Ethylene is a well-established negative regulator of nodulation, yet how ethylene biosynthesis and perception are spatially coordinated during early symbiotic signaling remains unresolved. Here, we investigate the dynamics of ethylene responses in Medicago (Medicago truncatula) using transcriptomics, promoter-reporter analyses, loss-of-function approaches and a synthetic reporter. We show that the activity of the ethylene-responsive EBSn reporter shifts from inner root tissues under nonsymbiotic conditions to the outer cortex and epidermis following rhizobial inoculation, revealing a spatial reprogramming of ethylene signaling. Among the 8 Medicago 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE (ACS) genes, MtACS3 is induced in outer root cell layers upon rhizobia application, while MtACS10 is repressed in the inner cortex and pericycle, mirroring the shift in ethylene perception. Functional analysis demonstrates that MtACS10 restricts nodule initiation, whereas MtACS3 modulates the number of infection threads, prevents nodule clustering, and contributes to the radial positioning of nodule primordia. Rhizobial induced ectopic ACS expression in the root interior counteracts MtACS10 repression and blocks nodulation, highlighting the requirement for spatially confined downregulation of ethylene biosynthesis. Together, these findings establish a framework in which localized shifts in ethylene biosynthesis, mediated by distinct Medicago ACS genes, balance infection and organogenesis while co-defining the spatial limits of the root susceptible zone.

MOTIVATION:In recent years, the availability of multi-omics data has increased substantially. Multi-omics data integration methods mainly aim to leverage different molecular layers to gain a complete molecular description of biological processes. An attractive integration approach is the reconstruction of multi-omics networks. However, the development of effective multi-omics network reconstruction strategies lags behind. RESULTS:In this study, we introduce collaborative graphical lasso, a novel approach that extends graphical lasso by incorporating collaboration between omics layers, thereby improving multi-omics data integration and enhancing network inference. Our method leverages a collaborative penalty term, which harmonizes the contribution of the omics layers to the reconstruction of the network structure. This promotes a cohesive integration of information across modalities, and it is introduced alongside a dual regularization scheme that separately controls sparsity within and between layers. To address the challenge of model selection in this framework, we propose XStARS, a stability-based criterion for multi-dimensional hyperparameter tuning. We assess the performance of collaborative graphical lasso and the corresponding model selection procedure through simulations, and we apply them to publicly available multi-omics data. This application demonstrated collaborative graphical lasso recovers established biological interactions while suggesting novel, biologically coherent connections. AVAILABILITY AND IMPLEMENTATION:We implemented collaborative graphical lasso as an R package, available on CRAN as coglasso. The results of the manuscript can be reproduced running the code available at https://github.com/DrQuestion/coglasso_reproducible_code, deposited on figshare with DOI: https://doi.org/10.6084/m9.figshare.32324376.

The phytohormone auxin plays a crucial role in the development of so-called syncytia induced by cyst nematodes upon feeding on plant roots. However, it is unknown whether nematode-derived auxin contributes to plant parasitism by cyst nematodes. To do so, plant-parasitic nematodes should be able to produce and release auxin into the environment. Here, we investigated whether this is the case. Mass-spectrometry analyses show that the cyst nematode Globodera pallida is able to synthesize and release the auxin indole-3-acetic acid (IAA) and indicates that its biosynthesis can be stimulated by root diffusate. Using a collection of known auxin biosynthesis genes from well-studied pathways in bacteria and plants we revealed that in the genome of G. pallida candidate genes are present for 5 major biosynthesis pathways, which could be responsible for auxin production. RNA-seq data supported the genomic analysis and revealed the upregulation of various candidate biosynthesis genes during parasitic life stages of the nematode, suggesting a possible role in plant parasitism. However, genomic analysis within the phylum Nematoda showed that the potential auxin biosynthesis genes are wide-spread and also occur in bacterivorous, fungivorous, entomopathogenic and animal-parasitic nematodes. Therefore, our data show that G. pallida is able to synthesize and release auxin into the environment, but that potential auxin biosynthesis pathways are not unique for plant-parasitic nematodes. So, auxin biosynthesis may play a broader role in the life history of nematodes. However, a more specialized role for nematode-produced auxin in plant parasitism cannot be excluded and needs further investigation.

