CORONATINE INSENSITIVE1 Research Articles

Transcriptional Mechanism of Jasmonate Receptor COI1-Mediated Delay of Flowering Time in Arabidopsis.

Flowering time of plants must be tightly regulated to maximize reproductive success. Plants have evolved sophisticated signaling network to coordinate the timing of flowering in response to their ever-changing environmental conditions. Besides being a key immune signal, the lipid-derived plant hormone jasmonate (JA) also regulates a wide range of developmental processes including flowering time. Here, we report that the CORONATINE INSENSITIVE1 (COI1)-dependent signaling pathway delays the flowering time of Arabidopsis thaliana by inhibiting the expression of the florigen gene FLOWERING LOCUS T (FT). We provide genetic and biochemical evidence that the APETALA2 transcription factors (TFs) TARGET OF EAT1 (TOE1) and TOE2 interact with a subset of JAZ (jasmonate-ZIM domain) proteins and repress the transcription of FT. Our results support a scenario that, when plants encounter stress conditions, bioactive JA promotes COI1-dependent degradation of JAZs. Degradation of the JAZ repressors liberates the transcriptional function of the TOEs to repress the expression of FT and thereby triggers the signaling cascades to delay flowering. Our study identified interacting pairs of TF and JAZ transcriptional regulators that underlie JA-mediated regulation of flowering, suggesting that JA signals are converted into specific context-dependent responses by matching pairs of TF and JAZ proteins.

The RING E3 Ligase KEEP ON GOING Modulates JASMONATE ZIM-DOMAIN12 Stability.

Jasmonate (JA) signaling in plants is mediated by the JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the activity of several transcription factors regulating JA-inducible gene expression. The hormone JA-isoleucine triggers the interaction of JAZ repressor proteins with the F-box protein CORONATINE INSENSITIVE1 (COI1), part of an S-phase kinase-associated protein1/Cullin1/F-box protein COI1 (SCF(COI1)) E3 ubiquitin ligase complex, and their degradation by the 26S proteasome. In Arabidopsis (Arabidopsis thaliana), the JAZ family consists of 13 members. The level of redundancy or specificity among these members is currently not well understood. Here, we characterized JAZ12, encoded by a highly expressed JAZ gene. JAZ12 interacted with the transcription factors MYC2, MYC3, and MYC4 in vivo and repressed MYC2 activity. Using tandem affinity purification, we found JAZ12 to interact with SCF(COI1) components, matching with observed in vivo ubiquitination and with rapid degradation after treatment with JA. In contrast to the other JAZ proteins, JAZ12 also interacted directly with the E3 RING ligase KEEP ON GOING (KEG), a known repressor of the ABSCISIC ACID INSENSITIVE5 transcription factor in abscisic acid signaling. To study the functional role of this interaction, we circumvented the lethality of keg loss-of-function mutants by silencing KEG using an artificial microRNA approach. Abscisic acid treatment promoted JAZ12 degradation, and KEG knockdown led to a decrease in JAZ12 protein levels. Correspondingly, KEG overexpression was capable of partially inhibiting COI1-mediated JAZ12 degradation. Our results provide additional evidence for KEG as an important factor in plant hormone signaling and a positive regulator of JAZ12 stability.

The role of plasma membrane H(+) -ATPase in jasmonate-induced ion fluxes and stomatal closure in Arabidopsis thaliana.

