The intragenic suppressor mutation Leu59Phe compensates for the effect of detrimental mutations in the jasmonate receptor COI1

The phytohormones jasmonates (JAs) control plant development, growth, and defense against insects and pathogens. The Arabidopsis JA receptor Coronatine Insensitive 1 (COI1) interacts with ARABIDOPSIS SKP-LIKE1 (ASK1)/ASK2 to form the SCF^COI1 E3 ligase and mediate JA responses. Here, we performed a genetic suppressor screen using the leaky coi1-2 (COI1^Leu245Phe) mutant for restored sensitivity to JA, and identified the intragenic suppressor mutation Leu59Phe, which was in the region connecting the F-box and leucine-rich repeats domains of COI1. The L59F substitution not only restores the COI1^L245F function, but also the COI1^Gly434Glu (coi1-22^rsp) function in JA responses, through recovering their interactions with ASK1 or ASK2 and their protein levels. The L59F change itself could not enhance the interactions between COI1 and ASK1/2, nor affect JA responses. The present study reveals that the Leu59Phe substitution compensates for the effect of some deleterious mutations in the JA receptor COI1.     

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 (COI1_G98D) 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).     

Oryza sativa COI Homologues Restore Jasmonate Signal Transduction in Arabidopsis coi1-1 Mutants

CORONATINE INSENSITIVE 1 (COI1) encodes an E3 ubiquitin ligase complex component that interacts with JAZ proteins and targets them for degradation in response to JA signaling. The Arabidopsis genome has a single copy of COI1, but the Oryza sativa genome has three closely related COI homologs. To examine the functions of the three OsCOIs, we used yeast two-hybrid assays to examine their interactions with JAZ proteins and found that OsCOIs interacted with OsJAZs and with JAZs, in a coronatine dependent manner. We also tested whether OsCOI1a and OsCOI1b could complement Arabidopsis coi1-1 mutants and found that overexpression of either gene in the coi1-1 mutant resulted in restoration of JA signal transduction and production of seeds, indicating successful complementation. Although OsCOI2 interacted with a few OsJAZs, we were not able to successfully complement the coi1-1 mutant with OsCOI2. Molecular modeling revealed that the three OsCOIs adopt 3D structures similar to COI1. Structural differences resulting from amino acid variations, especially among amino acid residues involved in the interaction with coronatine and JAZ proteins, were tested by mutation analysis. When His-391 in OsCOI2 was substituted with Tyr-391, OsCOI2 interacted with a wider range of JAZ proteins, including OsJAZ1, 2, 5∼9 and 11, and complemented coi1-1 mutants at a higher frequency than the other OsCOIs and COI1. These results indicate that the three OsCOIs are orthologues of COI1 and play key roles in JA signaling.     

Brassinosteroid negatively regulates jasmonate inhibition of root growth in Arabidopsis

