15N-labelling solution was deposited on the fungus-bearing trap. Finally, NanoSIMS imaging proved that nitrogen was transferred biotrophically from the fungus to the stem tissue. Conclusions: This study provides evidence that the fungi are connected endophytically to an ant-plant system and actively transfer nitrogen from 15N-labelling solution to the plant's stem tissues. Overall, this study underlines how complex the trophic structure of ant-plant interactions is due to the presence of the fungus and provides insight into the possibly important nutritional aspects and tradeoffs involved in myrmecophyte-ant mutualisms.

The current agriculture main challenge is to maintain food production while facing multiple threats such as increasing world population, temperature increase, lack of agrochemicals due to health issues and uprising of weeds resistant to herbicides. Developing novel, alternative, and safe methods is hence of paramount importance. Here, we show that complementary peptides (cPEPs) from any gene can be designed to target specifically plant coding genes. External application of synthetic peptides increases the abundance of the targeted protein, leading to related phenotypes. Moreover, we provide evidence that cPEPs can be powerful tools in agronomy to improve plant traits, such as growth, resistance to pathogen or heat stress, without the needs of genetic approaches. Finally, by combining their activity they can also be used to reduce weed growth.

The non-animal peroxidases belong to a superfamily of oxidoreductases that reduce hydrogen peroxide and oxidize numerous substrates. Since their initial characterization in 1992, a number of studies have provided an understanding of the origin and evolution of this protein family. Here, we report a comprehensive evolutionary analysis of non-animal peroxidases using integrated in silico and biochemical approaches. Thanks to the availability of numerous genomic sequences from more than 2500 species belonging to 14 kingdoms together with expert and comprehensive annotation of peroxidase sequences that have been centralized in a dedicated database, we have been able to use phylogenetic reconstructions to increase our understanding of the evolutionary processes underlying the diversification of non-animal peroxidases. We analysed the distribution of all non-animal peroxidases in more than 200 eukaryotic organisms in silico. First, we show that the presence or absence of non-animal peroxidases correlates with the presence or absence of certain organelles or with specific biological processes. Examination of almost 2000 organisms determined that ascorbate peroxidases (APxs) and cytochrome c peroxidases (CcPs) are present in those containing chloroplasts and mitochondria, respectively. Plants, which contain both organelles, are an exception and contain only APxs without CcP. Class II peroxidases (CII Prxs) are only found in fungi with wood-decay and plant-degradation abilities. Class III peroxidases (CIII Prxs) are only found in streptophyte algae and land plants, and have been subjected to large family expansion. Biochemical activities of APx, CcP, and CIII Prx assessed using protein extracts from 30 different eukaryotic organisms support the distribution of the sequences resulting from our in silico analysis. The biochemical results confirmed both the presence and classification of the non-animal peroxidase encoding sequences.

The rationale of this study is to compare and integrate two heterologous datasets intended to unravel the spatiotemporal specificities of gene expression in a rapidly growing and complex organ. We implemented medium-throughput RNA in situ hybridization (ISH) for 39 genes mainly corresponding to cell wall proteins for which we have particular interest, selected (i) on their sequence identity (24 class III peroxidase multigenic family members and 15 additional genes used as positive controls) and (ii) on their expression levels in a publicly available Arabidopsis thaliana seed tissue-specific transcriptomics study. The specificity of the hybridization signals was carefully studied, and ISH results obtained for the 39 selected genes were systematically compared with tissue-specific transcriptomics for 5 seed developmental stages. Integration of results illustrates the complementarity of both datasets. The tissue-specific transcriptomics provides high-throughput possibilities whereas ISH provides high spatial resolution. Moreover, depending on the tissues and the developmental stages considered, one or the other technique appears more sensitive than the other. For each tissue/developmental stage, we finally determined tissue-specific transcriptomic threshold values compatible with the spatiotemporally-specific detection limits of ISH for lists of hundreds to tens-of-thousands of genes.

