Signal Transduction Chain Research Articles

Hardware Development for Plant Cultivation Allowing In Situ Fluorescence Analysis of Calcium Fluxes in Plant Roots Under Microgravity and Ground-Control Conditions.

Maintaining an optimal leaf and stem orientation to yield a maximum photosynthetic output is accomplished by terrestrial plants using sophisticated mechanisms to balance their orientation relative to the Earth's gravity vector and the direction of sunlight. Knowledge of the signal transduction chains of both gravity and light perception and how they influence each other is essential for understanding plant development on Earth and plant cultivation in space environments. However, in situ analyses of cellular signal transduction processes in weightlessness, such as live cell imaging of signaling molecules using confocal fluorescence microscopy, require an adapted experimental setup that meets the special requirements of a microgravity environment. In addition, investigations under prolonged microgravity conditions require extensive resources, are rarely accessible, and do not allow for immediate sample preparation for the actual microscopic analysis. Therefore, supply concepts are needed that ensure both the viability of the contained plants over a longer period of time and an unhindered microscopic analysis in microgravity. Here, we present a customized supply unit specifically designed to study gravity-induced Ca2+ mobilization in roots of Arabidopsis thaliana. The unit can be employed for ground-based experiments, in parabolic flights, on sounding rockets, and probably also aboard the International Space Station.

Transcriptome, metabolome and suppressor analysis reveal an essential role for the ubiquitin-proteasome system in seedling chloroplast development

BackgroundMany regulatory circuits in plants contain steps of targeted proteolysis, with the ubiquitin proteasome system (UPS) as the mediator of these proteolytic events. In order to decrease ubiquitin-dependent proteolysis, we inducibly expressed a ubiquitin variant with Arg at position 48 instead of Lys (ubK48R). This variant acts as an inhibitor of proteolysis via the UPS, and allowed us to uncover processes that are particularly sensitive to UPS perturbation.ResultsExpression of ubK48R during germination leads to seedling death. We analyzed the seedling transcriptome, proteome and metabolome 24 h post ubK48R induction and confirmed defects in chloroplast development. We found that mutations in single genes can suppress seedling lethality, indicating that a single process in seedlings is critically sensitive to decreased performance of the UPS. Suppressor mutations in phototropin 2 (PHOT2) suggest that a contribution of PHOT2 to chloroplast protection is compromised by proteolysis inhibition.ConclusionsOverall, the results reveal protein turnover as an integral part of a signal transduction chain that protects chloroplasts during development.

RetS inhibits Pseudomonas aeruginosa biofilm formation by disrupting the canonical histidine kinase dimerization interface of GacS

Bacterial signaling histidine kinases (HKs) have long been postulated to function exclusively through linear signal transduction chains. However, several HKs have recently been shown to form complex multikinase networks (MKNs). The most prominent MKN, involving the enzymes RetS and GacS, controls the switch between the motile and biofilm lifestyles in the pathogenic bacterium Pseudomonas aeruginosa. While GacS promotes biofilm formation, RetS counteracts GacS using three distinct mechanisms. Two are dephosphorylating mechanisms. The third, a direct binding between the RetS and GacS HK regions, blocks GacS autophosphorylation. Focusing on the third mechanism, we determined the crystal structure of a cocomplex between the HK region of RetS and the dimerization and histidine phosphotransfer (DHp) domain of GacS. This is the first reported structure of a complex between two distinct bacterial signaling HKs. In the complex, the canonical HK homodimerization interface is replaced by a strikingly similar heterodimeric interface between RetS and GacS. We further demonstrate that GacS autophosphorylates in trans, thus explaining why the formation of a RetS-GacS complex inhibits GacS autophosphorylation. Using mutational analysis in conjunction with bacterial two-hybrid and biofilm assays, we not only corroborate the biological role of the observed RetS-GacS interactions, but also identify a residue critical for the equilibrium between the RetS-GacS complex and the respective RetS and GacS homodimers. Collectively, our findings suggest that RetS and GacS form a domain-swapped hetero-oligomer during the planktonic growth phase of P. aeruginosa before unknown signals cause its dissociation and a relief of GacS inhibition to promote biofilm formation.