Methyl jasmonate (MeJA) elicits stomatal closure in many plant species. Stomatal closure is accompanied by large ion fluxes across the plasma membrane (PM). Here, we recorded the transmembrane ion fluxes of H(+) , Ca(2+) and K(+) in guard cells of wild-type (Col-0) Arabidopsis, the CORONATINE INSENSITIVE1 (COI1) mutant coi1-1 and the PM H(+) -ATPase mutants aha1-6 and aha1-7, using a non-invasive micro-test technique. We showed that MeJA induced transmembrane H(+) efflux, Ca(2+) influx and K(+) efflux across the PM of Col-0 guard cells. However, this ion transport was abolished in coi1-1 guard cells, suggesting that MeJA-induced transmembrane ion flux requires COI1. Furthermore, the H(+) efflux and Ca(2+) influx in Col-0 guard cells was impaired by vanadate pre-treatment or PM H(+) -ATPase mutation, suggesting that the rapid H(+) efflux mediated by PM H(+) -ATPases could function upstream of the Ca(2+) flux. After the rapid H(+) efflux, the Col-0 guard cells had a longer oscillation period than before MeJA treatment, indicating that the activity of the PM H(+) -ATPase was reduced. Finally, the elevation of cytosolic Ca(2+) concentration and the depolarized PM drive the efflux of K(+) from the cell, resulting in loss of turgor and closure of the stomata.

An Arabidopsis Plasma Membrane Proton ATPase Modulates JA Signaling and Is Exploited by the Pseudomonas syringae Effector Protein AvrB for Stomatal Invasion.

Stomata are natural openings through which many pathogenic bacteria enter plants. Successful bacterial pathogens have evolved various virulence factors to promote stomatal opening. Here, we show that the Pseudomonas syringae type III effector protein AvrB induces stomatal opening and enhances bacterial virulence in a manner dependent on RPM1-INTERACTING4 (RIN4), which promotes stomatal opening by positively regulating the Arabidopsis plasma membrane H(+)-ATPase (AHA1), which is presumed to directly regulate guard cell turgor pressure. In support of a role of AHA1 in AvrB-induced stomatal opening, AvrB enhances ATPase activity in plants. Unexpectedly, AHA1 promotes the interaction between the jasmonate (JA) receptor CORONATINE INSENSITIVE1 (COI1) and JASMONATE ZIM-DOMAIN (JAZ) proteins and enhances JA signaling. JA signaling is required for optimum stomatal infection in AHA1-active plants. Similarly, AvrB also induces the COI1-JAZ9 interaction and the degradation of multiple JAZ proteins. AvrB-induced stomatal opening and virulence require the canonical JA signaling pathway, which involves the COI1 and NAC transcription factors. The findings thus point to a previously unknown pathway exploited by P. syringae that acts upstream of COI1 to regulate JA signaling and stomatal opening.

Tobacco nicotine uptake permease regulates the expression of a key transcription factor gene in the nicotine biosynthesis pathway.

The down-regulation of a tobacco (Nicotiana tabacum) plasma membrane-localized nicotine uptake permease, NUP1, was previously reported to reduce total alkaloid levels in tobacco plants. However, it was unclear how this nicotine transporter affected the biosynthesis of the alkaloid nicotine. When NUP1 expression was suppressed in cultured tobacco cells treated with jasmonate, which induces nicotine biosynthesis, the NICOTINE2-locus transcription factor gene ETHYLENE RESPONSE FACTOR189 (ERF189) and its target structural genes, which function in nicotine biosynthesis and transport, were strongly suppressed, resulting in decreased total alkaloid levels. Conversely, NUP1 overexpression had the opposite effect. In these experiments, the expression levels of the MYC2 transcription factor gene and its jasmonate-inducible target gene were not altered. Inhibiting tobacco alkaloid biosynthesis by suppressing the expression of genes encoding enzymes in the nicotine pathway did not affect the expression of ERF189 and other nicotine pathway genes, indicating that ERF189 is not regulated by cellular alkaloid levels. Suppressing the expression of jasmonate signaling components in cultured tobacco cells showed that NUP1 acts downstream of the CORONATINE INSENSITIVE1 receptor and MYC2, but upstream of ERF189. These results suggest that although jasmonate-activated expression of MYC2 induces the expression of both NUP1 and ERF189, expression of ERF189 may actually be mediated by NUP1. Furthermore, NUP1 overexpression in tobacco plants inhibited the long-range transport of nicotine from the roots to the aerial parts. Thus, NUP1 not only mediates the uptake of tobacco alkaloids into root cells, but also positively controls the expression of ERF189, a key gene in the biosynthesis of these alkaloids.

