Coronatine orchestrates ABI1-mediated stomatal opening to facilitate bacterial pathogen infection through importin ß protein SAD2.

Stomatal immunity plays an important role during bacterial pathogen invasion. Abscisic acid (ABA) induces plants to close their stomata and halt pathogen invasion, but many bacterial pathogens secrete phytotoxin coronatine (COR) to antagonize ABA signaling and reopen the stomata to promote infection at early stage of invasion. However, the underlining mechanism is not clear. SAD2 is an importin ß family protein, and the sad2 mutant shows hypersensitivity to ABA. We discovered ABI1, which negatively regulated ABA signaling and reduced plant sensitivity to ABA, was accumulated in the plant nucleus after COR treatment. This event required SAD2 to import ABI1 to the plant nucleus. Abolition of SAD2 undermined ABI1 accumulation. Our study answers the long-standing question of how bacterial COR antagonizes ABA signaling and reopens plant stomata during pathogen invasion.

XLG2 and CORI3 function additively to regulate plant defense against the necrotrophic pathogen Sclerotinia sclerotiorum.

The membrane-bound heterotrimeric G-proteins in plants play a crucial role in defending against a broad range of pathogens. This study emphasizes the significance of Extra-large Gα protein 2 (XLG2), a plant-specific G-protein, in mediating the plant response to Sclerotinia sclerotiorum, which infects over 600 plant species worldwide. Our analysis of Arabidopsis G-protein mutants showed that loss of XLG2 function increased susceptibility to S. sclerotiorum, accompanied by compromised accumulation of jasmonic acid (JA) during pathogen infection. Overexpression of the XLG2 gene in xlg2 mutant plants resulted in higher resistance and increased JA accumulation during S. sclerotiorum infection. Co-immunoprecipitation (co-IP) analysis on S. sclerotiorum infected Col-0 samples, using two different approaches, identified 201 XLG2-interacting proteins. The identified JA-biosynthetic and JA-responsive proteins had compromised transcript expression in the xlg2 mutant during pathogen infection. XLG2 was found to interact physically with a JA-responsive protein, Coronatine induced 1 (CORI3) in Co-IP, and confirmed using split firefly luciferase complementation and bimolecular fluorescent complementation assays. Additionally, genetic analysis revealed an additive effect of XLG2 and CORI3 on resistance against S. sclerotiorum, JA accumulation, and expression of the defense marker genes. Overall, our study reveals two independent pathways involving XLG2 and CORI3 in contributing resistance against S. sclerotiorum.

Deciphering physiological and transcriptional mechanisms of maize seed germination.

Maize is a valuable raw material for feed and food production. Healthy seed germination is important for improving the yield and quality of maize. Seed aging occurs relatively fast in crops and it is a process that delays germination as well as reduces its rate and even causes total loss of seed viability. However, the physiological and transcriptional mechanisms that regulate maize seeds, especially aging seed germination remain unclear. Coronatine (COR) which is a phytotoxin produced by Pseudomonas syringae and a new type of plant growth regulator can effectively regulate plant growth and development, and regulate seed germination. In this study, the physiological and transcriptomic mechanisms of COR-induced maize seed germination under different aging degrees were analyzed. The results showed that 0.001-0.01 µmol/L COR could promote the germination of aging maize seed and the growth of primary roots and shoots. COR treatment increased the content of gibberellins (GA3) and decreased the content of abscisic acid (ABA) in B73 seeds before germination. The result of RNA-seq analysis showed 497 differentially expressed genes in COR treatment compared with the control. Three genes associated with GA biosynthesis (ZmCPPS2, ZmD3, and ZmGA2ox2), and two genes associated with GA signaling transduction (ZmGID1 and ZmBHLH158) were up-regulated. Three genes negatively regulating GA signaling transduction (ZmGRAS48, ZmGRAS54, and Zm00001d033369) and two genes involved in ABA biosynthesis (ZmVP14 and ZmPCO14472) were down-regulated. The physiological test results also showed that the effects of GA and ABA on seed germination were similar to those of high and low-concentration COR, respectively, which indicated that the effect of COR on seed germination may be carried out through GA and ABA pathways. In addition, GO and KEGG analysis suggested that COR is also highly involved in antioxidant enzyme systems and secondary metabolite synthesis to regulate maize seed germination processes. These findings provide a valuable reference for further research on the mechanisms of maize seed germination.