A replication-incompetent CD154/40L recombinant vaccinia virus induces direct and macrophage-mediated antitumor effects in vitro and in vivo

ABSTRACTCD40 triggering may result in antitumor effects of potentially high clinical relevance. To gain insights important for patient selection and to identify adequate targeting techniques, we investigated CD40 expression in human cancer tissues and generated a replication-incompetent recombinant vaccinia virus expressing CD40 ligand (rVV40L). Its effects were explored in vitro and in vivo upon direct CD40 targeting on malignant cells or macrophage activation. CD40 expression was analyzed by immunohistochemistry in tumor and stromal cells in a multi-tumor array including 836 specimens from 27 different tumor types. Established tumor cell lines were used to explore the capacity of rVV40L to induce malignant cell apoptosis and modulate functional profiles of polarized macrophages. CD40 expression was detectable in significantly higher numbers of stromal as compared to malignant cells in lung and breast cancers. CD40 ligation following rVV40L infection induced apoptosis in CD40(+) cancer cells, but only in the presence of intact specific signal transduction chain. Importantly, rVV40L infection promoted the induction of TNF-α-dependent antitumor activity of M1-like macrophages directed against CD40(-) targets. CD40-activated M1-like macrophages also displayed enhanced ability to CXCL10-dependently recruit CD8+ T cells and to efficiently present cancer cell intracellular antigens through cross-priming. Moreover, rVV-driven CD40L expression partially “re-educated” M2-like macrophages, as suggested by detectable CXCL10 and IL-12 production. Most importantly, we observed that intra-tumoral injection of rVV40L-infected human macrophages inhibits progression of human CD40(-) tumors in vivo. First evidences of anticancer activity of rVV40L strongly encourage further evaluations.

Aureochromes - Blue Light Receptors.

A variety of living organisms including bacteria, fungi, animals, and plants use blue light (BL) to adapt to changing ambient light. Photosynthetic forms (plants and algae) require energy of light for photosynthesis, movements, development, and regulation of activity. Several complex light-sensitive systems evolved in eukaryotic cells to use the information of light efficiently with photoreceptors selectively absorbing various segments of the solar spectrum, being the first components in the light signal transduction chain. They are most diverse in algae. Photosynthetic stramenopiles, which received chloroplasts from red algae during secondary symbiosis, play an important role in ecosystems and aquaculture, being primary producers. These taxa acquired the ability to use BL for regulation of such processes as phototropism, chloroplast photo-relocation movement, and photomorphogenesis. A new type of BL receptor - aureochrome (AUREO) - was identified in Vaucheria frigida in 2007. AUREO consists of two domains: bZIP (basic-region leucine zipper) domain and LOV (light-oxygen-voltage-sensing) domain, and thus this photoreceptor is a BL-sensitive transcription factor. This review presents current data on the structure, mechanisms of action, and biochemical features of aureochromes.

Identification of a flagellar protein implicated in the gravitaxis in the flagellate Euglena gracilis

Flagellated cells are of great evolutionary importance across animal and plant species. Unlike higher plants, flagellated cells are involved in reproduction of macro-algae as well as in early diverging land plants. Euglena gracilis is an emerging flagellated model organism. The current study reports that a specific calmodulin (CaM2) involved in gravitaxis of E. gracilis interacts with an evolutionary conserved flagellar protein, EgPCDUF4201. The subsequent molecular analysis showed clearly that EgPCDUF4201 is also involved in gravitaxis. We performed subcellular localization of CaM2 using immunoblotting and indirect immunofluorescence. By employing yeast two-hybrid screen, EgPCDUF4201 was identified as an interaction partner of CaM2. The C-terminus of EgPCDUF4201 is responsible for the interaction with CaM2. Silencing of N- and C-terminus of EgPCDUF4201 using RNAi resulted in an impaired gravitaxis. Moreover, indirect immunofluorescence assay showed that EgPCDUF4201 is a flagella associated protein. The current study specifically addressed some important questions regarding the signal transduction chain of gravitaxis in E. gracilis. Besides the fact that it improved the current understanding of gravity sensing mechanisms in E. gracilis, it also gave rise to several interesting research questions regarding the function of the domain of unknown function 4201 in flagellated cells.