Jasmonoyl-L-isoleucine coordinates metabolic networks required for anthesis and floral attractant emission in wild tobacco (Nicotiana attenuata).

Jasmonic acid and its derivatives (jasmonates [JAs]) play central roles in floral development and maturation. The binding of jasmonoyl-L-isoleucine (JA-Ile) to the F-box of CORONATINE INSENSITIVE1 (COI1) is required for many JA-dependent physiological responses, but its role in anthesis and pollinator attraction traits remains largely unexplored. Here, we used the wild tobacco Nicotiana attenuata, which develops sympetalous flowers with complex pollination biology, to examine the coordinating function of JA homeostasis in the distinct metabolic processes that underlie flower maturation, opening, and advertisement to pollinators. From combined transcriptomic, targeted metabolic, and allometric analyses of transgenic N. attenuata plants for which signaling deficiencies were complemented with methyl jasmonate, JA-Ile, and its functional homolog, coronatine (COR), we demonstrate that (1) JA-Ile/COR-based signaling regulates corolla limb opening and a JA-negative feedback loop; (2) production of floral volatiles (night emissions of benzylacetone) and nectar requires JA-Ile/COR perception through COI1; and (3) limb expansion involves JA-Ile-induced changes in limb fresh mass and carbohydrate metabolism. These findings demonstrate a master regulatory function of the JA-Ile/COI1 duet for the main function of a sympetalous corolla, that of advertising for and rewarding pollinator services. Flower opening, by contrast, requires JA-Ile signaling-dependent changes in primary metabolism, which are not compromised in the COI1-silenced RNA interference line used in this study.

F-box蛋白COI1稳定性调控机制

<italic>Arabidopsis</italic> COI1 (coronatine insensitive 1), an F-box protein that physically interacts with ASK1 (<italic>Arabidopsis</italic> serine/Threonine kinase 1) protein to form SCF^COI1-E3 ligase complex, perceives jasmonate signals and modulates diverse aspects of the jasmonate-regulated plant defense and developmental processes. The COI1 homologs from various plant species have recently been identified, however, little is known about the molecular mechanism regulating protein stability of these COI1 homologs. In this study, we demonstrated that the COI1 homologs from vegetable crop <italic>Solanum lycopersicum</italic> and commercial plant <italic>Nicotiana attenuata</italic> remain stable via interacting with ASK1 protein, suggesting that assembly of SCF^COI1 complexes contributes to the stabilization of COI1 homologs. Meanwhile, the 26S proteasome inhibitor represses the degradation of COI1 homologs, demonstrating that ubiquitin-proteasome pathway plays a role in degradation of the COI1 homologs. These results suggest that two antagonistic pathways work together to maintain COI1 protein in tomato and tobacco at a suitable abundance for proper biological function.

Jasmonic acid and its precursor 12-oxophytodienoic acid control different aspects of constitutive and induced herbivore defenses in tomato.

