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.     

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).     

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.     

Defense against Sclerotinia sclerotiorum in Arabidopsis is dependent on jasmonic acid, salicylic acid, and ethylene signaling

Genotypic differences in susceptibility of Arabidopsis thaliana to Sclerotinia sclerotiorum have not been reported due to the extreme susceptibility of this cruciferous plant. To overcome this limitation, we have established inoculation conditions that enable evaluation of differences in susceptibility to S. sclerotiorum among Arabidopsis mutants and ecotypes. Two coil mutant alleles conferred hypersusceptibility to S. sclerotiorum. The plant defensin gene PDF1.2 was no longer induced after challenging the coi1-2 mutant with S. sclerotiorum. Hypersusceptibility of the coi1-2 mutant to S. sclerotiorum was not correlated with oxalate sensitivity. The mutants npr1 and ein2 were also hypersusceptible to S. sclerotiorum. Induction of PDF1.2 and the pathogenesis-related gene PR1 was reduced in ein2 and npr1 mutants, respectively. Actigard, a commercial formulation of the systemic acquired resistance inducer benzothiadiazole, reduced susceptibility to S. sclerotiorum. Based on histochemical analysis of oxalate-deficient and wild-type strains of S. sclerotiorum, oxalate caused a decrease in hydrogen peroxide production but no detectable changes in plant superoxide production or gene expression.     

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.     

Proteomics Study of COI1-regulated Proteins in Arabidopsis Flower

Jasmonates (JAs) are a new class of plant hormone that regulate expression of diverse genes to mediate various plant responses. The Arabidopsis F-box protein COM is required for plant defense and male fertility in JA signal pathway. To further investigate the regulatory role of COM in male fertility, we compared the proteomics profiles of Arabidopsis wild type (WT) flowers with coi1-1 mutant male-sterile flowers using two-dimensional difference gel electrophoresis coupled with matrix-assisted laser desoption/ionization-time-of-flight mass spectrometry. Sixteen proteins with potential function in specific biological processes such as metabolism processes and defense/stress responses were differentially expressed in WT and coi1-1 mutant flowers. Verification on a phi class glutathione transferase AtGSTF9, one out of these 16 identified proteins, revealed that the expression of AtGSTF9 was severely downregulated in flowers of coi1-1 mutant compared with that of WT. Further function analyses of these genes would provide new insights into the molecular basis of COI1-regulated male fertility.     

The jasmonate receptor COI1 plays a role in jasmonate-induced lateral root formation and lateral root positioning in Arabidopsis thaliana

Jasmonic acid (JA) regulates a broad range of plant defense and developmental responses. COI1 has been recently found to act as JA receptor. In this report, we show that low micromolar concentrations of JA inhibited primary root (PR) growth and promoted lateral root (LR) formation in Arabidopsis wild-type (WT) seedlings. It was observed that the coi1-1 mutant was less sensitive to JA on pericycle cell activation to induce lateral root primordia (LRP) formation and presented alterations in lateral root positioning and lateral root emergence on bends. To investigate JA-auxin interactions important for remodeling of root system (RS) architecture, we tested the expression of auxin-inducible markers DR5:uidA and BA3:uidA in WT and coi1-1 seedlings in response to indole-3-acetic acid (IAA) and JA and analyzed the RS architecture of a suite of auxin-related mutants under JA treatments. We found that JA did not affect DR5:uidA and BA3:uidA expression in WT and coi1-1 seedlings. Our data also showed that PR growth inhibition in response to JA was likely independent of auxin signaling and that the induction of LRP required ARF7, ARF19, SLR, TIR1, AFB2, AFB3 and AXR1 loci. We conclude that JA regulation of postembryonic root development involves both auxin-dependent and independent mechanisms.     

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.     

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.     

The COI1 and DFR Genes are Essential for Regulation of Jasmonate-Induced Anthocyanin Accumulation in Arabidopsis

Jasmonates （JAs） are a class of plant hormones that play important roles in the regulation of plant development and plant defense. It has been shown that Arabidopsis plants produce much higher levels of anthocyanins when treated exogenously with methyl jasmonate （MeJA）. However, a molecular link between the JA response and anthocyanin production has not been determined. The CORONATINE INSENTITIVE1 （COI1） gene is a key player in the regulation of many JA-related responses. In the present study, we demonstrate that the COI1 gene is also required for the JA-induced accumulation of anthocyanins in Arabidopsis. Furthermore, the MeJA-inducible expression of DIHYDROFLAVONOL REDUCTASE （DFR）, an essential component in the anthocyanin biosynthesis pathway, was completely eliminated in the coil mutant. Jasmonateinduced anthocyanin accumulation was found to be independent of auxin signaling. The present results indicate that the expression of both COI1 and DFR genes is required for the regulation of JA-induced anthocyanin accumulation and that DFR may be a key downstream regulator for this process.     