BACKGROUND: The parasitic plant Orobanche cumana is one of the most important threats to sunflower crops in Europe. Resistant sunflower varieties have been developed, but new O. cumana races have evolved and have overcome introgressed resistance genes, leading to the recurrent need for new resistance methods. Screening for resistance requires the phenotyping of thousands of sunflower plants to various O. cumana races. Most phenotyping experiments have been performed in fields at the later stage of the interaction, requiring time and space. A rapid phenotyping screening method under controlled conditions would need less space and would allow screening for resistance of many sunflower genotypes. Our study proposes a phenotyping tool for the sunflower/O. cumana interaction under controlled conditions through image analysis for broomrape tubercle analysis at early stages of the interaction. RESULTS: We optimized the phenotyping of sunflower/O. cumana interactions by using rhizotrons (transparent Plexiglas boxes) in a growth chamber to control culture conditions and Orobanche inoculum. We used a Raspberry Pi computer with a picamera for acquiring images of inoculated sunflower roots 3 weeks post inoculation. We set up a macro using ImageJ free software for the automatic counting of the number of tubercles. This phenotyping tool was named RhizOSun. We evaluated five sunflower genotypes inoculated with two O. cumana races and showed that automatic counting of the number of tubercles using RhizOSun was highly correlated with manual time-consuming counting and could be efficiently used for screening sunflower genotypes at the tubercle stage. CONCLUSION: This method is rapid, accurate and low-cost. It allows rapid imaging of numerous rhizotrons over time, and it enables image tracking of all the data with time kinetics. This paves the way toward automatization of phenotyping in rhizotrons that could be used for other root phenotyping, such as symbiotic nodules on legumes.

BACKGROUND: The ongoing adaptation of plants to their environment is the basis for their survival. In this adaptation, mechanoperception of gravity and local curvature plays a role of prime importance in finely regulating growth and ensuring a dynamic balance preventing buckling. However, the abiotic environment is not the exclusive cause of mechanical stimuli. Biotic interactions between plants and microorganisms also involve physical forces and potentially mechanoperception. Whether pathogens trigger mechanoperception in plants and the impact of mechanotransduction on the regulation of plant defense remains however elusive. RESULTS: Here, we found that the perception of pathogen-derived mechanical cues by microtubules potentiates the spatio-temporal implementation of plant immunity to fungus. By combining biomechanics modeling and image analysis of the post-invasion stage, we reveal that fungal colonization releases plant cell wall-born tension locally, causing fluctuations of tensile stress in walls of healthy cells distant from the infection site. In healthy cells, the pathogen-derived mechanical cues guide the reorganization of mechanosensing cortical microtubules (CMT). The anisotropic patterning of CMTs is required for the regulation of immunity-related genes in distal cells. The CMT-mediated mechanotransduction of pathogen-derived cues increases Arabidopsis disease resistance by 40% when challenged with the fungus Sclerotinia sclerotiorum. CONCLUSIONS: CMT anisotropic patterning triggered by pathogen-derived mechanical cues activates the implementation of early plant defense in cells distant from the infection site. We propose that the mechano-signaling triggered immunity (MTI) complements the molecular signals involved in pattern and effector-triggered immunity.

Recent studies have shown that hundreds of small proteins were occulted when protein-coding genes were annotated. These proteins, called alternative proteins, have failed to be annotated notably due to the short length of their open reading frame (less than 100 codons) or the enforced rule establishing that messenger RNAs (mRNAs) are monocistronic. Several alternative proteins were shown to be biologically active molecules and seem to be involved in a wide range of biological functions. However, genome-wide exploration of the alternative proteome is still limited to a few species. In the present article, we describe a deep peptidomics workflow which enabled the identification of 401 alternative proteins in Drosophila melanogaster . Subcellular localization, protein domains, and short linear motifs were predicted for 235 of the alternative proteins identified and point toward specific functions of these small proteins. Several alternative proteins had approximated abundances higher than their canonical counterparts, suggesting that these alternative proteins are actually the main products of their corresponding genes. Finally, we observed 14 alternative proteins with developmentally regulated expression patterns and 10 induced upon the heat-shock treatment of embryos, demonstrating stage or stress-specific production of alternative proteins.