The jasmonate family of growth regulators includes the isoleucine (Ile) conjugate of jasmonic acid (JA-Ile) and its biosynthetic precursor 12-oxophytodienoic acid (OPDA) as signaling molecules. To assess the relative contribution of JA/JA-Ile and OPDA to insect resistance in tomato (Solanum lycopersicum), we silenced the expression of OPDA reductase3 (OPR3) by RNA interference (RNAi). Consistent with a block in the biosynthetic pathway downstream of OPDA, OPR3-RNAi plants contained wild-type levels of OPDA but failed to accumulate JA or JA-Ile after wounding. JA/JA-Ile deficiency in OPR3-RNAi plants resulted in reduced trichome formation and impaired monoterpene and sesquiterpene production. The loss of these JA/JA-Ile -dependent defense traits rendered them more attractive to the specialist herbivore Manduca sexta with respect to feeding and oviposition. Oviposition preference resulted from reduced levels of repellant monoterpenes and sesquiterpenes. Feeding preference, on the other hand, was caused by increased production of cis-3-hexenal acting as a feeding stimulant for M. sexta larvae in OPR3-RNAi plants. Despite impaired constitutive defenses and increased palatability of OPR3-RNAi leaves, larval development was indistinguishable on OPR3-RNAi and wild-type plants, and was much delayed compared with development on the jasmonic acid-insensitive1 (jai1) mutant. Apparently, signaling through JAI1, the tomato ortholog of the ubiquitin ligase CORONATINE INSENSITIVE1 in Arabidopsis (Arabidopsis thaliana), is required for defense, whereas the conversion of OPDA to JA/JA-Ile is not. Comparing the signaling activities of OPDA and JA/JA-Ile, we found that OPDA can substitute for JA/JA-Ile in the local induction of defense gene expression, but the production of JA/JA-Ile is required for a systemic response.

Alternative functions of Arabidopsis Yellow Stripe-Like3: from metal translocation to pathogen defense.

YELLOW STRIPE-LIKE1 (YSL1) and YSL3 are involved in iron (Fe) and copper (Cu) translocation. Previously, we reported that upregulation of YSL1 and YSL3 under excess Cu caused high accumulation of Cu in the siz1 mutant, impaired in small ubiquitin-like modifier (SUMO) E3 ligase. Interestingly, the siz1 mutant contains high levels of salicylic acid (SA), involved in plant defense against biotrophic pathogens. In this study, we found that YSL1 and YSL3 were upregulated by SA. SA-regulated YSL3 but not YSL1 depended on NONEXPRESSOR OF PR1 (NPR1). Susceptibility to the pathogen Pseudomonas syringe pv. tomato (Pst) DC3000 was greater for ysl3 than the wild type. Also, during Pst DC3000 infection, YSL3 was positively regulated by SA signaling through NPR1 and the upregulation was enhanced in the coi1 mutant that defective in the jasmonic acid (JA) receptor, CORONATINE INSENSITIVE1. This line of evidence indicates that the regulation of YSL3 is downstream of SA signaling and interplays with JA signaling for involvement in pathogen-induced defense. We provide new insights into the biological function of the metal transporter YSL3 in plant pathogen defense.

THF1 mutations lead to increased basal and wound‐induced levels of oxylipins that stimulate anthocyanin biosynthesis via COI1 signaling in Arabidopsis

Mutants defective in chloroplast development or photosynthesis are liable to accumulate higher levels of anthocyanin in photo-oxidative stress. However, regulatory mechanisms of anthocyanin biosynthesis in the mutants remain unclear. Here, we investigated the mechanism by which the deletion of thylakoid formation1 (THF1) leads to an increased level of anthocyanin in Arabidopsis thaliana L. Physiological and genetic evidence showed that the increased level of anthocyanin in thf1 is dependent on coronatine-insensitive1 (COI1) signaling. Our data showed that thf1 had higher levels of basal α-linolenic acid (α-LeA), and methyl jasmonate (JA)-induced α-LeA and 12-oxophytodienoic acid (OPDA) than the wild type (WT). Consistently, expression levels of phospholipase genes including pPLAIIα and PLA-Iγ1 were elevated in thf1. Furthermore, inhibition of lipase activity by bromoenol lactone, a specific inhibitor of plant pPLA, led to producing identical levels of anthocyanins in WT and thf1 plants. Interestingly, OPDA biosynthesis was triggered by light illumination in isolated chloroplasts, indicating that new protein import into chloroplasts is not required for OPDA biosynthesis. Thus, we conclude that the elevated anthocyanin accumulation in thf1 is attributed to an increase in JA levels. This JA-mediated signaling to coordinate plant metabolism and growth in stress may be conserved in other photosensitive mutants.

The F-box protein COI1 functions upstream of MYB305 to regulate primary carbohydrate metabolism in tobacco (Nicotiana tabacum L. cv. TN90).