Jasmonate (JA) inhibits root growth of Arabidopsis thaliana seedlings. The mutation in COI1, that plays a central role in JA signaling, displays insensitivity to JA inhibition of root growth. To dissect JA signaling pathway, we recently isolated one mutant named psc1, which partially suppresses coi1 insensitivity to JA inhibition of root growth. As we identified the PSC1 gene as an allele of DWF4 that encodes a key enzyme in brassinosteroid (BR) biosynthesis, we hypothesized and demonstrated that BR is involved in JA signaling and negatively regulates JA inhibition of root growth. In our Plant Physiology paper, we analyzed effects of psc1 or exogenous BR on the inhibition of root growth by JA. Here we show that treatment with brassinazole (Brz), a BR biosynthesis inhibitor, increased JA sensitivity in both coi1-2 and wild type, which further confirms that BR negatively regulates JA inhibition of root growth. Since effects of psc1, Brz and exogenous BR on JA inhibition of root growth were mild, we suggests that BR negatively finely regulates JA inhibition of root growth in Arabidopsis.Key words: jasmonate signaling, root growth, brassinosteroid, brassinazole, arabidopsisJasmonate (JA) regulates many plant developmental processes and stress responses.^1,2 COI1 plays a central role in JA signaling and is required for all JA responses in Arabidopsis.^3,4 coi1-1, a strong mutation in COI1, is male sterile and exhibits loss of all JA responses tested to date, such as JA inhibition of root growth, the expression of JA-induced genes, and susceptibility to insect attack and pathogen infection, and coi1-2, a weak mutant of COI1, shows similar JA responses to coi1-1 except for partially fertile that makes it able to produce a small quantity of seeds.⁵To investigate COI1-mediated JA responses and dissect JA signaling pathway, we conducted genetic screens for suppressors of coi1-2. Previously, we identified cos1 that completely suppresses coil-2 insensitive to JA.⁶ Recently, we isolated the psc1 mutant that partially suppresses coi1-2 insensitivity to JA, and found that PSC1 is an allele of DWF4.⁷Since the DWF4 gene encodes a key enzyme in brassinosteroid (BR) biosynthesis,⁸ we hypothesized that BR is involved in JA signaling. By physiological analysis, we showed that psc1 partially restored JA inhibition of root growth in coi1-2 background and displayed JA hypersensitivity in wild-type COI1 background, the effects of psc1 were eliminated by exogenous BR, and that exogenous BR could attenuated JA inhibition of root growth in wild type. These findings demonstrated that BR is involved in JA signaling and indicated that BR negatively regulates JA inhibition of root growth.BR is a family of polyhydroxylated steroid hormones involved in many aspects of plant growth and development. The BR-deficient mutants exhibited severely retarded growth that was able to be rescued by exogenous BR.⁹ Brassinazole (Brz) is a BR biosynthesis inhibitor. The Arabidopsis seedlings treated with Brz displayed a BR deficient-mutant-like phenotype, which could be elimilated by exogenous BR.¹⁰To determine wether treatment with Brz affects JA inhibition of root growth, the seedlings of wild type and coi1-2 were grown in MS medium supplemented with MeJA and/or Brz. As shown in Figure 1, the relative root length was obviously reduced in both coi1-2 and wild type when treated with Brz relative to without Brz, indicating that the repression of BR biosynthesis by Brz could increase JA sensitivity. These results further confirm BR negatively regulates JA inhibition of root growth.Open in a separate windowFigure 1Effect of Brz on JA inhibition of root growth. Brz increased JA inhibition of root growth in both coi1-2 and wild type (WT). Root length of 7-day-old seedlings grown in MS medium containing 0, 5 and 10 μM MeJA without (−) or with (+) 0.5 μM Brz was expressed as a percentage of root length in MS without (−) or with (+) 0.5 µM Brz. Error bars represent SE (n > 30).It has been demonstrated that JA connects with other plant hormones including auxin, ethylene, abscisic acid, salicylic acid and gibberellin to form complex regulatory networks modulating plant developmental and stress responses.^11–15 We found that BR negatively regulates JA inhibition of root growth, suggesting that a cross talk between JA and BR exists in planta, which extends our understandings on the JA signal transduction.COI1 is a JA receptor¹⁶ and DWF4 catalyzes the rate-limiting step in BR-biosynthesis pathway.⁸ We found that JA inhibits DWF4 expression, this inhibition was dependent on COI1,⁷ indicating that DWF4 is downregulated by JA and is located downstream of COI1 in the JA signaling pathway.Since the effects of psc1, Brz, and exogenous BR on JA inhibition of root growth were mild, and the DWF4 expression was partially repressed by JA (Ren et al. 2009, Fig. 1), we suggest that BR negatively finely regulates JA inhibition of root growth, and propose a model for these regulations. As shown in Figure 2A, JA signal passes COI1 repressing substrates, such as JAZs,^17,18 i.e., JA activates degradation of substrates via SCF^COI1-26S proteasome,^16–18 whereas substrates positively regulate root growth through other regulators. JA also partially inhibits DWF4 expression through COI1, reducing BR that is required for root growth.^7,9 Mutation in COI1 interrupts JA signaling for failing in degradation of substrates and repression of DWF4 as well, resulting in JA-insensitivity (Fig. 2B). However, mutation in DWF4 or treatment with Brz causes a reduction in BR, which affects root growth, leading to JA-hypersensitivity in wild-type COI1 background (Fig. 2C and E) and partial restoration of JA sensitivity in coi1-2 background (Fig. 2D and F). Whereas, an application of exogenous BR could eliminate the effect of BR reduction resulted from repression of DWF4 by JA on root growth, attenuating JA sensitivity in wild type (Fig. 2G). Because the inhibition of DWF4 expression by JA is dependent on COI1, the coi1 mutant treated with exogenous BR do not show alteration in JA sensitivity (Fig. 2H).Open in a separate windowFigure 2A model for that BR negatively finely regulates JA inhibition of root growth in Arabidopsis. (A–D) Treatment with JA in wild type (A), coi1-2 (B), psc1 (C) and psc1coi1 (D). (E and F) Treatments with JA and Brz in wild type (E) and coi1-2 (F). (G and H) Treatments with JA and exogenous BR in wild type (G) and coi1-2 (H). Arrows indicate positive regulation or enhancement, whereas blunted lines indicate repression or negative regulation. Crosses indicate interruption or impairment. The letter “S” indicates substrates of SCF^COI1. Thicker arrows and blunted lines represent the central JA signaling pathway regulating JA inhibition of root growth. Broken arrows represent JA signaling pathway in which other regulators are involved. The intensity of gray boxes represents the degree of JA inhibition on root growth.     