Azospirillum brasilense FP2 modulates respiratory burst oxidase gene expression in maize seedlings

Azospirillum brasilense is free-living gram-negative bacteria isolated from the rhizosphere of grasses. The plant growth promoting rhizobacteria, such as A. brasilense, can promote plant growth through biological nitrogen fixation and phytohormones production. Our aim was to evaluate the expression of several maize genes involved in stress response of maize (Pioneer 30F53 variety) after inoculation with A. brasilense strain FP2. Samples were collected 1, 4, 7 and 10 days after inoculation (DAI). Significant increase of the transcript level was observed for Auxin transporter-like protein 1 (Zmaux1), Respiratory burst oxidase protein (ZmrbohA and ZmrbohB), MAP kinase 5 (Zmmpk5), Gibberellin 20 oxidase 4 (Zmga20ox4) and Ent-kaurene oxidase (Zmko1) when comparing inoculated to non-inoculated samples. For root samples, increasing transcripts levels for Zmmpk5 gene were observed from 1 to 7 DAI, and a decrease at 10 DAI in inoculated samples compared to non-inoculated. The modulation of gene expression observed for genes involved in the oxidative burst (ZmrbohA and ZmrbohB) and for the signal transduction chain (Zmmpk5), indicates that the plant respond to the presence of the bacterium.

Toward Unveiling the Mechanisms for Transcriptional Regulation of Proline Biosynthesis in the Plant Cell Response to Biotic and Abiotic Stress Conditions.

Proline accumulation occurs in plants following the exposure to a wide array of stress conditions, as well as during numerous physiological and adaptive processes. Increasing evidence also supports the involvement of proline metabolism in the plant response to pathogen attack. This requires that the biosynthetic pathway is triggered by components of numerous and different signal transduction chains. Indeed, several reports recently described activation of genes coding for enzymes of the glutamate pathway by transcription factors (TFs) belonging to various families. Here, we summarize some of these findings with special emphasis on rice, and show the occurrence of a plethora of putative TF binding sites in the promoter of such genes.

Long term stability of Oligo (dT) 25 magnetic beads for the expression analysis of Euglena gracilis for long term space projects

The unicellular freshwater flagellate Euglena gracilis has a highly developed sensory system. The cells use different stimuli such as light and gravity to orient themselves in the surrounding medium to find areas for optimal growth. Due to the ability to produce oxygen and consume carbon dioxide, Euglena is a suitable candidate for life support systems. Participation in a long-term space experiment would allow for the analysis of changes and adaptations to the new environment, and this could bring new insights into the mechanism of perception of gravity and the associated signal transduction chain. For a molecular analysis of transcription patterns, an automated system is necessary, capable of performing all steps from taking a sample, processing it and generating data. One of the developmental steps is to find long-term stable reagents and materials and test them for stability at higher-than-recommended temperature conditions during extended storage time. We investigated the usability of magnetic beads in an Euglena specific lysis buffer after addition of the RNA stabilizer Dithiothreitol over 360 days and the lysis buffer with the stabilizer alone over 455 days at the expected storage temperature of 19 °C. We can claim that the stability is not impaired at all after an incubation period of over one year. This might be an interesting result for researchers who have to work under non-standard lab conditions, as in biological or medicinal fieldwork.

New Insights on Signal Propagation by Sensory Rhodopsin II/Transducer Complex

The complex of two membrane proteins, sensory rhodopsin II (NpSRII) with its cognate transducer (NpHtrII), mediates negative phototaxis in halobacteria N. pharaonis. Upon light activation NpSRII triggers a signal transduction chain homologous to the two-component system in eubacterial chemotaxis. Here we report on crystal structures of the ground and active M-state of the complex in the space group I212121. We demonstrate that the relative orientation of symmetrical parts of the dimer is parallel (“U”-shaped) contrary to the gusset-like (“V”-shaped) form of the previously reported structures of the NpSRII/NpHtrII complex in the space group P21212, although the structures of the monomers taken individually are nearly the same. Computer modeling of the HAMP domain in the obtained “V”- and “U”-shaped structures revealed that only the “U”-shaped conformation allows for tight interactions of the receptor with the HAMP domain. This is in line with existing data and supports biological relevance of the “U” shape in the ground state. We suggest that the “V”-shaped structure may correspond to the active state of the complex and transition from the “U” to the “V”-shape of the receptor-transducer complex can be involved in signal transduction from the receptor to the signaling domain of NpHtrII.