Molecular mechanisms underlying quantitative variations of pathogenicity remain elusive. Here, we identified the Xanthomonas campestris XopJ6 effector that triggers disease resistance in cauliflower and Arabidopsis thaliana . XopJ6 is a close homolog of the Ralstonia pseudosolanacearum PopP2 YopJ family acetyltransferase. XopJ6 is recognized by the RRS1-R/RPS4 NLR pair that integrates a WRKY decoy domain mimicking effector targets. We identified a XopJ6 natural variant carrying a single residue substitution in XopJ6 WRKY-binding site that disrupts interaction with WRKY proteins. This mutation allows XopJ6 to evade immune perception while retaining some XopJ6 virulence functions. Interestingly, xopJ6 resides in a Tn 3 -family transposon likely contributing to xopJ6 copy number variation (CNV). Using synthetic biology, we demonstrate that xopJ6 CNV tunes pathogen virulence on Arabidopsis through gene dosage-mediated modulation of xopJ6 expression . Together, our findings highlight how sequence and structural genetic variations restricted at a particular effector gene contribute to bacterial host adaptation.

Sunflower broomrape (Orobanche cumana) is one of the main pests for sunflower crops. This holo-parasitic plant is specific to sunflower crops. Broomrape seeds perceive their host thanks to germination stimulants present in sunflower root exudates (Bouwmeester et al., 2021). Once germinated, the broomrape radicle grows toward the host root (Krupp et al., 2021) and develops papillae, which adhere to the host root and secrete mucilaginous compounds (Joel & Losner-Goshen, 1994). Subsequently, epidermal cells at the tip of the haustorium, a specific parasitic organ, differentiate into intrusive cells that penetrate the host root (Masumoto et al., 2021). This penetration combines physical pressure and degradation of sunflower root cell walls thanks to pectolytic activity enzymes released by the parasitic plant (Shomer-Ilan, 1993; Losner-Goshen et al., 1998). Intrusive cells make their way toward the host root vessels, crossing the successive host root tissues. Transcriptomic analyses showed that, in the case of a susceptible interaction, defense genes were activated only transiently and at a low level (Dos Santos et al., 2003a,b; Letousey et al., 2007). In addition, the expression of the putative defense suppressor gene Par1 of various parasitic plants at the early stages of interaction (Yang et al., 2020; Qiu et al., 2022), suggests manipulation of their host by parasitic plants. Once in contact with the host xylem vessels, intrusive cells differentiate into vessel elements and vascular connections are established (xylem as well as phloem), to insure the nutrient supply of the parasite (Krupp et al., 2019). Although numerous studies have been performed on parasitic seed germination and haustorium development (Yoshida et al., 2016), most of them were focused on the parasitic plants, and the host cellular mechanisms involved during the intrusive cell development were poorly described (Mutuku et al., 2021). How the host cells behave during the massive expansion of the haustorium tissues across the outer root cell layers remained quite unknown. A few studies published in the 1970–90s explored the host cellular reorganization during the early stages of the haustorium penetration of various Orobanchaceae parasitic plant species. It was shown that haustorium development is accompanied by unusual host cell proliferation (Dörr & Kollmann, 1974; Kuijt, 1977). Whether the penetration is intra- or intercellular in the root host was rarely stated. Dörr & Kollmann (1974) and Kuijt (1977) mentioned intercellular growth only of the haustorial cells, with no observations of plasmodesmata interconnecting host and parasite cells for the interactions O. crenata/Vicia faba and O. ramosa/Cannabis sativa. Intercellular penetration between two cortical cells was shown during the interaction between Striga gesnerioides (another Orobanchaceae species) and cowpea (Vigna unguiculata; Reiss & Bailey, 1998). By contrast, the work by Dörr (1969) on the stem parasitic plant species Cuscuta (Convolvulaceae family) on the host Pelargonium zonale revealed intracellular as well as intercellular penetrations preceding the vascular connection between the host and the parasite (Press et al., 1990). In addition, Musselman & Dickison (1975) showed an example of an intrusive cell of the parasitic plant Agalinis aphylla (Orobanchaceae family) penetrating intracellularly a cortical cell through a small opening in the