The tolerance of the Arabidopsis defense hormone receptor mutant coi1 against the vascular pathogen Verticillium longisporum is not due to increased levels of the active hormone jasmonoyl-isoleucine

Verticillium longisporum is a soil-borne vascular pathogen found primarily on oilseed rape in Northern Europe. Infection of the model plant Arabidopsis thaliana can be achieved under laboratory conditions. In the article related to this addendum, we have shown that Arabidopsis dde2–2 mutants that are compromised in their ability to synthesize the defense hormone jasmonoyl-isoleucine (JA-Ile) are slightly more susceptible than wild-type. Contrary to the expectation that hormone biosynthesis mutants and their respective receptor mutants should have the same phenotype, we found that plants that lack the JA-Ile receptor CORONATINE INSENSITIVE1 (COI1) are more tolerant to the disease. This addendum addressed the question whether the increased JA-Ile levels found in coi1 are responsible for its tolerance phenotype. Based on the evidence that the JA-Ile-deficient dde2–2 coi1-t double mutant is as tolerant as coi1-t, we conclude that increased JA-Ile levels do not protect Arabidopsis against the fungus in the absence of COI1.     

COS1: an Arabidopsis coronatine insensitive1 suppressor essential for regulation of jasmonate-mediated plant defense and senescence

The Arabidopsis thaliana CORONATINE INSENSITIVE1 (COI1) gene encodes an F-box protein to assemble SCF(COI1) complexes essential for response to jasmonates (JAs), which are a family of plant signaling molecules required for many essential functions, including plant defense and reproduction. To better understand the molecular basis of JA action, we screened for suppressors of coi1 and isolated a coi1 suppressor1 (cos1) mutant. The cos1 mutation restores the coi1-related phenotypes, including defects in JA sensitivity, senescence, and plant defense responses. The COS1 gene was cloned through a map-based approach and found to encode lumazine synthase, a key component in the riboflavin pathway that is essential for diverse yet critical cellular processes. We demonstrated a novel function for the riboflavin pathway that acts downstream of COI1 in the JA signaling pathway and is required for suppression of the COI1-mediated root growth, senescence, and plant defense.     