Impact of EHB1 and AGD12 on Root and Hypocotyl Phototropism in Arabidopsis thaliana

Previous findings concerning two proteins involved in gravity and light-induced signal transduction, (AGD12-ADP-RIBOSYLATION FACTOR GTPase-ACTIVATING PROTEIN 12 and EHB1 (ENHANCED BENDING 1)), in Arabidopsis provided evidence for an antagonistic regulation of a gravitropic stimulus on roots and shoots. Both proteins share high C-terminal homology containing a canonical calcium-binding C2-domain, but EHB1 lacks a functional N-terminally located GTP-binding domain. Physiological analysis using loss-of-function mutants under clinostat conditions, where any gravitropic stimulus could be ruled out, provided evidence for diminished phototropism of agd12 mutants, but enhanced phototropism of ehb1-mutants. Data could be confirmed by seedlings overexpressing either AGD12 or EHB1, showing enhanced bending in AGD12 and reduced bending in EHB1 overexpressing seedlings. We conclude that EHB1 and AGD12 are early signaling elements of the gravitropic phototropic bending response, because both of them bind to NPH3 (NON PHOTOTROPIC HYPOCOTYL), which was described as the first element of the phototropic signal transduction chain, directly interacting with phot1. Both proteins affect phototropic bending antagonistically. In contrast to the total incapability of agd12 and ehb1 mutants to respond to increased calcium concentration in response to gravity, agd12 nor ehb1 seedlings differ only gradually in their calcium induced bending response to blue light. This would indicate that EHB1 and AGD12 might be triggered by other signal elements within the phototropism signal transduction chain.

Arabidopsis thaliana G2-LIKE FLAVONOID REGULATOR and BRASSINOSTEROID ENHANCED EXPRESSION1 are low-temperature regulators of flavonoid accumulation.

Flavonoid synthesis is predominantly regulated at the transcriptional level through the MYB-basic helix-loop-helix (bHLH)-WD40 (MBW) (MYB: transcription factor of the myeloblastosis protein family, WD40: tanscription factor with a short structural motif of 40 amino acids which terminates in an aspartic acid-tryptophan dipeptide) complex, and responds to both environmental and developmental stimuli. Although the developmental regulation of flavonoid accumulation in Arabidopsis thaliana has been examined in great detail, the response of the flavonoid synthesis pathway to abiotic stress (particularly low temperature) remains unclear. A screen of a Dissociation element (Ds) transposon-induced mutation collection identified two lines which exhibited an altered profile of phenylpropanoid accumulation following exposure to low-temperature stress. One of the mutated genes (BRASSINOSTEROID ENHANCED EXPRESSION1 (BEE1)) encoded a brassinosteroid enhanced expression transcription factor, while the other (G2-LIKE FLAVONOID REGULATOR (GFR)) encoded a G2-like flavonoid regulator. Phenylpropanoid-targeted analysis was performed using high-performance LC-MS, and gene expression analysis using quantitative reverse transcription-PCR. In both mutants, the accumulation of quercetins and scopolin was reduced under low-temperature growing conditions, whereas that of anthocyanin was increased. BEE1 and GFR were both shown to negatively regulate anthocyanin accumulation by inhibiting anthocyanin synthesis genes via the suppression of the bHLH (TRANSPARENT TESTA8 (TT8) and GLABROUS3 (GL3)) and/or the MYB (PRODUCTION OF ANTHOCYANIN PIGMENTS2 (PAP2)) components of the MBW complex. Our results provide new insight into the regulatory control of phenylpropanoid metabolism at low temperatures, and reveal that BEE1 and GFR act as important components of the signal transduction chain.