cell wall. Thus, whether sunflower root penetration by the broomrape haustorium is intra-and/or intercellular remained an open question. This knowledge is required in the perspective of subsequently investigate and understand the sunflower cellular mechanisms associated with resistances to O. cumana. In this work, using an efficient selection of the early stages, and combining various microscopy approaches including live-cell imaging of transgenic fluorescent host tissues, we reinvestigated the relationships between host and parasitic tissues at the cell level during the early stages of haustorium penetration. The questions we addressed were as follows: (1) Do intrusive cells penetrate the host root inter or intracellularly? (2) Do the sunflower root cells in the vicinity of the intrusive cells die or stay alive? (3) Are sunflower cell divisions induced at the very early stages of the penetration, and which are the root tissues involved? To answer these questions, we needed to observe attachments at very early stages, that is, haustorium penetration sites sampled before the establishment of vessel connections. To this end, we used a dedicated growth and inoculation device called rhizotron, a plexiglass homemade box, which facilitates the observation of inoculated sunflower roots and selection of attachment sites (Le Ru et al., 2021; Supporting Information Notes S1). For large field and transmission electron microscopy (TEM) observation of stained longitudinal sections of attachments, we used root fragments from young inoculated wild-type sunflower plantlets (i.e. nontransformed). In addition, to get more information on the living status and the subcellular organization of the penetrated cells, we observed attachments using in vivo confocal imaging of living inoculated transgenic composite sunflower plants, that is, obtained by Agrobacterium rhizogenes-mediated transformation. This method generated plants with fluorescent roots, expressing the green fluorescent protein (GFP) targeted to the endoplasmic reticulum (ER) (Figs S1, S2; Table S1). Observation of attachments was performed from 4 to 8 d after inoculation (dai) (Table S2). Broomrape rarely penetrated the host root before 6 dai, while most of the haustoria had reached the inner root tissues (inner cortex to the vessels) at 8 dai. The kinetics were very similar whatever the type of plants and microscopy approach. Interestingly, similarly to our observations, Joel & Losner-Goshen (1994) observed the first stages of attachments at 5–7 dai. Germinated broomrape seeds developed papillae at the tip of the radicle when contacting the host root (Fig. S2i; Joel & Losner-Goshen, 1994). Mechanical pressure of the broomrape in contact with sunflower root epidermal cells led to cell wall deformation (Fig. 1a–c). Differentiated intrusive cells at the broomrape radicle tip were strongly stained by toluidine blue O. They displayed a very dense cytoplasm, a reduced vacuole and a large nucleus containing a darkly stained nucleolus, suggesting a high metabolic activity (Fig. 1a,d,g,j). Imaging early stages of broomrape penetration revealed that intrusive cells penetrated the epidermal layer as well as the successive outer cortical layers intracellularly (Fig. 1d–i). In our culture system, sunflower roots had 4–5 cortical cell layers between the epidermis and the endodermis (Fig. S3). Intracellular penetration of sunflower root cells was observed in all the analyzed penetration sites (21 sites for large field microscopy and 21 sites for confocal microscopy, Table S2). The use of the GFP-ER construct provided information about both the cytoplasmic organization and the nucleus position, thanks to the ER outline labeling the nuclear envelope (Genre et al., 2005). In many cases, the nucleus of the penetrated cell was strikingly positioned close to the intrusive cells (Fig. 1b,c,h,i). The nucleus repositioning close to the intruder is reminiscent of the cellular reorganization of plant cells during bacterial and fungal symbiotic or pathogenic interactions (Genre et al., 2005, 2008, 2009; Fournier et al., 2008). It suggests that the host nucleus perceives the intrusive cell, either through the exerted mechanical pressure (Genre et al., 2009) and/or through unknown chemical signals. However, in contrast to root penetration by symbiotic (Genre et al., 2005, 2008) or pathogenic biotrophic fungi (Koh et al., 2005; Kankanala et al., 2007; Genre et al., 2009), no cytoplasmic aggregation, nor specific ER reorganization were observed ahead of the