Nonhost resistance to Magnaporthe oryzae in Arabidopsis thaliana

Rice blast, caused by Magnaporthe oryzae, is a devastating disease of rice (Oryza sativa). The mechanisms involved in resistance of rice to blast have been studied extensively and the rice—M. oryzae pathosystem has become a model for plant—microbe interaction studies. However, the mechanisms involved in nonhost resistance (NHR) of other plants to rice blast are still poorly understood. We have recently demonstrated that AGB1 and PMR5 contribute to PEN2-mediated preinvasion resistance to M. oryzae in Arabidopsis thaliana, suggesting a complex genetic network regulating the resistance. To determine whether other defense factors: RAR1, SGT1 and NHO1, affected the A. thaliana-M. oryzae interactions, double mutants were generated between pen2 and these defense-related mutants. All these double mutants exhibited a level of penetration resistance similar to that of the pen2 mutant, suggesting that none of these mutants significantly compromised resistance to M. oryzae in a pen2 background.Key words: nonhost resistance, PEN2, RAR1, SGT1, NHO1Plants face microbial attacks and have evolved innate immunity systems to defend against these threats. The initial step of the immunity signaling pathway is recognition of intra- or extracellular pathogen-derived molecules. Externally oriented transmembrane-type proteins containing leucine-rich repeat (LRR) domains detect extracellular molecules, whereas cytoplasmic sensors possess nucleotide-binding (NB) and LRR domains (NLR).^1,2 The LRR domain serves as a pattern-recognition receptor to detect pathogen-derived molecules or host proteins that are targeted by pathogen peptides that have entered the cell, effectors.³ NLR-type sensors are the substrates of a structurally and functionally conserved chaperone complex that consists of HEAT SHOCK PROTEIN 90 (HSP90) and its cochaperone SUPPRESSOR OF THE G2 ALLELE OF SKP1 (SGT1). REQUIRED FOR MLA12 RESISTANCE 1 (RAR1) regulated the HSP90-SGT1 complex, resulting in the stabilization of NLR proteins. Thus, SGT1 and RAR1 are required for the function of multiple and distinct R genes that encode NLR immune sensors in plants.⁴ Experiments in RAR1-silenced transgenic rice lines showed that RAR1 is not essential for Pib, which encodes an NLR against rice blast fungus.⁵ In contrast, basal resistance to normally virulent races of rice blast fungus or bacterial blight is significantly reduced in RAR1-silenced lines. This result is consistent with earlier reports that RAR1 is involved in basal resistance to virulent Pseudomonas bacteria in Arabidopsis or blast fungus in barley.^6,7 The requirement of SGT1 for immunity in plants is shown mostly by transient silencing of a number of NLR proteins.^8,9 In addition, SGT1 is also required for immune responses triggered by non-NLR-type sensors.¹⁰ This requirement indicates that either SGT1 function is not limited to the NLR sensors, or some unknown SGT1-dependent NLR proteins also operate downstream of non NLR-type sensors. Furthermore, SGT1 is involved in nonhost resistance, indicating that SGT1 may be a general factor of disease resistance.¹⁰ An Arabidopsis mutant, nho1 (nonhost resistance 1), has been isolated on which Pseudomonas syringae pv. phaseolicola grows and causes disease symptoms.^11,12 It is significant that this mutant is also compromised in R-gene-mediated resistance to P. syringae.¹¹ Although NHO1 is the flagellin-induced glycerol kinase, whose exact function in NHR remains elusive.^12,13 A possible explanation might be that altered plant glycerol pools either directly or indirectly affect nutrient availability for P. syringae. NHO1 is also required for resistance to the fungal pathogen Botrytis cinerea, indicating that NHO1 is not limited to bacterial resistance.¹² However, these contributions to NHR to M. oryzae in A. thaliana have not been understood.To determine whether these factors were necessary for the resistance to M. oryzae in A. thaliana, the following A. thaliana mutants were inoculated with M. oryzae and monitored by microscopy: rar1-21;¹⁴ edm1-1;¹⁵ nho1-1,¹¹ (all Col-0 background). All these mutants exhibited a level of penetration resistance similar to that of the wild-type plants (data not shown), suggesting that none of these mutants significantly compromised resistance to M. oryzae. We have recently shown that among the penetration (pen) mutants, only the pen2,¹⁶ mutant allowed increased penetration into epidermal cells by M. oryzae.¹⁷ Thus, double mutants were generated between pen2 and these mutants to determine whether these factors were necessary for the resistance to M. oryzae in a pen2 background: pen2 rar1-21; pen2 edm1-1; pen2 nho1-1. All these double mutants exhibited a level of penetration resistance similar to that of the pen2 mutant (Fig. 1), suggesting that none of these mutants significantly compromised resistance to M. oryzae in a pen2 background. This might indicate that NHR against M. oryzae may not be conferred by RAR1- and SGT1-dependent NLR immune sensors. Alternatively, since there has been no report that RAR1 is required for any known transmembrane sensors, such as FLS2, EFR or Xa21, RAR1- and SGT1-independent transmembrane-type immune sensors may be required for NHR against M. oryzae. Future studies will be required to reveal the genetic and mechanistic requirements for NHR in A. thaliana-M. oryzae interactions.Open in a separate windowFigure 1Double mutant analysis to evaluate the role of the defense related genes on resistance to Magnaporthe oryzae in Arabidopsis thaliana. The frequency of M. oryzae penetration on double mutants at 3 days post-inoculation was expressed as a percentage of total appressoria. Data were collected from six independent plants per line. A minimum of 100 infection sites was inspected per leaf. Results represent mean ± standard error of three independent experiments.     