Jasmonic acid perception by COI1 involves inositol polyphosphates in Arabidopsis thaliana

Plant responses to wounding are part of their defense responses against insects, and are tightly regulated. The isoleucin conjugate of jasmonic acid (JA‐Ile) is a major regulatory molecule. We have previously shown that inositol polyphosphate signals are required for defense responses in Arabidopsis; however, the way in which inositol polyphosphates contribute to plant responses to wounding has so far remained unclear. Arabidopsis F‐box proteins involved in the perception of JA‐Ile (COI1) and auxin (TIR1) are structurally similar. Because TIR1 has recently been shown to contain inositol hexakisphosphate (InsP₆) as a co‐factor of unknown function, here we explored the possibility that InsP₆ or another inositol polyphosphate is required for COI1 function. In support of this hypothesis, COI1 variants with changes in putative inositol polyphosphate coordinating residues exhibited a reduced interaction with the COI1 target, JAZ9, in yeast two‐hybrid tests. The equivalent COI1 variants displayed a reduced capability to rescue jasmonate‐mediated root growth inhibition or silique development in Arabidopsis coi1 mutants. Yeast two‐hybrid tests using wild‐type COI1 in an ipk1Δ yeast strain exhibiting increased levels of inositol pentakisphosphate (InsP₅) and reduced levels of InsP₆ indicate an enhanced COI1/JAZ9 interaction. Consistent with these findings, Arabidopsis ipk1‐1 mutants, also with increased InsP₅ and reduced InsP₆ levels, showed increased defensive capabilities via COI1‐mediated processes, including wound‐induced gene expression, defense against caterpillars or root growth inhibition by jasmonate. The combined data from experiments using mutated COI1 variants, as well as yeast and Arabidopsis backgrounds altered in inositol polyphosphate metabolism, indicate that an inositol polyphosphate, and probably InsP₅, contributes to COI1 function.     

Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine-insensitive (coi1) mutation occurs through two distinct mechanisms

A new allele of the coronatine-insensitive locus (COI1) was isolated in a screen for Arabidopsis thaliana mutants with enhanced resistance to the bacterial pathogen Pseudomonas syringae. This mutant, designated coi1-20, exhibits robust resistance to several P. syringae isolates but remains susceptible to the virulent pathogens Erisyphe and cauliflower mosaic virus. Resistance to P. syringae strain PstDC3000 in coi1-20 plants is correlated with hyperactivation of PR-1 expression and accumulation of elevated levels of salicylic acid (SA) following infection, suggesting that the SA-mediated defense response pathway is sensitized in this mutant. Restriction of growth of PstDC3000 in coi1-20 leaves is partially dependent on NPR1 and fully dependent on SA, indicating that SA-mediated defenses are required for restriction of PstDC3000 growth in coi1-20 plants. Surprisingly, despite high levels of PstDC3000 growth in coi1-20 plants carrying the salicylate hydroxylase (nahG) transgene, these plants do not exhibit disease symptoms. Thus resistance to P. syringae in coi1-20 plants is conferred by two different mechanisms: (i) restriction of pathogen growth via activation of the SA-dependent defense pathway; and (ii) an SA-independent inability to develop disease symptoms. These findings are consistent with the hypotheses that the P. syringae phytotoxin coronatine acts to promote virulence by inhibiting host defense responses and by promoting lesion formation.     

Specific Missense Alleles of the Arabidopsis Jasmonic Acid Co-Receptor COI1 Regulate Innate Immune Receptor Accumulation and Function