penetration process. Interestingly, ER was surrounding the broomrape intrusive cells (Fig. 1e,f,h,i,k,l), showing active, though not massive, host intracellular reorganization along with the penetration process. These results suggested active membrane synthesis around intrusive cells requiring nucleus and ER activity in the host cell, and showed that the sunflower penetrated cells remained alive. Deeper root tissues (endodermis and pericycle) were also penetrated intracellularly by intrusive cells (Fig. 1j). In most cases, haustoria penetrated the host root with minimal host cell damage. However, the live-cell imaging approach revealed a few cases of cell death as shown by the absence of fluorescence (2 sites, Fig. S4a,b), or a severe ER disruption (1 site, Fig. S4c,d). Similarly, change of the vacuole structure was observed using large field microscopy for a few sites (7 sites among 21 penetration sites): appearing as a blue smear (Figs 1d, S4e) or light blue material filling the cell (Fig. S4e). One or a few penetrated cells only were affected, adjacent to the intrusive cells in outer root tissues. These results suggested that in some cases, penetration of the intrusive cells got out of control and synchronization of the penetration process and the sunflower cellular reorganization failed, leading to sunflower cell death. This phenomenon remained cell autonomous, without other defense reactions in the surrounding or the deepest root tissues. Furthermore, penetration could result in the separation of the host nucleus from the distal part of the penetrated cell, probably leading to cell death as well. Strikingly, broomrape intrusion was thicker in outer root tissues (Fig. 1) than in inner root tissues, in which only single elongated and separated intrusive cells were detected (Fig. 1e,f,h,i,k,l). Similarly, Dörr (1969) reported intracellular ‘searching hyphae’ for the Cuscuta stem parasite. Sunflower roots were known to swell locally at the site of broomrape attachment by means of cell division (Kuijt, 1977) and as early as 7 dai (Dörr & Kollmann, 1974). In the present study, sunflower root cell divisions were observed as early as 6 dai, close to attachments (9 and 7 sites for sections and live-cell imaging, respectively). Divisions were mostly anticlinal in the cortex and periclinal in the pericycle (Figs 1d,e,h,i, S5). The number of dividing root cell layers and the length of the dividing zone were highly variable (for example, from 1 cortical cell to > 30 cells in a row). These divisions may account for root hypertrophy that was previously observed at the site of 14-dai attachments in rhizotrons (Chabaud et al., 2022). These divisions could be induced indirectly (host hormonal regulation) or directly by the parasitic plant (hormonal release: such as auxin (Ishida et al., 2016) or cytokinin (Spallek et al., 2017)). Whether germinated broomrape seed exudates would be sufficient for the induction of host cell divisions remains an open question. The interface between intrusive cells and the sunflower penetrated root cells at early stages of the interaction was further characterized by TEM (Fig. 2). A 7 dai attachment with the haustorium reaching the 3rd cortical cell layer is illustrated Fig. 2(a,b). Starch grains, a sign of the transition from the autonomous (germination stage) to the parasitical stage (Joel & Losner-Goshen, 1994), were present in the central part of the attachment (Fig. 2a,c). In the outer root cell layers, the interface appears as a thick layer surrounding broomrape (Fig. 2b), as already described for Striga (Reiss & Bailey, 1998; Neumann et al., 1999). The intrusive cells were easily distinguished from sunflower root cortical cells thanks to their dense cytoplasm, containing Golgi stacks, large mitochondria as well as a reduced vacuole (Fig. 2b,e,f) as already reported by Kuijt & Toth (1976) and Kuijt (1977). By contrast, the host cortical cells whether penetrated or not, contained a large vacuole with a thin layer of surrounding cytoplasm (Fig. 2b,d). Mitochondria in the penetrated host cells were present all along the host plasmalemma, suggesting intense activity at the periphery of the host cell such as membrane biosynthesis (Fig. 2d). This dense cytoplasm confirmed that the penetrated host cells were alive at this stage. The parasitic cell wall was present all around the intrusive cells. By contrast, the presence of a host matrix along the anticlinal interface of the host penetrated cell was not always detectable and its appearance varied along the length