RESUMO
Plants are sessile and have to cope with environmentally induced damage through modification of growth and defense pathways. How tissue regeneration is triggered in such responses and whether this involves stem cell activation is an open question. The stress hormone jasmonate (JA) plays well-established roles in wounding and defense responses. JA also affects growth, which is hitherto interpreted as a trade-off between growth and defense. Here, we describe a molecular network triggered by wound-induced JA that promotes stem cell activation and regeneration. JA regulates organizer cell activity in the root stem cell niche through the RBR-SCR network and stress response protein ERF115. Moreover, JA-induced ERF109 transcription stimulates CYCD6;1 expression, functions upstream of ERF115, and promotes regeneration. Soil penetration and response to nematode herbivory induce and require this JA-mediated regeneration response. Therefore, the JA tissue damage response pathway induces stem cell activation and regeneration and activates growth after environmental stress.
Assuntos
Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Raízes de Plantas/metabolismo , Células-Tronco/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Ciclinas/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Herbivoria , Ácidos Indolacéticos/metabolismo , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Estresse Fisiológico , Fatores de Transcrição/metabolismoRESUMO
Small cell lung carcinoma (SCLC) is among the most lethal of all solid tumor malignancies. In an effort to identify novel therapeutic approaches for this recalcitrant cancer type, we applied genome-scale CRISPR/Cas9 inactivation screens to cell lines that we derived from a murine model of SCLC. SCLC cells were particularly sensitive to the deletion of NEDD8 and other neddylation pathway genes. Genetic suppression or pharmacological inhibition of this pathway using MLN4924 caused cell death not only in mouse SCLC cell lines but also in patient-derived xenograft (PDX) models of pulmonary and extrapulmonary small cell carcinoma treated ex vivo or in vivo. A subset of PDX models were exceptionally sensitive to neddylation inhibition. Neddylation inhibition suppressed expression of major regulators of neuroendocrine cell state such as INSM1 and ASCL1, which a subset of SCLC rely upon for cell proliferation and survival. To identify potential mechanisms of resistance to neddylation inhibition, we performed a genome-scale CRISPR/Cas9 suppressor screen. Deletion of components of the COP9 signalosome strongly mitigated the effects of neddylation inhibition in small cell carcinoma, including the ability of MLN4924 to suppress neuroendocrine transcriptional program expression. This work identifies neddylation as a regulator of neuroendocrine cell state and potential therapeutic target for small cell carcinomas.
Assuntos
Carcinoma de Células Pequenas/terapia , Ciclopentanos , Neoplasias Pulmonares/terapia , Proteína NEDD8/metabolismo , Pirimidinas , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Complexo do Signalossomo COP9/genética , Carcinoma de Células Pequenas/fisiopatologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclopentanos/farmacologia , Ciclopentanos/uso terapêutico , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Neoplasias Pulmonares/fisiopatologia , Camundongos , Proteína NEDD8/genética , Células Neuroendócrinas/citologia , Células Neuroendócrinas/efeitos dos fármacos , Proteínas/genética , Proteínas/metabolismo , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Proteínas Repressoras/genética , Deleção de SequênciaRESUMO
Extracellular 2'3'-cyclic-GMP-AMP (cGAMP) is an immunotransmitter exported by diseased cells and imported into host cells to activate the innate immune STING pathway. We previously identified SLC19A1 as a cGAMP importer, but its use across human cell lines is limited. Here, we identify LRRC8A heteromeric channels, better known as volume-regulated anion channels (VRAC), as widely expressed cGAMP transporters. LRRC8A forms complexes with LRRC8C and/or LRRC8E, depending on their expression levels, to transport cGAMP and other 2'3'-cyclic dinucleotides. In contrast, LRRC8D inhibits cGAMP transport. We demonstrate that cGAMP is effluxed or influxed via LRRC8 channels, as dictated by the cGAMP electrochemical gradient. Activation of LRRC8A channels, which can occur under diverse stresses, strongly potentiates cGAMP transport. We identify activator sphingosine 1-phosphate and inhibitor DCPIB as chemical tools to manipulate channel-mediated cGAMP transport. Finally, LRRC8A channels are key cGAMP transporters in resting primary human vasculature cells and universal human cGAMP transporters when activated.