Auxin is a signaling molecule that regulates multiple processes in the growth and development of land plants. Research gathered from model species, particularly Arabidopsis thaliana, has revealed that the nuclear auxin pathway controls many of these processes through transcriptional regulation. Recently, a non-transcriptional pathway based on rapid phosphorylation mediated by kinases has been described, complementing the understanding of the complexity of auxin-regulated processes. Phylogenetic inferences of both pathways indicate that only some of these components are conserved beyond land plants. This raises fundamental questions about the evolutionary origin of auxin responses and whether algal sisters share mechanistic features with land plants. Here, we explore auxin responses in the unicellular streptophyte alga Penium margaritaceum. By assessing physiological, transcriptomic, and cellular responses, we found that auxin triggers cell proliferation, gene regulation, and acceleration of cytoplasmic streaming. Notably, all these responses are also triggered by the structurally related tryptophan. These results identify shared auxin response features among land plants and algae and suggest that less chemically specific responses preceded the emergence of auxin-specific regulatory networks in land plants.

The importance of auxin in plant organ development, including root nodule formation, is well known. The spatiotemporal distribution pattern of auxin during nodule development has been illustrated using auxin reporter constructs. However, our understanding of how this pattern is established and maintained remains elusive. Here, we studied how the auxin gradient is associated with the spatiotemporal expression patterns of known auxin biosynthesis and transport genes at different stages of nodule development in Medicago (Medicago truncatula). In addition, we examined the Medicago PIN-FORMED10 (MtPIN10) expression pattern and polar positioning on the cell membrane during nodule primordium development to investigate auxin flux. RNA interference and the application of auxin biosynthesis inhibitors were used to demonstrate the importance of auxin biosynthesis and transport at the initial stages of nodulation. Our results show that upon rhizobium inoculation before the first cell divisions, a specific subset of Medicago YUCCA (MtYUC) and MtPIN genes, as well as Medicago LIKE AUXIN RESISTANT2 (MtLAX2), are expressed in the pericycle and contribute to the creation of an auxin maximum. Overall, we demonstrate that the dynamic spatiotemporal expression of both MtYUC and MtPIN genes results in specific auxin outputs during the different stages of nodule primordia and nodule meristem formation.

The hallmark of the legume lncRNA EARLY NODULIN40 (ENOD40), involved in rhizobium-induced nodulation, is the presence of a highly conserved stretch of 24 nucleotides, designated box2, preceded by a small open-reading-frame (sORF) coding for a peptide of 12 to 13 amino acids. Although there is a well-established link between ENOD40 and nodulation, it is not fully clear by which mechanism ENOD40 functions in this process. Here, we show that a region harboring box2 can complement nodule formation in an ENOD40 knock-out mutant (enod40-1-2/1). The sequence of box2 bears the characteristics of a miR169 target mimic. We show that the artificial target mimic MIM169defg can indeed complement the reduced capability of nodule formation in enod40-1-2/1, and that box2 exhibits target mimic activity in a transient luciferase assay. In addition, the introduction of a miR169-resistant form of MtNF-YA1 also elevates the capacity to form nodules in enod40-1-2/1. We conclude that ENOD40 effectuates nodule initiation by posttranscriptional upregulation of the miR169 target NF-YA1, which encodes an essential transcription factor in this step of the nodulation process.

Plants have a remarkable regenerative capacity, but this varies widely among species and tissue types. Important crop cultivars show regenerative recalcitrance, which is a major obstacle for the application of modern plant propagation and breeding techniques. Regeneration generally involves empirically determined tissue culture methods that are based on the principle of inducing totipotency. Cells are first persuaded to change fate toward root stem cell-like identity and then are reprogrammed to acquire shoot fate. Alternatively, pluri- or totipotent cells can lead to the formation of a complete plantlet through somatic embryogenesis. We applied our knowledge of root stem cell niche biology to directly use the implicated stem cell factors, including RETINOBLASTOMA (RBR), SCARECROW (SCR), SHORT ROOT (SHR), and members of the AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) and WUSCHEL-related homeobox (WOX) gene families, as a tool to induce regeneration in a way similar to the principle of induced pluripotent stem cells in the animal field. We show that stem cell factors synergistically induce regeneration involving the somatic embryogenesis pathway and can break recalcitrance in Arabidopsis (Arabidopsis thaliana) and pepper (Capsicum annuum).