of the haustorium. On the outermost anticlinal side of the host penetrated cell, the host interface with the haustorium appeared as a dark thick layer, in continuity with the existing periclinal host cell wall (Fig. 2b,d). It could partly result from the invagination of the existing periclinal host cell wall pushed in by the penetrating intrusive cells. The discontinuity of the staining suggests disorganization of this host cell wall/matrix. On the innermost side of the cell, the host matrix was either too thin to be visible (Fig. 2e,f) or appeared as a low-density material (stars in Fig. 2g,h) separating the host cell plasmalemma from the parasitic cell wall, and differing from the existing darker periclinal host cell wall. The use of various fluorescent dyes to distinguish host cell wall from newly made matrix would be interesting, as done for the symbiotic nitrogen-fixing bacterial infection thread (Rae et al., 2021). At the frontline of the haustorium penetration the existing periclinal host cell wall seemed also disorganized (Fig. 2i), suggesting progressive local enzymatic degradation of the host cell wall. This apparent dissolution of the nearby host cell walls (Kuijt, 1977) or a partial digestion of the cell wall at the interface (Kurotani et al., 2020, in the case of the interaction Phtheirospermum japonicum/Arabidopsis thaliana) had been reported previously. While cell wall degrading enzymes which might contribute to this process have been identified from the parasite (Shomer-Ilan, 1993; Losner-Goshen et al., 1998), there is no evidence at the moment of the direct involvement of host enzyme activities involved in host cell wall degradation in this context (Mitsumasu et al., 2015; Yang et al., 2020). Nevertheless, the host cell plasmalemma seemed to remain undisturbed and continuous (Figs 2g, S6b). Both host and parasitic plasma membranes were highly convoluted at the front line of the haustorium (Fig. S6b), suggesting membrane synthesis for the haustorium accommodation (host) and haustorium expansion (parasite). No plasmodesmata were observed on the interface at these early stages, indicating that molecular exchanges between the parasite and the host happened at later stages, or through vessel connections, as interspecific plasmodesmata have been shown in the phloem (Krupp et al., 2019). In some cases, the penetration led to disaggregation of the vacuole of the host cell (Fig. S6c,d), with disruption of the host cell plasmalemma, leading to cell necrosis, as for Striga (Neumann et al., 1999). However, as mentioned above, this was not very common and remained cell autonomous. In addition, no evidence of cell death was observed at the later stages (Chabaud et al., 2022). Altogether, these results showed that the parasitic intrusive cells penetrate the host root cells intracellularly, as a result of degradation of the host cell wall and formation of a new host trans-cellular apoplastic compartment for haustorium accommodation. Most striking among our findings has been the observation of intracellular haustorium penetration of host root tissues, in contrast to most studies on Orobanchaceae. These studies relied mainly on the observation of transverse sections, in contrast to the longitudinal sections used in this work, which made it easier to distinguish between intra- and intercellular processes. Our work showed the intimate broomrape penetration into its host, through the formation of a new apoplastic compartment. It suggested that although host cell wall integrity has been damaged by parasitic cell wall degrading enzymes (Shomer-Ilan, 1993; Losner-Goshen et al., 1998), only minor defense reactions were induced as previously reported for biotrophic pathogenic fungi (Mendgen & Hahn, 2002; Bellincampi et al., 2014). In that respect, genes encoding inhibitors of cell wall degrading enzymes could be good candidates for increasing resistance to broomrape. In addition, as HaOr7 (Duriez et al., 2019) and HaOrDeb2 (Fernandez-Aparicio et al., 2022) encode Leucine-Rich-Repeat Receptors Like Proteins, providing resistance to various O. cumana races, it would be of outstanding interest to characterize the cellular processes involved in these incompatible interactions. Comparing the cellular processes for various O. cumana races could highlight common or different mechanisms. Finally, using these approaches on other major parasitic plant species such as Striga will be of great interest for future resistance development in a larger host range. We thank P. Gresshoff (University of