Gating of Acoustic Transducer Channels Is Shaped by Biomechanical Filter Processes

Mechanoelectrical transduction of acoustic signals is the fundamental process for hearing in all ears across the animal kingdom. Here, we performed in vivo laser-vibrometric and electrophysiological measurements at the transduction site in an insect ear (Mecopoda elongata) to relate the biomechanical tonotopy along the hearing organ to the frequency tuning of the corresponding sensory cells. Our mechanical and electrophysiological map revealed a biomechanical filter process that considerably sharpens the neuronal response. We demonstrate that the channel gating, which acts on chordotonal stretch receptor neurons, is based on a mechanical directionality of the sound-induced motion. Further, anatomical studies of the transduction site support our finding of a stimulus-relevant tilt. In conclusion, we were able to show, in an insect ear, that directionality of channel gating considerably sharpens the neuronal frequency selectivity at the peripheral level and have identified a mechanism that enhances frequency discrimination in tonotopically organized ears.

A Putative Non-Canonical Ras-Like GTPase from P. falciparum: Chemical Properties and Characterization of the Protein.

During its development the malaria parasite P. falciparum has to adapt to various different environmental contexts. Key cellular mechanisms involving G-protein coupled signal transduction chains are assumed to act at these interfaces. Heterotrimeric G-proteins are absent in Plasmodium. We here describe the first cloning and expression of a putative, non-canonical Ras-like G protein (acronym PfG) from Plasmodium. PfG reveals an open reading frame of 2736 bp encoding a protein of 912 amino acids with a theoretical pI of 8.68 and a molecular weight of 108.57 kDa. Transcript levels and expression are significantly increased in the erythrocytic phase in particular during schizont and gametocyte formation. Most notably, PfG has GTP binding capacity and GTPase activity due to an EngA2 domain present in small Ras-like GTPases in a variety of Bacillus species and Mycobacteria. By contrast, plasmodial PfG is divergent from any human alpha-subunit. PfG was expressed in E. coli as a histidine-tagged fusion protein and was stable only for 3.5 hours. Purification was only possible under native conditions by Nickel-chelate chromatography and subsequent separation by Blue Native PAGE. Binding of a fluorescent GTP analogue BODIPY® FL guanosine 5’O-(thiotriphosphate) was determined by fluorescence emission. Mastoparan stimulated GTP binding in the presence of Mg2+. GTPase activity was determined colorimetrically. Activity expressed as absolute fluorescence was 50% higher for the human paralogue than the activity of the parasitic enzyme. The PfG protein is expressed in the erythrocytic stages and binds GTP after immunoprecipitation. Immunofluorescence using specific antiserum suggests that PfG localizes to the parasite cytosol. The current data suggest that the putitative, Ras-like G-protein might be involved in a non-canonical signaling pathway in Plasmodium. Research on the function of PfG with respect to pathogenesis and antimalarial chemotherapy is currently under way.

Border control--a membrane-linked interactome of Arabidopsis.

Cellular membranes act as signaling platforms and control solute transport. Membrane receptors, transporters, and enzymes communicate with intracellular processes through protein-protein interactions. Using a split-ubiquitin yeast two-hybrid screen that covers a test-space of 6.4 × 10(6) pairs, we identified 12,102 membrane/signaling protein interactions from Arabidopsis. Besides confirmation of expected interactions such as heterotrimeric G protein subunit interactions and aquaporin oligomerization, >99% of the interactions were previously unknown. Interactions were confirmed at a rate of 32% in orthogonal in planta split-green flourescent protein interaction assays, which was statistically indistinguishable from the confirmation rate for known interactions collected from literature (38%). Regulatory associations in membrane protein trafficking, turnover, and phosphorylation include regulation of potassium channel activity through abscisic acid signaling, transporter activity by a WNK kinase, and a brassinolide receptor kinase by trafficking-related proteins. These examples underscore the utility of the membrane/signaling protein interaction network for gene discovery and hypothesis generation in plants and other organisms.