Jasmonate (JA) plays an important role in regulating plant male fertility and secondary metabolism, but its role in regulating primary metabolism remains unclear. The F-box protein CORONATINE INSENSITIVE 1 (COI1) is a critical component of the JA receptor, and mediates JA-signalling by targeting JASMONATE ZIM-domain (JAZ) proteins for proteasomal degradation in response to JA perception. Here, we found that RNA interference-mediated knockdown of NtCOI1 in tobacco (Nicotiana tabacum L. cv. TN90) recapitulated many previously observed phenotypes in coi1 mutants, including male sterility, JA insensitivity, and loss of floral anthocyanin production. It also affected starch metabolism in the pollen, anther wall, and floral nectary, leading to pollen abortion and loss of floral nectar. Transcript levels of genes encoding starch metabolism enzymes were significantly altered in the pollen, anther wall, and floral nectary of NtCOI1-silenced tobacco. Changes in leaf primary metabolism were also observed in the NtCOI1-silenced tobacco. The expression of NtMYB305, an orthologue of MYB305 previously identified as a flavonoid metabolic regulator in Antirrhinum majus flowers and as a floral-nectar regulator mediating starch synthesis in ornamental tobacco, was extremely downregulated in NtCOI1-silenced tobacco. These findings suggest that NtCOI1 functions upstream of NtMYB305 and plays a fundamental role in coordinating plant primary carbohydrate metabolism and correlative physiological processes.

The Activated SA and JA Signaling Pathways Have an Influence on flg22-Triggered Oxidative Burst and Callose Deposition

The first line of defense in plants against pathogens is induced by the recognition of microbe-associated molecular patterns (MAMP). Perception of bacterial flagellin (flg22) by the pattern recognition receptor flagellin-sensing 2 (FLS2) is the best characterized MAMP response, although the underlying molecular mechanisms are not fully understood. Here we studied the relationship between salicylic acid (SA) or jasmonic acid (JA) signaling and FLS2-mediated signaling by monitoring flg22-triggered responses in known SA or JA related mutants of Arabidopsis thaliana (L.) Heynh. The sid2 mutant, impaired in SA biosynthesis, had less basal FLS2 mRNA accumulation than the wild type, which correlated with suppression of early flg22 responses such as ROS production and induction of marker genes, WRKY29 and FRK1. The JA-signaling mutants, jar1 and coi1, exhibited an enhanced flg22-triggered oxidative burst and more callose accumulation than the wild type, and pretreatment with SA or coronatine (COR), a structural mimic of JA-isoleucine, altered these flg22-induced responses. Nonexpressor of pathogenesis-related genes 1 (NPR1) acted downstream of SID2 and required SA-dependent priming for the enhanced flg22-triggered oxidative burst and callose deposition. Activation of JA signaling by COR pretreatment suppressed the flg22-triggered oxidative burst and callose accumulation in a coronatine insensitive 1 (COI1) dependent manner. COR had a negative effect on flg22 responses but only the flg22-triggered oxidative burst depended on SA-JA/COR signaling antagonism. Thus the activated SA and JA signaling pathways have an influence on flg22-triggered oxidative burst and callose deposition. These results may explain how SA and JA signaling are cross talked for regulation of flg22-triggered responses.

Wound-induced expression of DEFECTIVE IN ANTHER DEHISCENCE1 and DAD1-like lipase genes is mediated by both CORONATINE INSENSITIVE1-dependent and independent pathways in Arabidopsis thaliana