The final steps of ethylene biosynthesis involve the consecutive activity of two enzymes, 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO). These enzymes are encoded by small gene families, which, in the case of legumes, have not been systematically characterized at the level of gene family membership or phylogenetic relationship. Moreover, the absence of consensus nomenclature complicates comparisons within the scientific literature, where authors are addressing the roles of these genes in planta. In this study, we provide a framework in which the ACS and ACO gene family members of several legume species, including the two model legumes Medicago truncatula and Lotus japonicus, were systematically annotated, named, and analysed relative to genes from other dicot and monocot model species. A combination of phylogenetic and reciprocal BLAST analyses was used to identify evolutionary relationships among genes, including the identification of orthologous relationships that can inform hypotheses about function. Given the role of ethylene as a negative regulator of the legume-rhizobium symbiosis, we queried publicly available RNA-seq expression datasets to obtain an overview of the expression profiles of these genes in the interaction between M. truncatula and its nitrogen-fixing microsymbiont. The resulting evolutionary framework, as well as structural and expression analyses, are intended to facilitate ongoing functional studies in legumes.

stem cuttings are the primary method for propagation in medicinal cannabis, yet propagation has not been studied as extensively as the later stages of crop cultivation. This study examined how mother plant age and photosynthetic photon flux density (PPFD) during mother plant cultivation and propagation affect rooting, growth, and development of apical stem cuttings. Mother plants (Cannabis sativa 'Original Blitz' and 'King Harmony') were grown in climate-controlled chambers under two light intensities (400 and 800 lmolm-2s-1) for up to 6 months. Apical stem cuttings were excised every 3 weeks and subsequently propagated without externally applied auxin for 3 weeks under three light intensities (50, 150, and 250 lmolm-2s-1). Mother plant age did not affect rooting (root dry mass and fraction of rooted cuttings). However, older mother plants exhibited decreased cutting dry mass at severance, which coincided with a reduced leaf area. The light intensity during mother plant cultivation had genotype-specific effects, with rooting either reduced or unaffected for the higher light intensity. This reduction coincided with an accumulation of starch and soluble sugar at the stem base at severance, while auxin concentrations in the apex, leaf, and stem base were unaffected by light intensity during mother plant cultivation. In contrast, light intensity during propagation did not affect the fraction of rooted cuttings. However, higher light intensity increased root and cutting dry mass. These findings indicate that mother plant age, up to 6 months, does not impact rooting in stem cuttings. However, higher light intensity during mother plant cultivation reduced rooting genotype-dependently, whereas higher light intensity during propagation increased root dry mass without affecting fraction of rooted cuttings.

In sweet pepper (Capsicum annuum L.), the fruit yield is often negatively affected by fruit abortion. Here we investigated whether fruit abortion is affected by the blue:red light ratio (B:R) and the possible underlying physiological mechanisms related to carbohydrates and hormones. Sweet pepper plants were grown at B:R of 1:10, 1:3, 1:1 or 9:1 with a total photosynthetic photon flux density of 200 mu mol m- 2 s- 1, resulting in a phytochrome photostationary state (PSS) of 0.88, 0.88, 0.86 and 0.72, respectively. For fruit set observations, each plant was allowed to retain 12 flowers on 4 main stems. Sweet pepper plants grown at the highest B:R (9:1) showed a low fruit set (around 3 fruits per plant), whereas the other three treatments resulted in higher fruit set (6-7 fruits per plant). This response matched with the changes in PSS, suggesting the B:R effect on fruit set might be controlled by phytochrome signaling, which requires further investigation. Plant shoot biomass and leaf area were reduced at B:R of 1:1 and 9:1. The reduced fruit set was associated with a drop in starch content and sucrose synthases activity; and a low auxin, high salicylic acid and high cis-Zeatin type levels in flowers. Flowers in the low fruit set treatment also failed to reduce the abscisic acid and ethylene levels after anthesis. We concluded that both the reduced starch content and the hormonal changes in flowers play a role in triggering fruit abortion at the high B:R of 9:1.

Legume plants can acquire mineral nitrogen (N) either through their roots or via a symbiotic interaction with N-fixing rhizobia bacteria housed in root nodules. To identify shoot-to-root systemic signals acting in Medicago truncatula plants at N deficit or N satiety, plants were grown in a split-root experimental design in which either high or low N was provided to half of the root system, allowing the analysis of systemic pathways independently of any local N response. Among the plant hormone families analyzed, the cytokinin trans-zeatin accumulated in plants at N satiety. Cytokinin application by petiole feeding led to inhibition of both root growth and nodulation. In addition, an exhaustive analysis of miRNAs revealed that miR2111 accumulates systemically under N deficit in both shoots and non-treated distant roots, whereas a miRNA related to inorganic phosphate (Pi) acquisition, miR399, accumulates in plants grown under N satiety. These two accumulation patterns are dependent on Compact Root Architecture 2 (CRA2), a receptor required for C-terminally Encoded Peptide (CEP) signaling. Constitutive ectopic expression of miR399 reduced nodule numbers and root biomass depending on Pi availability, suggesting that the miR399-dependent Pi-acquisition regulatory module controlled by N availability affects the development of the whole legume plant root system. Cytokinins and the phosphate-related microRNA miR399 accumulate in plants at N satiety in a CRA2 pathway-dependent manner, and systemically inhibit symbiotic nodulation and root development.