The Arabidopsis gain-of-function mutant ssi4 requires RAR1 and SGT1b differentially for defense activation and morphological alterations

A gain-of-function mutation in resistance (R) gene SSI4 causes constitutive activation of defense responses, spontaneous necrotic lesion formation, enhanced resistance against virulent pathogens, and a severe dwarf phenotype. Genetic analysis revealed that ssi4-induced H(2)O(2) accumulation and spontaneous cell death require RAR1, whereas ssi4-mediated stunting is dependent on SGT1b. By contrast, both RAR1 and SGT1b are required in a genetically additive manner for ssi4-induced disease resistance, SA accumulation, and lesion formation after pathogen infection. These data point to cooperative yet distinct functions of RAR1 and SGT1b in responses conditioned by a deregulated nucleotide-binding leucine-rich repeat protein. We also found that RAR1 and SGT1b together contribute to basal resistance because an ssi4 rar1 sgt1b triple mutant exhibited enhanced susceptibility to virulent pathogen infection compared with wild-type SSI4 plants. All ssi4-induced phenotypes were suppressed when plants were grown at 22 degrees C under high relative humidity. However, low temperature (16 degrees C) triggered ssi4-mediated cell death via an RAR1-dependent pathway even in the presence of high humidity. Thus, multiple environmental factors impact on ssi4 signaling, as has been observed for other constitutive defense mutants and R gene-triggered pathways.     

Epidermal jasmonate perception is sufficient for all aspects of jasmonate‐mediated male fertility in Arabidopsis

Jasmonate (JA) signaling is essential for several environmental responses and reproductive development in many plant species. In Arabidopsis thaliana, the most obvious phenotype of JA biosynthetic and perception mutants is profound sporophytic male sterility characterized by failure of stamen filament elongation, severe delay of anther dehiscence and pollen inviability. The site of action of JA in the context of reproductive development has been discussed, but the ideas have not been tested experimentally. To this end we used targeted expression of a COI1‐YFP transgene in the coi1‐1 mutant background. As COI1 is an essential component of the JA co‐receptor complex, the null coi1‐1 mutant is male sterile due to lack of JA perception. We show that expression of COI1‐YFP in the epidermis of the stamen filament and anther in coi1 mutant plants is sufficient to rescue filament elongation, anther dehiscence and pollen viability. In contrast, filament expression alone or expression in the tapetum do not restore dehiscence and pollen viability. These results demonstrate that epidermal JA perception is sufficient for anther function and pollen viability, and suggest the presence of a JA‐dependent non‐autonomous signal produced in the anther epidermis to synchronize both anther dehiscence and pollen maturation.     

The role of RAR1 in Agrobacterium-mediated plant transformation

RAR1 is identified as a critical protein involved in plant innate immunity. We investigated the role of RAR1 in Agrobacterium-mediated plant transformation based on the previous findings that accessory proteins associated with the E3 ligase complex such as SGT1, which tightly interacts with RAR1, play a role in the transformation process. RAR1 gene silencing in Nicotiana benthamiana and Arabidopsis rar1 mutant analysis suggested that RAR1 is required for early stages of Agrobacterium-mediated plant transformation. This finding further illustrates that RAR1, along with SGT1, that serve as a HSP90 co-chaperone is important for Agrobacterium-mediated plant transformation.     

Point mutations in Arabidopsis Cullin1 reveal its essential role in jasmonate response