Spermine deficiency shifts the balance between jasmonic acid and salicylic acid-mediated defence responses in Arabidopsis.

Polyamines are small aliphatic polycations present in all living organisms. In plants, the most abundant polyamines are putrescine (Put), spermidine (Spd) and spermine (Spm). Polyamine levels change in response to different pathogens, including Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). However, the regulation of polyamine metabolism and their specific contributions to defence are not fully understood. Here we report that stimulation of Put biosynthesis by Pst DC3000 is dependent on coronatine (COR) perception and jasmonic acid (JA) signalling, independently of salicylic acid (SA). Conversely, lack of Spm in spermine synthase (spms) mutant stimulated galactolipids and JA biosynthesis, and JA signalling under basal conditions and during Pst DC3000 infection, whereas compromised SA-pathway activation and defence outputs through SA-JA antagonism. The dampening of SA responses correlated with COR and Pst DC3000-inducible deregulation of ANAC019 expression and its key SA-metabolism gene targets. Spm deficiency also led to enhanced disease resistance to the necrotrophic fungal pathogen Botrytis cinerea and stimulated endoplasmic reticulum (ER) stress signalling in response to Pst DC3000. Overall, our findings provide evidence for the integration of polyamine metabolism in JA- and SA-mediated defence responses, as well as the participation of Spm in buffering ER stress during defence.

Deciphering transcriptional mechanisms of maize internodal elongation by regulatory network analysis.

The lengths of the basal internodes is an important factor for lodging resistance of maize (Zea mays). In this study, foliar application of coronatine (COR) to 10 cultivars at the V8 growth stage had different suppression effects on the length of the eighth internode, with three being categorized as strong-inhibition cultivars (SC), five as moderate (MC), and two as weak (WC). RNA-sequencing of the eighth internode of the cultivars revealed a total of 7895 internode elongation-regulating genes, including 777 transcription factors (TFs). Genes related to the hormones cytokinin, gibberellin, auxin, and ethylene in the SC group were significantly down-regulated compared to WC, and more cell-cycle regulatory factors and cell wall-related genes showed significant changes, which severely inhibited internode elongation. In addition, we used EMSAs to explore the direct regulatory relationship between two important TFs, ZmABI7 and ZmMYB117, which regulate the cell cycle and cell wall modification by directly binding to the promoters of their target genes ZmCYC1, ZmCYC3, ZmCYC7, and ZmCPP1. The transcriptome reported in this study will provide a useful resource for studying maize internode development, with potential use for targeted genetic control of internode length to improve the lodging resistance of maize.

Complete Genome Sequencing of Three Clade-1 Xanthomonads Reveals Genetic Determinants for a Lateral Flagellin and the Biosynthesis of Coronatine-Like Molecules in Xanthomonas.

Evolutionarily, early-branching xanthomonads, also referred to as clade-1 xanthomonads, include major plant pathogens, most of which colonize monocotyledonous plants. Seven species have been validly described, among them the two sugarcane pathogens Xanthomonas albilineans and Xanthomonas sacchari, as well as Xanthomonas translucens, which infects small-grain cereals and diverse grasses but also asparagus and pistachio trees. Single-gene sequencing and genomic approaches have indicated that this clade likely contains more, yet-undescribed species. In this study, we sequenced representative strains of three novel species using long-read sequencing technology. Xanthomonas campestris pv. phormiicola strain CFBP 8444 causes bacterial streak on New Zealand flax, another monocotyledonous plant. Xanthomonas sp. strain CFBP 8443 has been isolated from common bean, and Xanthomonas sp. strain CFBP 8445 originated from banana. Complete assemblies of the chromosomes confirmed their unique phylogenetic position within clade 1 of Xanthomonas. Genome mining revealed novel genetic features, hitherto undescribed in other members of the Xanthomonas genus. In strain CFBP 8444, we identified genes related to the synthesis of coronatine-like compounds, a phytotoxin produced by several pseudomonads, which raises interesting questions about the evolution and pathogenicity of this pathogen. Furthermore, strain CFBP 8444 was found to contain a second, atypical flagellar gene cluster in addition to the canonical flagellar gene cluster. Overall, this research represents an important step toward better understanding the evolutionary history and biology of early-branching xanthomonads.