Queensland, Australia) for the A. rhizogenes strain K599. This study was supported by the ‘Laboratoires d'Excellences (LABEX)’ ‘Towards a Unified theory of biotic interactions: roLe of environmental Pertubations’ (TULIP; ANR-10-LABX-41) and/or by the ‘École Universitaire de Recherche (EUR)’ TULIP-GS (ANR-18-EURE-0019). Slide scanning was performed using the Nanozoomer from the Imagery Platform of the Federated Research Institute AgroBiosciences-Interactions-Biodiversité (FRAIB; Castanet-Tolosan, France). This study was performed in the frame of a 2-year project (SunOrCell), funded by ‘Promosol/SeleoPro’ (the association of French Sunflower and Rapeseed Breeders for promoting these crops). The International Consortium of Sunflower Genomics (ICSG) supported the grants for the training students CG and ARS. None declared. M-CA performed cytological experiments (large field microscopy and TEM). CG, AR-S and AL established sunflower transformation experiments. M-CB produced sunflower and broomrape resources. SM coordinated the ICSG, assisted with the construction of the project and the writing of the manuscript. JF gave technical and scientific advice and assisted with the construction and the writing of the manuscript. MC designed the experiments, carried out confocal microscopy and cytology experiments and wrote the manuscript. Fig. S1 Schedule of sunflower transformation experiments, broomrape inoculation of composite plants for confocal microscopy observations. Fig. S2 The various steps of transformation of sunflower plants via Agrobacterium rhizogenes, transfer of composite plants in rhizotrons, broomrape inoculation and observation using confocal microscopy. Fig. S3 Longitudinal section of a sunflower root. Fig. S4 Sunflower cell death or endoplasmic reticulum destructuring associated with haustorium penetration. Fig. S5 Multiple divisions in the host root at the site of broomrape intrusion. Fig. S6 Transmission electron microscopy of a 7 dai attachment. Notes S1 Details of the materials and methods. Table S1 Efficiency of sunflower transformation via Agrobacterium rhizogenes. Table S2 Overview of observed sites. 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Tyrosine-specific protein tyrosine phosphatases (Tyr-specific PTPases) are key signaling enzymes catalyzing the removal of the phosphate group from phosphorylated tyrosine residues on target proteins. This post-translational modification notably allows the regulation of mitogen-activated protein kinase (MAPK) cascades during defense reactions. Arabidopsis thaliana protein tyrosine phosphatase 1 ( At PTP1), the only Tyr-specific PTPase present in this plant, acts as a repressor of H 2 O 2 production and regulates the activity of MPK3/MPK6 MAPKs by direct dephosphorylation. Here, we report that recombinant histidine (His)- At PTP1 protein activity is directly inhibited by H 2 O 2 and nitric oxide (NO) exogenous treatments. The effects of NO are exerted by S-nitrosation, i.e., the formation of a covalent bond between NO and a reduced cysteine residue. This post-translational modification targets the catalytic cysteine C265 and could protect the At PTP1 protein from its irreversible oxidation by H 2 O 2 . This mechanism of protection could be a conserved mechanism in plant PTPases.

Plant cell walls constitute complex polysaccharidic/proteinaceous networks whose biosynthesis and dynamics implicate several cell compartments. The synthesis and remodeling of homogalacturonan pectins involve Golgi-localized methylation/acetylation and subsequent cell wall-localized demethylation/deacetylation. So far, TRICHOME BIREFRINGENCE-LIKE (TBL) family members have been described as Golgi-localized acetyltransferases targeting diverse hemicelluloses or pectins. Using seed mucilage secretory cells (MSCs) from Arabidopsis thaliana , we demonstrate the atypical localization of TBL38 restricted to a cell wall microdomain. A tbl38 mutant displays an intriguing homogalacturonan immunological phenotype in this cell wall microdomain and in an MSC surface-enriched abrasion powder. Mass spectrometry oligosaccharide profiling of this fraction reveals an increased homogalacturonan acetylation phenotype. Finally, TBL38 displays pectin acetylesterase activity in vitro . These results indicate that TBL38 is an atypical cell wall-localized TBL that displays a homogalacturonan acetylesterase activity rather than a Golgi-localized acetyltransferase activity as observed in previously studied TBLs. TBL38 function during seed development is discussed.