The SKP1-Cullin/Cdc53-F-box protein ubiquitin ligases (SCF) target many important regulatory proteins for degradation and play vital roles in diverse cellular processes. In Arabidopsis there are 11 Cullin members (AtCUL). AtCUL1 was demonstrated to assemble into SCF complexes containing COI1, an F-box protein required for response to jasmonates (JA) that regulate plant fertility and defense responses. It is not clear whether other Cullins also associate with COI1 to form SCF complexes, thus, it is unknown whether AtCUL1, or another Cullin that assembles into SCF(COI1) (even perhaps two or more functionally redundant Cullins), plays a major role in JA signaling. We present genetic and physiological data to directly demonstrate that AtCUL1 is necessary for normal JA responses. The homozygous AtCUL1 mutants axr6-1 and axr6-2, the heterozygous mutants axr6/AXR6, and transgenic plants expressing mutant AtCUL1 proteins containing a single amino acid substitution from phenylalanine-111 to valine, all exhibit reduced responses to JA. We also demonstrate that ax6 enhances the effect of coi1 on JA responses, implying a genetic interaction between COI1 and AtCUL1 in JA signaling. Furthermore, we show that the point mutations in AtCUL1 affect the assembly of COI1 into SCF, thus attenuating SCF(COI1) formation.     

New Clothes for the Jasmonic Acid Receptor COI1: Delayed Abscission,Meristem Arrest and Apical Dominance

In a screen for delayed floral organ abscission in Arabidopsis, we have identified a novel mutant of CORONATINE INSENSITIVE 1 (COI1), the F-box protein that has been shown to be the jasmonic acid (JA) co-receptor. While JA has been shown to have an important role in senescence, root development, pollen dehiscence and defense responses, there has been little focus on its critical role in floral organ abscission. Abscission, or the detachment of organs from the main body of a plant, is an essential process during plant development and a unique type of cell separation regulated by endogenous and exogenous signals. Previous studies have indicated that auxin and ethylene are major plant hormones regulating abscission; and here we show that regulation of floral organ abscission is also controlled by jasmonic acid in Arabidopsis thaliana. Our characterization of coi1-1 and a novel allele (coi1-37) has also revealed an essential role in apical dominance and floral meristem arrest. In this study we provide genetic evidence indicating that delayed abscission 4 (dab4-1) is allelic to coi1-1 and that meristem arrest and apical dominance appear to be evolutionarily divergent functions for COI1 that are governed in an ecotype-dependent manner. Further characterizations of ethylene and JA responses of dab4-1/coi1-37 also provide new information suggesting separate pathways for ethylene and JA that control both floral organ abscission and hypocotyl growth in young seedlings. Our study opens the door revealing new roles for JA and its interaction with other hormones during plant development.     

Fusarium oxysporum hijacks COI1-mediated jasmonate signaling to promote disease development in Arabidopsis

Although defense responses mediated by the plant oxylipin jasmonic acid (JA) are often necessary for resistance against pathogens with necrotrophic lifestyles, in this report we demonstrate that jasmonate signaling mediated through COI1 in Arabidopsis thaliana is responsible for susceptibility to wilt disease caused by the root-infecting fungal pathogen Fusarium oxysporum . Despite compromised JA-dependent defense responses, the JA perception mutant coronatine insensitive 1 ( coi1 ), but not JA biosynthesis mutants, exhibited a high level of resistance to wilt disease caused by F. oxysporum . This response was independent from salicylic acid-dependent defenses, as coi1/NahG plants showed similar disease resistance to coi1 plants. Inoculation of reciprocal grafts made between coi1 and wild-type plants revealed that coi1 -mediated resistance occurred primarily through the coi1 rootstock tissues. Furthermore, microscopy and quantification of fungal DNA during infection indicated that coi1 -mediated resistance was not associated with reduced fungal penetration and colonization until a late stage of infection, when leaf necrosis was highly developed in wild-type plants. In contrast to wild-type leaves, coi1 leaves showed no necrosis following the application of F. oxysporum culture filtrate, and showed reduced expression of senescence-associated genes during disease development, suggesting that coi1 resistance is most likely achieved through the inhibition of F. oxysporum -incited lesion development and plant senescence. Together, our results indicate that F. oxysporum hijacks non-defensive aspects of the JA-signaling pathway to cause wilt-disease symptoms that lead to plant death in Arabidopsis.     