Coronatine promotes maize water uptake by directly binding to the aquaporin ZmPIP2;5 and enhancing its activity.

Water uptake is crucial for crop growth and development and drought stress tolerance. The water channel aquaporins (AQP) play important roles in plant water uptake. Here, we discovered that a jasmonic acid analog, coronatine (COR), enhanced maize (Zea mays) root water uptake capacity under artificial water deficiency conditions. COR treatment induced the expression of the AQP gene Plasma membrane intrinsic protein 2;5 (ZmPIP2;5). In vivo and in vitro experiments indicated that COR also directly acts on ZmPIP2;5 to improve water uptake in maize and Xenopus oocytes. The leaf water potential and hydraulic conductivity of roots growing under hyperosmotic conditions were higher in ZmPIP2;5-overexpression lines and lower in the zmpip2;5 knockout mutant, compared to wild-type plants. Based on a comparison between ZmPIP2;5 and other PIP2s, we predicted that COR may bind to the functional site in loop E of ZmPIP2;5. We confirmed this prediction by surface plasmon resonance technology and a microscale thermophoresis assay, and showed that deleting the binding motif greatly reduced COR binding. We identified the N241 residue as the COR-specific binding site, which may activate the channel of the AQP tetramer and increase water transport activity, which may facilitate water uptake under hyperosmotic stress.

The jasmonoyl-isoleucine receptor CORONATINE INSENSITIVE1 suppresses defense gene expression in Arabidopsis roots independently of its ligand.

The F-box protein CORONANTINE INSENSITIVE1 (COI1) serves as the receptor for the plant hormone jasmonoyl-isoleucine (JA-Ile). COI1, its co-receptors of the JASMONATE ZIM-domain (JAZ) protein family, and JA-Ile form a functional unit that regulates growth or defense mechanisms in response to various stress cues. Strikingly, COI1, but not JA-Ile, is required for susceptibility of Arabidopsis thaliana towards the soil-borne vascular pathogen Verticillium longisporum. In order to obtain marker genes for further analysis of this JA-Ile-independent COI1 function, transcriptome analysis of roots of coi1 and allene oxide synthase (aos) plants (impaired in JA biosynthesis) was performed. Intriguingly, nearly all of the genes that are differentially expressed in coi1 versus aos and wild type are constitutively more highly expressed in coi1. To support our notion that COI1 acts independently of its known downstream signaling components, coi1 plants were complemented with a COI1 variant (COI1AA ) that is compromised in its interaction with JAZs. As expected, these plants showed only weak induction of the expression of the JA-Ile marker gene VEGETATIVE STORAGE PROTEIN2 after wounding and remained sterile. On the other hand, genes affected by COI1 but not by JA-Ile were still strongly repressed by COI1AA . We suggest that COI1 has a potential moonlighting function that serves to repress gene expression in a JA-Ile- and JAZ-independent manner.

Reference-aided full-length transcript assembly, cDNA cloning, and molecular characterization of coronatine-insensitive 1b (COI1b) gene in coconut (Cocos nucifera L.).

BACKGROUND: In the Philippines, 26% of the total agricultural land is devoted to coconut production making coconut one of the most valuable industrial crop in the country. However, the country's multimillion-dollar coconut industry is threatened by the outbreak of coconut scale insect (CSI) and other re-emerging insect pests promoting national research institutes to work jointly on developing new tolerant coconut varieties. Here, we report the cloning and characterization of coronatine-insensitive 1 (COI1) gene, one of the candidate insect defense genes, using 'Catigan Green Dwarf' (CATD) genome sequence assembly as reference. METHODS AND RESULTS: Two (2) splicing variants were identified and annotated-CnCOI1b-1 and CnCOI1b-2. The full-length cDNA of CnCOI1b-1 was 7919 bp with an ORF of 1176 bp encoding for a deduced protein of 391 amino acids while CnCOI1b-2 has 2360 bp full-length cDNA with an ORF of 1743 bp encoding a deduced protein of 580 amino acids. The 3D structural model for the two (2) isoforms were generated through homology modelling. Functional analysis revealed that both isoforms are involved in various physiological and developmental plant processes including defense response of plants to insects and pathogens. Phylogenetic analysis confirms high degree of COI1 protein conservation during evolution, especially among monocot species. Differential gene expression via qRT-PCR analysis revealed a seven-fold increase of COI1 gene expression in coconut post introduction of CSI relative to base levels. CONCLUSION: This study provided the groundwork for further research on the actual role of COI1 in coconut in response to insect damage. The findings of this study are also vital to facilitate the development of improved insect-resistant coconut varieties for vibrant coconut industry.