Assuntos
Sistemas CRISPR-Cas , Proteínas de Membrana/metabolismo , Nucleotídeos Cíclicos/metabolismo , Transporte Biológico , Ciclopentanos/farmacologia , Humanos , Indanos/farmacologia , Lisofosfolipídeos/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Células U937RESUMO
Proper anther dehiscence is essential for successful pollination and reproduction in angiosperms, and jasmonic acid (JA) is crucial for the process. However, the mechanisms underlying the tight regulation of JA biosynthesis during anther development remain largely unknown. Here, we demonstrate that the rice (Oryza sativa L.) ethylene-response factor-associated amphiphilic repression (EAR) motif-containing protein TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORS (TCP) INTERACTOR CONTAINING EAR MOTIF PROTEIN1 (OsTIE1) tightly regulates JA biosynthesis by repressing TCP transcription factor OsTCP1/PCF5 during anther development. The loss of OsTIE1 function in Ostie1 mutants causes male sterility. The Ostie1 mutants display inviable pollen, early stamen filament elongation, and precocious anther dehiscence. In addition, JA biosynthesis is activated earlier and JA abundance is precociously increased in Ostie1 anthers. OsTIE1 is expressed during anther development, and OsTIE1 is localized in nuclei and has transcriptional repression activity. OsTIE1 directly interacts with OsTCP1, and overexpression of OsTCP1 caused early anther dehiscence resembling that of Ostie1. JA biosynthesis genes including rice LIPOXYGENASE are regulated by the OsTIE1-OsTCP1 complex. Our findings reveal that the OsTIE1-OsTCP1 module plays a critical role in anther development by finely tuning JA biosynthesis and provide a foundation for the generation of male sterile plants for hybrid seed production.
Assuntos
Ciclopentanos , Flores , Regulação da Expressão Gênica de Plantas , Oryza , Oxilipinas , Infertilidade das Plantas , Proteínas de Plantas , Oryza/genética , Oryza/metabolismo , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Flores/genética , Flores/metabolismo , Flores/crescimento & desenvolvimento , Flores/fisiologia , Infertilidade das Plantas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/metabolismo , Plantas Geneticamente Modificadas , MutaçãoRESUMO
Bacterial pathogens deliver effectors into host cells to suppress immunity. How host cells target these effectors is critical in pathogen-host interactions. SUMOylation, an important type of posttranslational modification in eukaryotic cells, plays a critical role in immunity, but its effect on bacterial effectors remains unclear in plant cells. In this study, using bioinformatic and biochemical approaches, we found that at least 16 effectors from the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 are SUMOylated by the enzyme cascade from Arabidopsis thaliana. Mutation of SUMOylation sites on the effector HopB1 enhances its function in the induction of plant cell death via stability attenuation of a plant receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1)-ASSOCIATED RECEPTOR KINASE 1. By contrast, SUMOylation is essential for the function of another effector, HopG1, in the inhibition of mitochondria activity and jasmonic acid signaling. SUMOylation of both HopB1 and HopG1 is increased by heat treatment, and this modification modulates the functions of these 2 effectors in different ways in the regulation of plant survival rates, gene expression, and bacterial infection under high temperatures. Therefore, the current work on the SUMOylation of effectors in plant cells improves our understanding of the function of dynamic protein modifications in plant-pathogen interactions in response to environmental conditions.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Resposta ao Choque Térmico , Pseudomonas syringae , Sumoilação , Arabidopsis/microbiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Morte Celular , Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas , Resposta ao Choque Térmico/genética , Interações Hospedeiro-Patógeno , Temperatura Alta , Células Vegetais/metabolismo , Células Vegetais/microbiologia , Doenças das Plantas/microbiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Pseudomonas syringae/patogenicidade , Pseudomonas syringae/fisiologia , Transdução de SinaisRESUMO
Coronatine and related bacterial phytotoxins are mimics of the hormone jasmonyl-L-isoleucine (JA-Ile), which mediates physiologically important plant signalling pathways1-4. Coronatine-like phytotoxins disrupt these essential pathways and have potential in the development of safer, more selective herbicides. Although the biosynthesis of coronatine has been investigated previously, the nature of the enzyme that catalyses the crucial coupling of coronafacic acid to amino acids remains unknown1,2. Here we characterize a family of enzymes, coronafacic acid ligases (CfaLs), and resolve their structures. We found that CfaL can also produce JA-Ile, despite low similarity with the Jar1 enzyme that is responsible for ligation of JA and L-Ile in plants5. This suggests that Jar1 and CfaL evolved independently to catalyse similar reactions-Jar1 producing a compound essential for plant development4,5, and the bacterial ligases producing analogues toxic to plants. We further demonstrate how CfaL enzymes can be used to synthesize a diverse array of amides, obviating the need for protecting groups. Highly selective kinetic resolutions of racemic donor or acceptor substrates were achieved, affording homochiral products. We also used structure-guided mutagenesis to engineer improved CfaL variants. Together, these results show that CfaLs can deliver a wide range of amides for agrochemical, pharmaceutical and other applications.