The co-chaperone HOP3 participates in jasmonic acid signaling by regulating CORONATINE-INSENSITIVE 1 activity

HOPs (HSP70–HSP90 organizing proteins) are a highly conserved family of HSP70 and HSP90 co-chaperones whose role in assisting the folding of various hormonal receptors has been extensively studied in mammals. In plants, HOPs are mainly associated with stress response, but their potential involvement in hormonal networks remains completely unexplored. In this article we describe that a member of the HOP family, HOP3, is involved in the jasmonic acid (JA) pathway and is linked to plant defense responses not only to pathogens, but also to a generalist herbivore. The JA pathway regulates responses to Botrytis cinerea infection and to Tetranychus urticae feeding; our data demonstrate that the Arabidopsis (Arabidopsis thaliana) hop3-1 mutant shows an increased susceptibility to both. The hop3-1 mutant exhibits reduced sensitivity to JA derivatives in root growth assays and downregulation of different JA-responsive genes in response to methyl jasmonate, further revealing the relevance of HOP3 in the JA pathway. Interestingly, yeast two-hybrid assays and in planta co-immunoprecipitation assays found that HOP3 interacts with COI1, suggesting that COI1 is a target of HOP3. Consistent with this observation, COI1 activity is reduced in the hop3-1 mutant. All these data strongly suggest that, specifically among HOPs, HOP3 plays a relevant role in the JA pathway by regulating COI1 activity in response to JA and, consequently, participating in defense signaling to biotic stresses.

One-sentence summary: The co-chaperone protein HOP3 (HSP70-HSP90 ORGANIZING PROTEIN 3) regulates the activity of jasmonic acid co-receptor CORONATINE INSENSITIVE 1 and functions in plant defense.     

Plastidial fatty acid signaling modulates salicylic acid- and jasmonic acid-mediated defense pathways in the Arabidopsis ssi2 mutant

A mutation in the Arabidopsis gene ssi2/fab2, which encodes stearoyl–acyl carrier protein desaturase (S-ACP-DES), results in the reduction of oleic acid (18:1) levels in the mutant plants and also leads to the constitutive activation of NPR1-dependent and -independent defense responses. By contrast, ssi2 plants are compromised in the induction of the jasmonic acid (JA)–responsive gene PDF1.2 and in resistance to the necrotrophic pathogen Botrytis cinerea. Although S-ACP-DES catalyzes the initial desaturation step required for JA biosynthesis, a mutation in ssi2 does not alter the levels of the JA precursor linolenic acid (18:3), the perception of JA or ethylene, or the induced endogenous levels of JA. This finding led us to postulate that the S-ACP-DES–derived fatty acid (FA) 18:1 or its derivative is required for the activation of certain JA-mediated responses and the repression of the salicylic acid (SA) signaling pathway. Here, we report that alteration of the prokaryotic FA signaling pathway in plastids, leading to increased levels of 18:1, is required for the rescue of ssi2-triggered phenotypes. 18:1 levels in ssi2 plants were increased by performing epistatic analyses between ssi2 and several mutants in FA pathways that cause an increase in the levels of 18:1 in specific compartments of the cell. A loss-of-function mutation in the soluble chloroplastic enzyme glycerol-3-phosphate acyltransferase (ACT1) completely reverses SA- and JA-mediated phenotypes in ssi2. In contrast to the act1 mutation, a loss-of-function mutation in the endoplasmic reticulum–localized ω6 oleate desaturase (FAD2) does not alter SA- or JA-related phenotypes of ssi2. However, a mutation in the plastidial membrane–localized ω6 desaturase (FAD6) mediates a partial rescue of ssi2-mediated phenotypes. Although ssi2 fad6 plants are rescued in their morphological phenotypes, including larger size, absence of visible lesions, and straight leaves, these plants continue to exhibit microscopic cell death and express the PR-1 gene constitutively. In addition, these plants are unable to induce the expression of PDF1.2 in response to the exogenous application of JA. Because the act1 mutation rescues all of these phenotypes in ssi2 fad6 act1 triple-mutant plants, act1-mediated reversion may be mediated largely by an increase in the free 18:1 content within the chloroplasts. The reversion of JA responsiveness in ssi2 act1 plants is abolished in the ssi2 act1 coi1 triple-mutant background, suggesting that both JA- and act1-generated signals are required for the expression of the JA-inducible PDF1.2 gene. Our conclusion that FA signaling in plastids plays an essential role in the regulation of SSI2-mediated defense signaling is further substantiated by the fact that overexpression of the N-terminal–deleted SSI2, which lacks the putative plastid-localizing transit peptide, is unable to rescue ssi2-triggered phenotypes, as opposed to overexpression of the full-length protein.     