Lipopolysaccharides elicit an oxidative burst as a component of the innate immune system in the seagrass Thalassia testudinum

This study represents the first report characterizing the biological effects of a lipopolysaccharide (LPS) immune modulator on a marine vascular plant. LPS was shown to serve as a strong elicitor of the early defense response in the subtropical seagrass Thalassia testudinum Banks ex König and was capable of inducing an oxidative burst identified at the single cell level. The formation of reactive oxygen species (ROS), detected by a redox-sensitive fluorescent probe and luminol-based chemiluminescence, included a diphenyleneiodonium sensitive response, suggesting the involvement of an NADPH oxidase. A 900bp cDNA fragment coding for this enzyme was sequenced and found to encode a NAD binding pocket domain with extensive homology to the Arabidopsis thaliana rbohF (respiratory burst oxidase homolog) gene. The triggered release of ROS occurred at 20min post-elicitation and was dose-dependent, requiring a minimal threshold of 50μg/mL LPS. Pharmacological dissection of the early events preceding ROS emission indicated that the signal transduction chain of events involved extracellular alkalinization, G-proteins, phospholipase A2, as well as K(+), Ca(2+), and anion channels. Despite exclusively thriving in a marine environment, seagrasses contain ROS-generating machinery and signal transduction components that appear to be evolutionarily conserved with the well-characterized defense response systems found in terrestrial plants.

Ground state structure of D75N mutant of sensory rhodopsin II in complex with its cognate transducer

The complex of sensory rhodopsin II (NpSRII) with its cognate transducer (NpHtrII) mediates negative phototaxis in halobacteria Natronomonas pharaonis. Upon light activation NpSRII triggers, by means of NpHtrII, a signal transduction chain homologous to the two component system in eubacterial chemotaxis. Here we report on the crystal structure of the ground state of the mutant NpSRII-D75N/NpHtrII complex in the space group I212121. Mutations of this aspartic acid in light-driven proton pumps dramatically modify or/and inhibit protein functions. However, in vivo studies show that the similar D75N mutation retains functionality of the NpSRII/NpHtrII complex. The structure provides the molecular basis for the explanation of the unexpected observation that the wild and the mutant complexes display identical physiological response on light excitation.

Brassinosteroid signalling

The brassinosteroid (BR) class of steroid hormones regulates plant development and physiology. The BR signal is transduced by a receptor kinase-mediated signal transduction pathway, which is distinct from animal steroid signalling systems. Recent studies have fully connected the BR signal transduction chain and have identified thousands of BR target genes, linking BR signalling to numerous cellular processes. Molecular links between BR and several other signalling pathways have also been identified. Here, we provide an overview of the highly integrated BR signalling network and explain how this steroid hormone functions as a master regulator of plant growth, development and metabolism.

Factors involved in the rise of phosphoenolpyruvate carboxylase-kinase activity caused by salinity in sorghum leaves

Salinity increases phosphoenolpyruvate carboxylase kinase (PEPCase-k) activity in sorghum leaves. This work has been focused on the mechanisms responsible for this phenomenon. The light-triggered expression of SbPPCK1 gene, accountable for the photosynthetic C4-PEPCase-k, is controlled by a complex signal transduction chain involving phospholipases C and D (PLC and PLD). These two phospholipase-derived signalling pathways were functional in salinized plants. Pharmacological agents that act on PLC (U-73122, neomycin) or PLD (n-butanol) derived signals, blocked the expression of SbPPCK1, but had little effect on PEPCase-k activity. This discrepancy was further noticed when SbPPCK1-3 gene expression and PEPCase-k activity were studied in parallel. At 172mM, the main effect of NaCl was to decrease the rate of PEPCase-k protein turnover. Meanwhile, 258mM NaCl significantly increased both SbPPCK1 and SbPPCK2 gene expression and/or mRNA stability. The combination of these factors contributed to maintain a high PEPCase-k activity in salinity. LiCl increased calcium-dependent protein kinase (CDPK) activity in illuminated sorghum leaves while it decreased the rate of PEPCase-k degradation. The latter effect was restrained by W7, an inhibitor of CDPK activity. Recombinant PEPCase-k protein was phosphorylated in vitro by PKA. A conserved phosphorylation motif, which can be recognized by PKA and by plant CDPKs, is present in the three PEPCase-ks proteins. Thus, it is possible that a phosphorylation event could be controlling (increasing) the stability of PEPCase-k in salinity. These results propose a new mechanism of regulation of PEPCase-k levels, and highlight the relevance of the preservation of key metabolic elements during the bulk degradation of proteins, which is commonly associated to stress.

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.