In recent years, the availability of multi-omics data has increased substantially. Multi-omics data integration methods mainly aim to leverage different molecular data sets to gain a complete molecular description of biological processes. An attractive integration approach is the reconstruction of multi-omics networks. However, the development of effective multi-omics network reconstruction strategies lags behind. This hinders maximizing the potential of multi-omics data sets. With this study, we advance the frontier of multi-omics network reconstruction by introducing "collaborative graphical lasso" as a novel strategy. Our proposed algorithm synergizes "graphical lasso" with the concept of "collaboration", effectively harmonizing multi-omics data sets integration, thereby enhancing the accuracy of network inference. Besides, to tackle model selection in this framework, we designed an ad hoc procedure based on network stability. We assess the performance of collaborative graphical lasso and the corresponding model selection procedure through simulations, and we apply them to publicly available multi-omics data. This demonstrated collaborative graphical lasso is able to reconstruct known biological connections and suggest previously unknown and biologically coherent interactions, enabling the generation of novel hypotheses. We implemented collaborative graphical lasso as an R package, available on CRAN as coglasso.

C-terminally encoded peptides (CEPs) are small secreted signaling peptides that promote nitrogen-fixing root nodulation symbiosis in legumes, depending on soil mineral nitrogen availability.1 In Medicago truncatula, their action is mediated by the leucine-rich repeat receptor-like protein kinase COMPACT ROOT ARCHITECTURE 2 (CRA2).2,3,4 Like most land plants, under inorganic phosphate limitation, M. truncatula establishes another root endosymbiotic interaction with arbuscular fungi, the arbuscular mycorrhizal symbiosis (AMS). Because this interaction is beneficial for the plant but has a high energetic cost, it is tightly controlled by host plants to limit fungal infections mainly depending on phosphate availability.5 We show in this study that the expression of a subset of CEP-encoding genes is enhanced in the low-phosphate conditions and that overexpression of the low-phosphate-induced MtCEP1 gene, previously shown to promote the nitrogen-fixing root nodulation symbiosis, enhances AMS from the initial entry point of the fungi. Conversely, a loss-of-function mutation of the CRA2 receptor required for mediating CEP peptide action2 decreases the endomycorrhizal interaction from the same initial fungal entry stage. Transcriptomic analyses revealed that the cra2 mutant is negatively affected in the regulation of key phosphate transport and response genes as well as in the biosynthesis of strigolactone hormones that are required for establishing AMS. Accordingly, strigolactone contents were drastically decreased in cra2 mutant roots. Overall, we showed that the CEP/CRA2 pathway promotes both root nodulation and AMS in legume plants, depending on soil mineral nutrient availability.

Early sprouting is a main cause of onion spoilage during storage. However, limited knowledge is available on which factors trigger sprouting. Here, this was studied in the Hyfive and Exhibition cultivars, which largely differ in sprouting time. Sprouting progress was compared to the fructan and abscisic acid (ABA) profiles in the bulb scales and basal plates. Fructan concentrations decreased in the scales from harvest time onwards in the late-sprouting cultivar Hyfive, while remaining constant in the cultivar Exhibition until internal sprouting. In the basal plates, fructan concentrations increased in both cultivars from approximately one month after harvest, but reached maximum concentrations at moments that could not be related to the difference in internal sprouting. ABA levels generally decreased in the scales of both cultivars, while increasing in their basal plates. Nevertheless, for fructans, the measured variation in ABA concentrations was not consistently associated with differences in internal sprouting. A subsequent perturbation of internal sprouting by Maleic Hydrazide treatment in the cultivar Hyfive confirmed a lack of correlation. Altogether, this indicates that fructan and ABA levels in the scales and basal plate tissue change independent of internal sprouting and cannot be regarded as predictive markers for sprouting and storability.