Endogenous JA production is not necessary for wound-induced expression of JA-biosynthetic lipase genes such as DAD1 in Arabidopsis. However, the JA-Ile receptor COI1 is often required for their JA-independent induction. Wounding is a serious event in plants that may result from insect feeding and increase the risk of pathogen infection. Wounded plants produce high amounts of jasmonic acid (JA), which triggers the expression of insect and pathogen resistance genes. We focused on the transcriptional regulation of DEFECTIVE IN ANTHER DEHISCENCE1 and six of its homologs including DONGLE (DGL) in Arabidopsis, which encode lipases involved in JA biosynthesis. Plants constitutively expressing DAD1 accumulated a higher amount of JA than control plants after wounding, indicating that the expression of these lipase genes contributes to determining JA levels. We found that the expression of DAD1, DGL, and other DAD1-LIKE LIPASE (DALL) genes is induced upon wounding. Some DALLs were also expressed in unwounded leaves. Further experiments using JA-biosynthetic and JA-response mutants revealed that the wound induction of these genes is regulated by several distinct pathways. DAD1 and most of its homologs other than DALL4 were fully induced without relying on endogenous JA-Ile production and were only partly affected by JA deficiency, indicating that positive feedback by JA is not necessary for induction of these genes. However, DAD1 and DGL required CORONATINE INSENSITIVE1 (COI1) for their expression, suggesting that a molecule other than JA might act as a regulator of COI1. Wound induction of DALL1, DALL2, and DALL3 did not require COI1. This differential regulation of DAD1 and its homologs might explain their functions at different time points after wounding.

Amino acid substitutions of GLY98, LEU245 and GLU543 in COI1 distinctively affect jasmonate-regulated male fertility in Arabidopsis

Jasmonate (JA) regulates various plant defense and developmental processes. The F-box protein CORONATINE INSENSITIVE 1 (COI1) perceives JA signals to mediate diverse plant responses including male fertility, root growth, anthocyanin accumulation, and defense against abiotic and biotic stresses. In this study, we carried out genetic, physiological and biochemical analysis on a series of coi1 mutant alleles, and found that different amino acid mutations in COI1 distinctively affect JA-regulated male fertility in Arabidopsis. All the JA responses are disrupted by the COI1 mutations W467 in coi1-1, Q343 (coi1-6), G369E (coi1-4), G98D (coi1-5), G155E (coi1-7), D452A (coi1-9) and L490A (coi1-10), though the coi1-5 mutant (COI1G98D) contains adequate COI1 protein (~ 60% of wild-type). Interestingly, the low basal level of COI1(E543K) in the coi1-8 mutant (~ 10% of wild-type COI1 level) is sufficient for maintaining male fertility ( ~50% of wild-type fertility); the coi1-2 mutant with low level of COI1(L245F) (~ 10% of wild-type) is male sterile under normal growth condition (22°C) but male fertile (~ 80% of wild-type fertility) at low temperature (16°C); however, both coi1-2 and coi1-8 are defective in the other JA responses (root growth, anthocyanin accumulation, and plant response to the pathogen Pst DC3000 infection).

Arabidopsis mutants affecting oxylipin signaling in photo-oxidative stress responses

Plant oxylipins derive from oxygenation of polyunsaturated fatty acids in thylakoid membranes and oxylipins such as jasmonic acid (JA) and 12-oxo-phytodienoic acid (OPDA) play important roles in adaptation to photo-oxidative stress. OPDA functions both as a JA precursor and as a biologically active signaling molecule that induces expression of a specific set of genes. These genes can be induced by OPDA in the JA-insensitive coronatine insensitive1 (coi1) mutant, suggesting that there is an alternative pathway for OPDA signaling, independent of COI1-dependent JA signaling. However, little is known about OPDA signaling in photo-oxidative stress responses. In this study, we isolated Arabidopsis mutants with constitutively enhanced expression from the OPDA-responsive HsfA2 promoter. We used deletion mapping and complementation analysis to identify one responsible gene as CATALASE2. Our results thus indicate that ROS-producing cellular metabolism links to OPDA signaling.