Characterization of the early response of Arabidopsis to Alternaria brassicicola infection using expression profiling

All tested accessions of Arabidopsis are resistant to the fungal pathogen Alternaria brassicicola. Resistance is compromised by pad3 or coi1 mutations, suggesting that it requires the Arabidopsis phytoalexin camalexin and jasmonic acid (JA)-dependent signaling, respectively. This contrasts with most well-studied Arabidopsis pathogens, which are controlled by salicylic acid-dependent responses and do not benefit from absence of camalexin or JA. Here, mutants with defects in camalexin synthesis (pad1, pad2, pad3, and pad5) or in JA signaling (pad1, coi1) were found to be more susceptible than wild type. Mutants with defects in salicylic acid (pad4 and sid2) or ethylene (ein2) signaling remained resistant. Plant responses to A. brassicicola were characterized using expression profiling. Plants showed dramatic gene expression changes within 12 h, persisting at 24 and 36 h. Wild-type and pad3 plants responded similarly, suggesting that pad3 does not have a major effect on signaling. The response of coi1 plants was quite different. Of the 645 genes induced by A. brassicicola in wild-type and pad3 plants, 265 required COI1 for full expression. It is likely that some of the COI1-dependent genes are important for resistance to A. brassicicola. Responses to A. brassicicola were compared with responses to Pseudomonas syringae infection. Despite the fact that these pathogens are limited by different defense responses, approximately 50% of the induced genes were induced in response to both pathogens. Among these, requirements for COI1 were consistent after infection by either pathogen, suggesting that the regulatory effect of COI1 is similar regardless of the initial stimulus.     

A conditionally fertile coi1 allele indicates cross-talk between plant hormone signalling pathways in Arabidopsis thaliana seeds and young seedlings

Jasmonates (JAs) regulate Arabidopsis thaliana (L.) Heynh. wound and defense responses, pollen development, and stress-related growth inhibition. Significantly, each of these responses requires COI1, an F-box protein. We fused firefly luciferase as a reporter to the JA-responsive promoter for the vegetative storage protein gene (VSP) and used this to screen for mutants that failed to express luciferase in the presence of JA, isolating a mutant designated coi1-16. Comparisons with coi1-1 and jar1-1 plants indicated that coi1-16 was only slightly more sensitive to JA than coi1-1 plants. However, whilst coi1-16 plants failed to produce viable pollen at 22 degrees C, they were fertile at 16 degrees C. Therefore, unlike the other coi1 mutants, coi1-16 could be maintained as a pure line and did not require selection. We have used coi1-16 seeds to define novel interactions between JA and other hormone signalling pathways in seed germination and in the development of young seedlings.     

The vascular pathogen Verticillium longisporum requires a jasmonic acid-independent COI1 function in roots to elicit disease symptoms in Arabidopsis shoots

Verticillium longisporum is a soil-borne vascular pathogen that causes reduced shoot growth and early senescence in Arabidopsis (Arabidopsis thaliana). Here, we report that these disease symptoms are less pronounced in plants that lack the receptor of the plant defense hormone jasmonic acid (JA), CORONATINE INSENSITIVE1 (COI1). Initial colonization of the roots was comparable in wild-type and coi1 plants, and fungal DNA accumulated to almost similar levels in petioles of wild-type and coi1 plants at 10 d post infection. Completion of the fungal life cycle was impaired in coi1, as indicated by the reduced number of plants with microsclerotia, which are detected on dead plant material at late stages of the disease. Contrary to the expectation that the hormone receptor mutant coi1 should display the same phenotype as the corresponding hormone biosynthesis mutant delayed dehiscence2 (dde2), dde2 plants developed wild-type-like disease symptoms. Marker genes of the JA and the JA/ethylene defense pathway were induced in petioles of wild-type plants but not in petioles of dde2 plants, indicating that fungal compounds that would activate the known COI1-dependent signal transduction chain were absent. Grafting experiments revealed that the susceptibility-enhancing COI1 function acts in the roots. Moreover, we show that the coi1-mediated tolerance is not due to the hyperactivation of the salicylic acid pathway. Together, our results have unraveled a novel COI1 function in the roots that acts independently from JA-isoleucine or any JA-isoleucine mimic. This COI1 activity is required for a yet unknown root-to-shoot signaling process that enables V. longisporum to elicit disease symptoms in Arabidopsis.