Assuntos
Amidas/metabolismo , Ligases/química , Ligases/metabolismo , Amidas/química , Aminoácidos/biossíntese , Aminoácidos/química , Azospirillum lipoferum/enzimologia , Azospirillum lipoferum/genética , Ácidos Carboxílicos/metabolismo , Ciclopentanos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Herbicidas/química , Herbicidas/metabolismo , Indenos/química , Isoleucina/análogos & derivados , Isoleucina/biossíntese , Isoleucina/química , Cinética , Modelos Moleculares , Pectobacterium/enzimologia , Pectobacterium/genética , Pseudomonas syringae/enzimologia , Pseudomonas syringae/genéticaRESUMO
SLC7A11 is a cystine transporter and ferroptosis inhibitor. How the stability of SLC7A11 is coordinately regulated in response to environmental cystine by which E3 ligase and deubiquitylase (DUB) remains elusive. Here, we report that neddylation inhibitor MLN4924 increases cystine uptake by causing SLC7A11 accumulation, via inactivating Cullin-RING ligase-3 (CRL-3). We identified KCTD10 as the substrate-recognizing subunit of CRL-3 for SLC7A11 ubiquitylation, and USP18 as SLC7A11 deubiquitylase. Upon cystine deprivation, the protein levels of KCTD10 or USP18 are decreased or increased, respectively, contributing to SLC7A11 accumulation. By destabilizing or stabilizing SLC7A11, KCTD10, or USP18 inversely regulates the cystine uptake and ferroptosis. Biologically, MLN4924 combination with SLC7A11 inhibitor Imidazole Ketone Erastin (IKE) enhanced suppression of tumor growth. In human breast tumor tissues, SLC7A11 levels were negatively or positively correlated with KCTD10 or USP18, respectively. Collectively, our study defines how SLC7A11 and ferroptosis is coordinately regulated by the CRL3KCTD10/E3-USP18/DUB axis, and provides a sound rationale of drug combination to enhance anticancer efficacy.
Assuntos
Cistina , Ferroptose , Pirimidinas , Ubiquitina Tiolesterase , Animais , Feminino , Humanos , Camundongos , Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ciclopentanos/metabolismo , Ciclopentanos/farmacologia , Cistina/metabolismo , Células HEK293 , Piperazinas/farmacologia , Pirimidinas/farmacologia , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
ABSTRACT: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis and limited treatment options. Programmed cell death ligand 1(PD-L1) is recognized to be involved in the pathobiology of ATLL. However, what molecules control PD-L1 expression and whether genetic or pharmacological intervention might modify PD-L1 expression in ATLL cells are still unknown. To comprehend the regulatory mechanisms of PD-L1 expression in ATLL cells, we performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening in this work. In ATLL cells, we discovered that the neddylation-associated genes NEDD8, NAE1, UBA3, and CUL3 negatively regulated PD-L1 expression, whereas STAT3 positively did so. We verified, in line with the genetic results, that treatment with the JAK1/2 inhibitor ruxolitinib or the neddylation pathway inhibitor pevonedistat resulted in a decrease in PD-L1 expression in ATLL cells or an increase in it, respectively. It is significant that these results held true regardless of whether ATLL cells had the PD-L1 3' structural variant, a known genetic anomaly that promotes PD-L1 overexpression in certain patients with primary ATLL. Pevonedistat alone showed cytotoxicity for ATLL cells, but compared with each single modality, pevonedistat improved the cytotoxic effects of the anti-PD-L1 monoclonal antibody avelumab and chimeric antigen receptor (CAR) T cells targeting PD-L1 in vitro. As a result, our work provided insight into a portion of the complex regulatory mechanisms governing PD-L1 expression in ATLL cells and demonstrated the in vitro preliminary preclinical efficacy of PD-L1-directed immunotherapies by using pevonedistat to upregulate PD-L1 in ATLL cells.