Jasmonic acid negatively regulates resistance to Tobacco mosaic virus in tobacco

Nicotiana tabacum (tobacco) cultivars possessing the N resistance gene to Tobacco mosaic virus (TMV) induce a hypersensitive response, which is accompanied by the production of phytohormones such as salicylic acid (SA) and jasmonic acid (JA), to enclose the invaded virus at the initial site of infection, which inhibits viral multiplication and spread. SA functions as a positive regulator of TMV resistance. However, the role of JA in TMV resistance has not been fully elucidated. Exogenously applied methyl jasmonate, a methyl ester of JA, reduced local resistance to TMV and permitted systemic viral movement. Furthermore, in contrast to a previous finding, we demonstrated that silencing of CORONATINE-INSENSITIVE 1 (COI1), a JA receptor, reduced viral accumulation in a tobacco cultivar possessing the N gene, as did that of allene oxide synthase, a JA biosynthetic enzyme. The reduction in viral accumulation in COI1-silenced tobacco plants was correlated with an increase in SA, and lowering SA levels by introducing an SA hydroxylase gene attenuated this reduction. Viral susceptibility did not change in a COI1-silenced tobacco cultivar lacking the N gene. These results suggest that JA signaling is not directly responsible for susceptibility to TMV, but is indirectly responsible for viral resistance through the partial inhibition of SA-mediated resistance conferred by the N gene, and that a balance between endogenous JA and SA levels is important for determining the degree of resistance.

Jasmonate ZIM-Domain (JAZ) Protein Regulates Host and Nonhost Pathogen-Induced Cell Death in Tomato and Nicotiana benthamiana

The nonhost-specific phytotoxin coronatine (COR) produced by several pathovars of Pseudomonas syringae functions as a jasmonic acid-isoleucine (JA-Ile) mimic and contributes to disease development by suppressing plant defense responses and inducing reactive oxygen species in chloroplast. It has been shown that the F-box protein CORONATINE INSENSITIVE 1 (COI1) is the receptor for COR and JA-Ile. JASMONATE ZIM DOMAIN (JAZ) proteins act as negative regulators for JA signaling in Arabidopsis. However, the physiological significance of JAZ proteins in P. syringae disease development and nonhost pathogen-induced hypersensitive response (HR) cell death is not completely understood. In this study, we identified JAZ genes from tomato, a host plant for P. syringae pv. tomato DC3000 (Pst DC3000), and examined their expression profiles in response to COR and pathogens. Most JAZ genes were induced by COR treatment or inoculation with COR-producing Pst DC3000, but not by the COR-defective mutant DB29. Tomato SlJAZ2, SlJAZ6 and SlJAZ7 interacted with SlCOI1 in a COR-dependent manner. Using virus-induced gene silencing (VIGS), we demonstrated that SlJAZ2, SlJAZ6 and SlJAZ7 have no effect on COR-induced chlorosis in tomato and Nicotiana benthamiana. However, SlJAZ2-, SlJAZ6- and SlJAZ7-silenced tomato plants showed enhanced disease-associated cell death to Pst DC3000. Furthermore, we found delayed HR cell death in response to the nonhost pathogen Pst T1 or a pathogen-associated molecular pattern (PAMP), INF1, in SlJAZ2- and SlJAZ6-silenced N. benthamiana. These results suggest that tomato JAZ proteins regulate the progression of cell death during host and nonhost interactions.

Basic Helix-Loop-Helix Transcription Factors JASMONATE-ASSOCIATED MYC2-LIKE1 (JAM1), JAM2, and JAM3 Are Negative Regulators of Jasmonate Responses in Arabidopsis