C-terminally encoded peptides (CEPs) are small secreted signalling peptides that promote in legumes the root nitrogen-fixing nodulation symbiosis depending on soil mineral nitrogen availability[1][1]. In Medicago truncatula, their action is mediated by the Leucine-rich repeat receptor-like protein kinase COMPACT ROOT ARCHITECTURE 2 (CRA2)[2][2]–[4][3]. As most land plants, under inorganic phosphate (Pi) limitation, M. truncatula establishes another root endosymbiotic interaction with arbuscular fungi, the arbuscular mycorrhizal symbiosis (AMS). Because this interaction is beneficial for the plant but has a high energetic cost, it is tightly controlled by host plants to limit AMS infections mainly depending on Pi availability[5][4]. We show in this study that the expression of a subset of CEP encoding genes is enhanced in the low Pi conditions that favour AMS colonization, and that overexpression of the low Pi-induced MtCEP1 gene, previously shown to promote the nitrogen-fixing root nodulation symbiosis, enhances the AMS colonization from the initial entry point of the fungi. Conversely, a loss-of-function mutation of the CRA2 receptor required for mediating CEP peptides action[2][2] decreases the AMS interaction capacity from the same initial fungal entry stage. Transcriptomic analyses revealed that the cra2 mutant is negatively affected in the regulation of key Pi transport and response genes as well as in the biosynthesis of strigolactone (SL) hormones that are required for establishing the AMS interaction. Accordingly, SL contents were drastically decreased in cra2 mutant roots. Overall, we showed that the CEP/CRA2 pathway promotes, in legume plants, both root nodulation and AMS depending on soil mineral nutrients availability. In brief The establishment and maintenance of root nodule and arbuscular mycorrhizal symbioses (AMS) in legume plants is tightly regulated respectively by nitrogen and phosphate availability. Pedinotti and Teyssendier de la Serve et al. show that the CEP/CRA2 pathway, previously known to promote root competence to nodulate under low nitrogen conditions, also enhances AMS establishment by controlling the expression of Pi homeostasis and strigolactone (SL) hormone biosynthesis genes, as well as SL accumulation. Highlights ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2 [3]: #ref-4 [4]: #ref-5

In this study, we explore the interplay between the plant hormones gibberellins (GA), brassinosteroids (BR), and Indole-3-Acetic Acid (IAA) in their collective impact on plant shade avoidance elongation under varying light conditions. We focus particularly on low Red:Far-red (R:FR) light conditions achieved by supplementing the background light with FR. We characterized the tomato internode response to low R:FR and, with RNA-seq analysis, we were able to identify some of the potential regulatory hormonal pathways. Through a series of exogenous pharmacological modulations of GA, IAA, and BR, we demonstrate that GA and BR are sufficient but also necessary for inducing stem elongation under low R:FR light conditions. Intriguingly, while IAA alone shows limited effects, its combination with GA yields significant elongation, suggesting a nuanced hormonal balance. Furthermore, we unveil the complex interplay of these hormones under light with low R:FR, where the suppression of one hormone's effect can be compensated by the others. This study provides insights into the hormonal mechanisms governing plant adaptation to light, highlighting the intricate and adaptable nature of plant growth responses. Our findings have far-reaching implications for agricultural practices, offering potential strategies for optimizing plant growth and productivity in various lighting environments.

Stem cuttings are used in the commercial cultivation of many crops, including medicinal cannabis, to produce large numbers of uniform and genetically identical plants. Light is an important environmental factor determining the success of the rooting of stem cuttings. The aim of this study was to investigate the influence of different fractions of far-red and blue during the adventitious rooting phase of medicinal cannabis stem cuttings on rooting and whether these effects are related to changes in endogenous auxin and/or carbohydrates. Two experiments were conducted in climate chambers with sole LED lighting (blue, red, far-red) using two cannabis cultivars. In Experiment 1, four light treatments were applied: 100 mu mol m(-2)s(-1) red:blue (88:12) with additional 0, 50 or 100 mu mol m(-2)s(-1) far-red and a fourth treatment with 100 mu mol m(-2)s(-1) sole red with additional 50 mu mol m(-2)s(-1) far-red. In Experiment 2, the following four light treatments were applied: 90 mu mol m(-2)s(-1) red:blue (45:45) with additional 0 or 45 mu mol m(-2)s(-1) far-red, a third treatment with 45 mu mol m(-2)s(-1) sole red with additional 45 mu mol m(-2)s(-1) far-red, and a fourth dynamic treatment which was 45 mu mol m(-2)s(-1) sole red with additional 45 mu mol m(-2)s(-1) far-red for 7 days followed by 90 mu mol m(-2)s(-1) red:blue (45:45) for 14 days. The effects on rooting in both experiments were measured after 21 days of light treatments. In Experiment 2, periodic measurements of auxin and carbohydrates were performed. Far-red improved adventitious rooting only in Experiment 2, where both cultivars responded similarly. Adding far-red only during the initial stage (7 days) of rooting was sufficient to improve rooting, while it did not result in excessive stem elongation. The presence or absence of blue did not significantly affect rooting. Although the positive effects of far-red on auxin and carbohydrate concentrations in stem cuttings are a likely explanation for the observed effects of far-red on rooting, we did not find a correlation between auxin or carbohydrates and rooting.