Coronatine Enhances Chilling Tolerance of Tomato Plants by Inducing Chilling-Related Epigenetic Adaptations and Transcriptional Reprogramming.

Low temperature is an important environmental factor limiting the widespread planting of tropical and subtropical crops. The application of plant regulator coronatine, which is an analog of Jasmonic acid (JA), is an effective approach to enhancing crop's resistance to chilling stress and other abiotic stresses. However, the function and mechanism of coronatine in promoting chilling resistance of tomato is unknown. In this study, coronatine treatment was demonstrated to significantly increase tomato chilling tolerance. Coronatine increases H3K4me3 modifications to make greater chromatin accessibility in multiple chilling-activated genes. Corresponding to that, the expression of CBFs, other chilling-responsive transcription factor (TF) genes, and JA-responsive genes is significantly induced by coronatine to trigger an extensive transcriptional reprogramming, thus resulting in a comprehensive chilling adaptation. These results indicate that coronatine enhances the chilling tolerance of tomato plants by inducing epigenetic adaptations and transcriptional reprogramming.

JAZ4 is involved in plant defense, growth, and development in Arabidopsis.

Jasmonate zim-domain (JAZ) proteins comprise a family of transcriptional repressors that modulate jasmonate (JA) responses. JAZ proteins form a co-receptor complex with the F-box protein coronatine insensitive1 (COI1) that recognizes both jasmonoyl-l-isoleucine (JA-Ile) and the bacterial-produced phytotoxin coronatine (COR). Although several JAZ family members have been placed in this pathway, the role of JAZ4 in this model remains elusive. In this study, we observed that the jaz4-1 mutant of Arabidopsis is hyper-susceptible to Pseudomonas syringae pv. tomato (Pst) DC3000, while Arabidopsis lines overexpressing a JAZ4 protein lacking the Jas domain (JAZ4∆Jas) have enhanced resistance to this bacterium. Our results show that the Jas domain of JAZ4 is required for its physical interaction with COI1, MYC2 or MYC3, but not with the repressor complex adaptor protein NINJA. Furthermore, JAZ4 degradation is induced by COR in a proteasome- and Jas domain-dependent manner. Phenotypic evaluations revealed that expression of JAZ4∆Jas results in early flowering and increased length of root, hypocotyl, and petiole when compared with Col-0 and jaz4-1 plants, although JAZ4∆Jas lines remain sensitive to MeJA- and COR-induced root and hypocotyl growth inhibition. Additionally, jaz4-1 mutant plants have increased anthocyanin accumulation and late flowering compared with Col-0, while JAZ4∆Jas lines showed no alteration in anthocyanin production. These findings suggest that JAZ4 participates in the canonical JA signaling pathway leading to plant defense response in addition to COI1/MYC-independent functions in plant growth and development, supporting the notion that JAZ4-mediated signaling may have distinct branches.

Coronatine Contributes to Pseudomonas cannabina pv. alisalensis Virulence by Overcoming Both Stomatal and Apoplastic Defenses in Dicot and Monocot Plants.