Assuntos
Ciclopentanos , Leucemia-Linfoma de Células T do Adulto , Linfoma , Pirimidinas , Adulto , Humanos , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/patologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Antígeno B7-H1/metabolismo , Linfoma/genéticaRESUMO
CONSTANS (CO) is a master flowering-time regulator that integrates photoperiodic and circadian signals in Arabidopsis thaliana. CO is expressed in multiple tissues, including young leaves and seedling roots, but little is known about the roles and underlying mechanisms of CO in mediating physiological responses other than flowering. Here, we show that CO expression is responsive to jasmonate. CO negatively modulated jasmonate-imposed root-growth inhibition and anthocyanin accumulation. Seedlings from co mutants were more sensitive to jasmonate, whereas overexpression of CO resulted in plants with reduced sensitivity to jasmonate. Moreover, CO mediated the diurnal gating of several jasmonate-responsive genes under long-day conditions. We demonstrate that CO interacts with JASMONATE ZIM-DOMAIN (JAZ) repressors of jasmonate signaling. Genetic analyses indicated that CO functions in a CORONATINE INSENSITIVE1 (COI1)-dependent manner to modulate jasmonate responses. Furthermore, CO physically associated with the basic helix-loop-helix (bHLH) subgroup IIId transcription factors bHLH3 and bHLH17. CO acted cooperatively with bHLH17 in suppressing jasmonate signaling, but JAZ proteins interfered with their transcriptional functions and physical interaction. Collectively, our results reveal the crucial regulatory effects of CO on mediating jasmonate responses and explain the mechanism by which CO works together with JAZ and bHLH subgroup IIId factors to fine-tune jasmonate signaling.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Plântula/genética , Plântula/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ciclopentanos/farmacologia , Ciclopentanos/metabolismo , Oxilipinas/farmacologia , Oxilipinas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica de Plantas/genéticaRESUMO
F-box proteins have diverse functions in eukaryotic organisms, including plants, mainly targeting proteins for 26S proteasomal degradation. Here, we demonstrate the role of the F-box protein SKP1-INTERACTING PARTNER 31 (SKIP31) from Arabidopsis (Arabidopsis thaliana) in regulating late seed maturation events, seed vigor, and viability through biochemical and genetic studies using skip31 mutants and different transgenic lines. We show that SKIP31 is predominantly expressed in seeds and that SKIP31 interacts with JASMONATE ZIM DOMAIN (JAZ) proteins, key repressors in jasmonate (JA) signaling, directing their ubiquitination for proteasomal degradation independently of coronatine/jasmonic acid-isoleucine (JA-Ile), in contrast to CORONATINE INSENSITIVE 1, which sends JAZs for degradation in a coronatine/JA-Ile dependent manner. Moreover, JAZ proteins interact with the transcription factor ABSCISIC ACID-INSENSITIVE 5 (ABI5) and repress its transcriptional activity, which in turn directly or indirectly represses the expression of downstream genes involved in the accumulation of LATE EMBRYOGENESIS ABUNDANT proteins, protective metabolites, storage compounds, and abscisic acid biosynthesis. However, SKIP31 targets JAZ proteins, deregulates ABI5 activity, and positively regulates seed maturation and consequently seed vigor. Furthermore, ABI5 positively influences SKIP31 expression, while JAZ proteins repress ABI5-mediated transactivation of SKIP31 and exert feedback regulation. Taken together, our findings reveal the role of the SKIP31-JAZ-ABI5 module in seed maturation and consequently, establishment of seed vigor.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas F-Box , Arabidopsis/genética , Arabidopsis/metabolismo , Isoleucina/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ácido Abscísico/farmacologia , Ácido Abscísico/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Proteínas F-Box/genética , Sementes/genética , Sementes/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
The rhizosphere is a niche surrounding plant roots, where soluble and volatile molecules mediate signaling between plants and the associated microbiota. The preferred lifestyle of soil microorganisms is in the form of biofilms. However, less is known about whether root volatile organic compounds (rVOCs) can influence soil biofilms beyond the 2-10 mm rhizosphere zone influenced by root exudates. We report that rVOCs shift the microbiome composition and growth dynamics of complex soil biofilms. This signaling is evolutionarily conserved from ferns to higher plants. Methyl jasmonate (MeJA) is a bioactive signal of rVOCs that rapidly triggers both biofilm and microbiome changes. In contrast to the planktonic community, the resulting biofilm community provides ecological benefits to the host from a distance via growth enhancement. Thus, a volatile host defense signal, MeJA, is co-opted for assembling host-beneficial biofilms in the soil microbiota and extending the sphere of host influence in the rhizosphere.