Plants utilize proteins containing nucleotide binding site (NB) and leucine-rich repeat (LRR) domains as intracellular innate immune receptors to recognize pathogens and initiate defense responses. Since mis-activation of defense responses can lead to tissue damage and even developmental arrest, proper regulation of NB–LRR protein signaling is critical. RAR1, SGT1, and HSP90 act as regulatory chaperones of pre-activation NB–LRR steady-state proteins. We extended our analysis of mutants derived from a rar1 suppressor screen and present two allelic rar1 suppressor (rsp) mutations of Arabidopsis COI1. Like all other coi1 mutations, coi1^rsp missense mutations impair Jasmonic Acid (JA) signaling resulting in JA–insensitivity. However, unlike previously identified coi1 alleles, both coi1^rsp alleles lack a male sterile phenotype. The coi1^rsp mutants express two sets of disease resistance phenotypes. The first, also observed in coi1-1 null allele, includes enhanced basal defense against the virulent bacterial pathogen Pto DC3000 and enhanced effector-triggered immunity (ETI) mediated by the NB–LRR RPM1 protein in both rar1 and wild-type backgrounds. These enhanced disease resistance phenotypes depend on the JA signaling function of COI1. Additionally, the coi1^rsp mutants showed a unique inability to properly regulate RPM1 accumulation and HR, exhibited increased RPM1 levels in rar1, and weakened RPM1-mediated HR in RAR1. Importantly, there was no change in the steady-state levels or HR function of RPM1 in coi1-1. These results suggest that the coi1^rsp proteins regulate NB–LRR protein accumulation independent of JA signaling. Based on the phenotypic similarities and genetic interactions among coi1^rsp, sgt1b, and hsp90.2^rsp mutants, our data suggest that COI1 affects NB–LRR accumulation via two NB–LRR co-chaperones, SGT1b and HSP90. Together, our data demonstrate a role for COI1 in disease resistance independent of JA signaling and provide a molecular link between the JA and NB–LRR signaling pathways.     

The COP9 signalosome interacts physically with SCF COI1 and modulates jasmonate responses

The COP9 signalosome (CSN) is an evolutionarily conserved, nucleus-enriched multiprotein complex. CSN plays roles in photomorphogenesis, auxin response, and floral organ formation, possibly via the regulation of ubiquitin-proteasome-mediated protein degradation. COI1 encodes an F-box protein, which is a subunit of SCF(COI1) E3 ubiquitin ligase, and is required for jasmonate (JA) responses. Here, we demonstrate using coimmunoprecipitation and gel-filtration analyses that endogenous as well as epitope-tagged COI1 forms SCF(COI1) and associates directly with CSN in vivo. Like the coi1-1 mutant, CSN reduction-of-function plants exhibited a JA-insensitive root elongation phenotype and an absence of JA-induced-specific gene expression. Genome expression profile analyses indicated that JA-triggered genome expression is critically dependent on COI1 dosage. More importantly, most of the COI1-dependent JA-responsive genes also required CSN function, and CSN abundance was shown to be important for JA responses. Furthermore, we showed that both COI1 and CSN are essential for modulating the expression of genes in most cellular pathways responsive to JA. Thus, CSN and SCF(COI1) work together to control genome expression and promote JA responses.     

Ethylene and jasmonic acid signaling affect the NPR1-independent expression of defense genes without impacting resistance to Pseudomonas syringae and Peronospora parasitica in the Arabidopsis ssi1 mutant

Salicylic acid (SA), ethylene, and jasmonic acid (JA) are important signaling molecules in plant defense to biotic stress. An intricate signaling network involving SA, ethylene, and JA fine tunes plant defense responses. SA-dependent defense responses in Arabidopsis thaliana are mediated through NPR1-dependent and -independent mechanisms. We have previously shown that activation of an NPR1-independent defense mechanism confers enhanced disease resistance and constitutive expression of the pathogenesis-related (PR) genes in the Arabidopsis ssi1 mutant. In addition, the ssi1 mutant constitutively expresses the defensin gene PDF1.2. Moreover, SA is required for the ssi1-conferred constitutive expression of PDF1.2 in addition to PR genes. Hence, the ssi1 mutant appears to target a step common to SA- and ethylene- or JA-regulated defense pathways. In the present study, we show that, in addition to SA, ethylene and JA signaling also are required for the ssi1-conferred constitutive expression of PDF1.2 and the NPR1-independent expression of PR-1. Furthermore, the ethylene-insensitive ein2 and JA-insensitive jar1 mutants enhance susceptibility of ssi1 plants to the necrotrophic fungus Botrytis cinerea. However, defects in either the ethylene- or JA-signaling pathways do not compromise ssi1-conferred resistance to the bacterial pathogen Pseudomonas synringae pv. maculicola and the oomycete pathogen Peronospora parasitica. Interestingly, ssi1 exhibits a marginal increase in the levels of ethylene and JA, suggesting that low endogenous levels of these phytohormones are sufficient to activate expression of defense genes. Taken together, our results indicate that although cross talk in ssi1 renders expression of ethylene- or JA-responsive defense genes sensitive to SA and vice versa, it does not affect downstream signaling leading to resistance.     

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.     

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.