Jasmonates regulate transcriptional reprogramming during growth, development, and defense responses. Jasmonoyl-isoleucine, an amino acid conjugate of jasmonic acid (JA), is perceived by the protein complex composed of the F-box protein CORONATINE INSENSITIVE1 (COI1) and JASMONATE ZIM DOMAIN (JAZ) proteins, leading to the ubiquitin-dependent degradation of JAZ proteins. This activates basic helix-loop-helix-type MYC transcription factors to regulate JA-responsive genes. Here, we show that the expression of genes encoding other basic helix-loop-helix transcription factors, JASMONATE ASSOCIATED MYC2-LIKE1 (JAM1), JAM2, and JAM3, is positively regulated in a COI1- and MYC2-dependent manner in Arabidopsis (Arabidopsis thaliana). However, contrary to myc2, the jam1jam2jam3 triple mutant exhibited shorter roots when treated with methyl jasmonate (MJ), indicating enhanced responsiveness to JA. Our genome-wide expression analyses revealed that key jasmonate metabolic genes as well as a set of genes encoding transcription factors that regulate the JA-responsive metabolic genes are negatively regulated by JAMs after MJ treatment. Consistently, loss of JAM genes resulted in higher accumulation of anthocyanin in MJ-treated plants as well as higher accumulation of JA and 12-hydroxyjasmonic acid in wounded plants. These results show that JAMs negatively regulate the JA responses in a manner that is mostly antagonistic to MYC2.

A mutation in the expansin‐like A2 gene enhances resistance to necrotrophic fungi and hypersensitivity to abiotic stress in Arabidopsis thaliana

Expansins are cell wall loosening agents, known for their endogenous function in cell wall extensibility. The Arabidopsis expansin-like A2 (EXLA2) gene was identified by its down-regulation in response to infection by the necrotrophic pathogen Botrytis cinerea, and by the reduced susceptibility of an exla2 mutant to the same pathogen. The exla2 mutant was equally susceptible to Pseudomonas syringae pv. tomato, but was more resistant to the necrotrophic fungus Alternaria brassicicola, when compared with the wild-type or with transgenic, ectopic EXLA2-overexpressing lines. The exla2 mutants also enhanced tolerance to the phytoprostane-A1 . This suggests that the absence or down-regulation of EXLA2 leads to increased resistance to B. cinerea in a CORONATINE INSENSITIVE 1 (COI1)-dependent manner, and this down-regulation can be achieved by phytoprostane-A1 treatment. EXLA2 is induced significantly by salinity and cold, and by the exogenous application of abscisic acid. The exla2 mutant also showed hypersensitivity towards increased salt and cold, and this hypersensitivity required a functional abscisic acid pathway. The differential temporal expression of EXLA2 and the phenotypes in transgenic plants with altered expression of EXLA2 indicate that plant cell wall structure is an important player during Arabidopsis developmental stages. Our results indicate that EXLA2 appears to be important in response to various biotic and abiotic stresses, particularly in the pathogenesis of necrotrophic pathogens and in the tolerance to abiotic stress.

Nicotiana attenuata MPK4 suppresses a novel jasmonic acid (JA) signaling‐independent defense pathway against the specialist insect Manduca sexta, but is not required for the resistance to the generalist Spodoptera littoralis

How plants tailor their defense responses to attack from different insects remains largely unknown. Here, we studied the role of a mitogen-activated protein kinase (MAPK), MPK4, in the resistance of a wild tobacco Nicotiana attenuata to two herbivores, the specialist Manduca sexta and the generalist Spodoptera littoralis. Stably transformed N. attenuata plants silenced in MPK4 (irMPK4) were generated and characterized for traits important for defense against herbivores. Only the oral secretions (OS) from M. sexta, but not the OS from S. littoralis or mechanical wounding, induced elevated levels of jasmonic acid (JA) in irMPK4 plants relative to the wild-type plants. Moreover, silencing of MPK4 strongly increased the resistance of N. attenuata to M. sexta in a fashion that was independent of COI1 (CORONATINE INSENSITIVE1)-mediated JA signaling. Untargeted metabolomic screening identified several new MPK4-dependent putative defensive compounds against M. sexta. By contrast, silencing of MPK4 did not affect the growth of the generalist insect S. littoralis, and we propose that this was because of the very low levels of fatty acid-amino acid conjugates (FACs) in S. littoralis OS. Thus, MPK4 is likely to be a key signaling element that enables plants to tailor defense responses to different attackers.