Pseudomonas cannabina pv. alisalensis is a causative agent of bacterial blight of crucifers including cabbage, radish, and broccoli. Importantly, P. cannabina pv. alisalensis can infect not only a wide range of Brassicaceae spp. but, also, green manure crops such as oat. However, P. cannabina pv. alisalensis virulence mechanisms have not been investigated and are not fully understood. We focused on coronatine (COR) function, which is one of the well-known P. syringae pv. tomato DC3000 virulence factors, in P. cannabina pv. alisalensis infection processes on both dicot and monocot plants. Cabbage and oat plants dip-inoculated with a P. cannabina pv. alisalensis KB211 COR mutant (ΔcmaA) exhibited reduced virulence compared with P. cannabina pv. alisalensis wild type (WT). Moreover, ΔcmaA failed to reopen stomata on both cabbage and oat, suggesting that COR facilitates P. cannabina pv. alisalensis entry through stomata into both plants. Furthermore, cabbage and oat plants syringe-infiltrated with ΔcmaA also showed reduced virulence, suggesting that COR is involved in overcoming not only stomatal-based defense but also apoplastic defense. Indeed, defense-related genes, including PR1 and PR2, were highly expressed in plants inoculated with ΔcmaA compared with WT, indicating that COR suppresses defense-related genes of both cabbage and oat. Additionally, salicylic acid accumulation increases after ΔcmaA inoculation compared with WT. Taken together, COR contributes to causing disease by suppressing stomatal-based defense and apoplastic defense in both dicot and monocot plants. Here, we investigated COR functions in the interaction of P. cannabina pv. alisalensis and different host plants (dicot and monocot plants), using genetically and biochemically defined COR deletion mutants.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2021.

Transcriptome dynamic landscape underlying the improvement of maize lodging resistance under coronatine treatment.

BACKGROUND: Lodging is one of the important factors causing maize yield. Plant height is an important factor in determining plant architecture in maize (Zea mays L.), which is closely related to lodging resistance under high planting density. Coronatine (COR), which is a phytotoxin and produced by the pathogen Pseudomonas syringae, is a functional and structural analogue of jasmonic acid (JA). RESULTS: In this study, we found COR, as a new plant growth regulator, could effectively reduce plant height and ear height of both hybrids (ZD958 and XY335) and inbred (B73) maize by inhibiting internode growth during elongation, thus improve maize lodging resistance. To study gene expression changes in internode after COR treatment, we collected spatio-temporal transcriptome of inbred B73 internode under normal condition and COR treatment, including the three different regions of internode (fixed, meristem and elongation regions) at three different developmental stages. The gene expression levels of the three regions at normal condition were described and then compared with that upon COR treatment. In total, 8605 COR-responsive genes (COR-RGs) were found, consist of 802 genes specifically expressed in internode. For these COR-RGs, 614, 870, 2123 of which showed expression changes in only fixed, meristem and elongation region, respectively. Both the number and function were significantly changed for COR-RGs identified in different regions, indicating genes with different functions were regulated at the three regions. Besides, we found more than 80% genes of gibberellin and jasmonic acid were changed under COR treatment. CONCLUSIONS: These data provide a gene expression profiling in different regions of internode development and molecular mechanism of COR affecting internode elongation. A putative schematic of the internode response to COR treatment is proposed which shows the basic process of COR affecting internode elongation. This research provides a useful resource for studying maize internode development and improves our understanding of the COR regulation mechanism based on plant height.

Genome-Wide Characterization of Jasmonates Signaling Components Reveals the Essential Role of ZmCOI1a-ZmJAZ15 Action Module in Regulating Maize Immunity to Gibberella Stalk Rot.

Gibberella stalk rot (GSR) by Fusarium graminearum causes significant losses of maize production worldwide. Jasmonates (JAs) have been broadly known in regulating defense against pathogens through the homeostasis of active JAs and COI-JAZ-MYC function module. However, the functions of different molecular species of JAs and COI-JAZ-MYC module in maize interactions with Fusarium graminearum and regulation of diverse metabolites remain unknown. In this study, we found that exogenous application of MeJA strongly enhanced resistance to GSR. RNA-seq analysis showed that MeJA activated multiple genes in JA pathways, which prompted us to perform a genome-wide screening of key JA signaling components in maize. Yeast Two-Hybrid, Split-Luciferase, and Pull-down assays revealed that the JA functional and structural mimic coronatine (COR) functions as an essential ligand to trigger the interaction between ZmCOIa and ZmJAZ15. By deploying CRISPR-cas9 knockout and Mutator insertional mutants, we demonstrated that coi1a mutant is more resistant, whereas jaz15 mutant is more susceptible to GSR. Moreover, JA-deficient opr7-5opr8-2 mutant displayed enhanced resistance to GSR compared to wild type. Together, these results provide strong evidence that ZmJAZ15 plays a pivotal role, whereas ZmCOIa and endogenous JA itself might function as susceptibility factors, in maize immunity to GSR.