Plant scientists are rapidly integrating single-cell RNA sequencing (scRNA-seq) into their workflows. Maximizing the potential of scRNA-seq requires a proper understanding of the spatiotemporal context of cells. However, positional information is inherently lost during scRNA-seq, limiting its potential to characterize complex biological systems. In this review we highlight how current single-cell analysis pipelines cannot completely recover spatial information, which confounds biological interpretation. Various strategies exist to identify the location of RNA, from classical RNA in situ hybridization to spatial transcriptomics. Herein we discuss the possibility of utilizing this spatial information to supervise single-cell analyses. An integrative approach will maximize the potential of each technology, and lead to insights which go beyond the capability of each individual technology.

In this study, we explore the dynamic interplay between the plant hormones gibberellins (GA), brassinosteroids (BR), and Indole-3-Acetic Acid (IAA) in their collective impact on plant shade avoidance elongation under varying light conditions. We focus particularly on low Red: Far-red (R:FR) light conditions achieved by supplementing the background light with FR. Our research delves into how these hormones individually and synergistically influence stem elongation in tomato plants. Through meticulous experimental modulations of GA, IAA, and BR, we demonstrate that GA and BR are sufficient but also necessary for inducing stem elongation under low R:FR light conditions. Intriguingly, while IAA alone shows limited effects, its combination with GA yields significant elongation, suggesting a nuanced hormonal balance. Furthermore, we unveil the complex interplay of these hormones under light with low R:FR, where the suppression of one hormone’s effect can be compensated by the others. This study provides insights into the hormonal mechanisms governing plant adaptation to light, highlighting the intricate and adaptable nature of plant growth responses. Our findings have far-reaching implications for agricultural practices, offering potential strategies for optimizing plant growth and productivity in various lighting environments.Highlight This study unveils the interplay of brassinosteroids and gibberellins in shade avoidance elongation, revealing how tomatoes acclimate in response to far-red enriched light conditions.### Competing Interest StatementThe authors have declared no competing interest.* SAS : Shade avoidance syndrome GA : Gibberellins BR : Brassinosteroids IAA : Indole-3-Acetic Acid R : red light FR : Far-red light WL : White light DEG : Differentially expressed genes GO : Gene Ontology NPA : Naphthylphthalamic acid BBo : 4-biphenylboronic acid PEO-IAA : 2-(1H-Indol-3-yl)-4-oxo-4-phenyl-butyric acid PBZ : Paclobutrazol BZ : Brassinazole

Tomato bacterial canker caused by Clavibacter michiganensis (Cm) is considered to be one of the most destructive bacterial diseases of tomato. To date, no resistance to the pathogen has been identified. While several molecular studies have identified (Cm) bacterial factors involved in disease development, the plant genes and mechanisms associated with susceptibility of tomato to the bacterium remain largely unknown. Here, we show for the first time that tomato gene SlWAT1 is a susceptibility gene to Cm. We inactivated the gene SlWAT1 through RNAi and CRISPR/Cas9 to study changes in tomato susceptibility to Cm. Furthermore, we analysed the role of the gene in the molecular interaction with the pathogen. Our findings demonstrate that SlWAT1 functions as an S gene to genetically diverse Cm strains. Inactivation of SlWAT1 reduced free auxin contents and ethylene synthesis in tomato stems and suppressed the expression of specific bacterial virulence factors. However, CRISPR/Cas9 slwat1 mutants exhibited severe growth defects. The observed reduced susceptibility is possibly a result of downregulation of bacterial virulence factors and reduced auxin contents in transgenic plants. This shows that inactivation of an S gene may affect the expression of bacterial virulence factors.