New Insight into Justicidin B Pathway and Production in Linum austriacum.

Lignans are the main secondary metabolites synthetized by Linum species as plant defense compounds but they are also valuable for human health, in particular, for novel therapeutics. In this work, Linum austriacum in vitro cultures, cells (Cc), adventitious roots (ARc) and hairy roots (HRc) were developed for the production of justicidin B through elicitation with methyl jasmonate (MeJA) and coronatine (COR). The performances of the cultures were evaluated for their stability, total phenols content and antioxidant ability. NMR was used to identify justicidin B and isojusticidin B and HPLC to quantify the production, highlighting ARc and HRc as the highest productive tissues. MeJA and COR treatments induced the synthesis of justicidin B more than three times and the synthesis of other compounds. RNA-sequencing and a de novo assembly of L. austriacum ARc transcriptome was generated to identify the genes activated by MeJA. Furthermore, for the first time, the intracellular localization of justicidin B in ARc was investigated through microscopic analysis. Then, HRc was chosen for small-scale production in a bioreactor. Altogether, our results improve knowledge on justicidin B pathway and cellular localization in L. austriacum for future scale-up processes.

Enhanced Aryltetralin Lignans Production in Linum Adventi-Tious Root Cultures.

Lignans are the main secondary metabolites synthetized by Linum species as plant defense molecules. They are also valuable for human health, in particular, for their potent antiviral and antineoplastic properties. In this study, the adventitious root cultures of three Linum species (L. flavum, L. mucronatum and L. dolomiticum) were developed to produce aryltetralin lignans. The effect of two elicitors, methyl jasmonate and coronatine, on aryltetralin lignans production was also evaluated. The adventitious root cultures from L. dolomiticum were obtained and analyzed for the first time and resulted as the best producer for all the aryltetralins highlighted in this system: Podophyllotoxin, 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7-O-ß-glucoside, the last showing a productivity of 92.6 mg/g DW. The two elicitors differently affected the production of the 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7-O-ß-glucoside.

Systematic identification of genes associated with plant growth-defense tradeoffs under JA signaling in Arabidopsis.

MAIN CONCLUSION: Co-expression and regulatory networks yield important insights into the growth-defense tradeoffs mechanism under jasmonic acid (JA) signals in Arabidopsis. Elevated defense is commonly associated with growth inhibition. However, a comprehensive atlas of the genes associated with the plant growth-defense tradeoffs under JA signaling is lacking. To gain an insight into the dynamic architecture of growth-defense tradeoffs, a coexpression network analysis was employed on publicly available high-resolution transcriptomes of Arabidopsis treated with coronatine (COR), a mimic of jasmonoyl-l-isoleucine. The genes involved in JA-mediated growth-defense tradeoffs were systematically revealed. Promoter enrichment analysis revealed the core regulatory module in which the genes underwent rapid activation, sustained upregulation after COR treatment, and mediated the growth-defense tradeoffs. Several transcription factors (TFs), including RAP2.6L, MYB44, WRKY40, and WRKY18, were identified as instantly activated components associated with pathogen and insect resistance. JA might rapidly activate RAV1 and KAN1 to repress brassinosteroid (BR) response genes, upregulate KAN1, the C2H2 TF families ZF2, ZF3, ZAT6, and STZ/ZAT10 to repress the biosynthesis, transport, and signaling of auxin to arrest growth. Independent datasets and preserved analyses validated the reproducibility of the results. Our study provided a comprehensive snapshot of genes that respond to JA signals and provided valuable resources for functional studies on the genetic modification of breeding population that exhibit robust growth and defense simultaneously.

A scalable synthesis of (+)-coronafacic acid.

A facile, efficient, and scalable synthesis of optically pure coronafacic acid by resolution of racemic coronafacic acid obtained using an improved version of Watson's method has been developed. By optimizing the boron-mediated aldol reaction of Watson, we were able to prepare 2.1 g of racemic coronafacic acid. This was coupled with (S)-4-isopropyl-2-oxazolidinone to give a mixture of diastereomeric coronafacyl oxazolidinones, which were readily separable by silica-gel column chromatography to give 630 mg of optically pure (+)-coronafacic acid.

Pathogen exploitation of an abscisic acid- and jasmonate-inducible MAPK phosphatase and its interception by Arabidopsis immunity.

Phytopathogens promote virulence by, for example, exploiting signaling pathways mediated by phytohormones such as abscisic acid (ABA) and jasmonate (JA). Some plants can counteract pathogen virulence by invoking a potent form of immunity called effector-triggered immunity (ETI). Here, we report that ABA and JA mediate inactivation of the immune-associated MAP kinases (MAPKs), MPK3 and MPK6, in Arabidopsis thaliana ABA induced expression of genes encoding the protein phosphatases 2C (PP2Cs), HAI1, HAI2, and HAI3 through ABF/AREB transcription factors. These three HAI PP2Cs interacted with MPK3 and MPK6 and were required for ABA-mediated MPK3/MPK6 inactivation and immune suppression. The bacterial pathogen Pseudomonas syringae pv. tomato (Pto) DC3000 activates ABA signaling and produces a JA-mimicking phytotoxin, coronatine (COR), that promotes virulence. We found that Pto DC3000 induces HAI1 through COR-mediated activation of MYC2, a master transcription factor in JA signaling. HAI1 dephosphorylated MPK3 and MPK6 in vitro and was necessary for COR-mediated suppression of MPK3/MPK6 activation and immunity. Intriguingly, upon ETI activation, A. thaliana plants overcame the HAI1-dependent virulence of COR by blocking JA signaling. Finally, we showed conservation of induction of HAI PP2Cs by ABA and JA in other Brassicaceae species. Taken together, these results suggest that ABA and JA signaling pathways, which are hijacked by the bacterial pathogen, converge on the HAI PP2Cs that suppress activation of the immune-associated MAPKs. Also, our data unveil interception of JA-signaling activation as a host counterstrategy against the bacterial suppression of MAPKs during ETI.

Design of a bacterial speck resistant tomato by CRISPR/Cas9-mediated editing of SlJAZ2.

Due to their different lifestyles, effective defence against biotrophic pathogens normally leads to increased susceptibility to necrotrophs, and vice versa. Solving this trade-off is a major challenge for obtaining broad-spectrum resistance in crops and requires uncoupling the antagonism between the jasmonate (JA) and salicylate (SA) defence pathways. Pseudomonas syringae pv. tomato (Pto) DC3000, the causal agent of tomato bacterial speck disease, produces coronatine (COR) that stimulates stomata opening and facilitates bacterial leaf colonization. In Arabidopsis, stomata response to COR requires the COR co-receptor AtJAZ2, and dominant AtJAZ2Δjas repressors resistant to proteasomal degradation prevent stomatal opening by COR. Here, we report the generation of a tomato variety resistant to the bacterial speck disease caused by PtoDC3000 without compromising resistance to necrotrophs. We identified the functional ortholog of AtJAZ2 in tomato, found that preferentially accumulates in stomata and proved that SlJAZ2 is a major co-receptor of COR in stomatal guard cells. SlJAZ2 was edited using CRISPR/Cas9 to generate dominant JAZ2 repressors lacking the C-terminal Jas domain (SlJAZ2Δjas). SlJAZ2Δjas prevented stomatal reopening by COR and provided resistance to PtoDC3000. Water transpiration rate and resistance to the necrotrophic fungal pathogen Botrytis cinerea, causal agent of the tomato gray mold, remained unaltered in Sljaz2Δjas plants. Our results solve the defence trade-off in a crop, by spatially uncoupling the SA-JA hormonal antagonism at the stomata, entry gates of specific microbes such as PtoDC3000. Moreover, our results also constitute a novel CRISPR/Cas-based strategy for crop protection that could be readily implemented in the field.