Assuntos
Acetatos , Ciclopentanos , Microbiota , Oxilipinas , Solo , Raízes de Plantas , Microbiologia do Solo , Rizosfera , BiofilmesRESUMO
Cullin-RING ligases (CRLs) represent the largest E3 ubiquitin ligase family in eukaryotes, and the identification of their substrates is critical to understanding regulation of the proteome. Using genetic and pharmacologic Cullin inactivation coupled with genetic (GPS) and proteomic (QUAINT) assays, we have identified hundreds of proteins whose stabilities or ubiquitylation status are regulated by CRLs. Together, these approaches yielded many known CRL substrates as well as a multitude of previously unknown putative substrates. We demonstrate that one substrate, NUSAP1, is an SCF(Cyclin F) substrate during S and G2 phases of the cell cycle and is also degraded in response to DNA damage. This collection of regulated substrates is highly enriched for nodes in protein interaction networks, representing critical connections between regulatory pathways. This demonstrates the broad role of CRL ubiquitylation in all aspects of cellular biology and provides a set of proteins likely to be key indicators of cellular physiology.
Assuntos
Genoma Humano , Proteoma/análise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ciclopentanos/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Pirimidinas/farmacologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
UBE2M and UBE2F are two family members of neddylation E2 conjugating enzyme that, together with E3s, activate CRLs (Cullin-RING Ligases) by catalyzing cullin neddylation. However, whether and how two E2s cross-talk with each other are largely unknown. Here, we report that UBE2M is a stress-inducible gene subjected to cis-transactivation by HIF-1 and AP1, and MLN4924, a small molecule inhibitor of E1 NEDD8-activating enzyme (NAE), upregulates UBE2M via blocking degradation of HIF-1α and c-JUN. UBE2M is a dual E2 for targeted ubiquitylation and degradation of UBE2F, acting as a neddylation E2 to activate CUL3-Keap1 E3 under physiological conditions but as a ubiquitylation E2 for Parkin-DJ-1 E3 under stressed conditions. UBE2M-induced UBE2F degradation leads to CRL5 inactivation and subsequent NOXA accumulation to suppress the growth of lung cancer cells. Collectively, our study establishes a negative regulatory axis between two neddylation E2s with UBE2M ubiquitylating UBE2F, and two CRLs with CRL3 inactivating CRL5.
Assuntos
Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proteínas Culina/metabolismo , Ciclopentanos/farmacologia , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pirimidinas/farmacologia , Estresse Fisiológico/fisiologia , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/biossíntese , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Ubiquitinas/metabolismoRESUMO
Insect herbivory causes severe damage to plants and threatens the world's food production. During evolutionary adaptation, plants have evolved sophisticated mechanisms to rapidly accumulate a key defense hormone, jasmonate (JA), that triggers plant defense against herbivory. However, little is known about how plants initially activate JA biosynthesis at encounter with herbivory. Here, we uncover that a novel JAV1-JAZ8-WRKY51 (JJW) complex controls JA biosynthesis to defend against insect attack. In healthy plants, the JJW complex represses JA biosynthesis to restrain JA at a low basal level to ensure proper plant growth. When plants are injured by insect attack, injury rapidly triggers calcium influxes to activate calmodulin-dependent phosphorylation of JAV1, which disintegrates JJW complex and activates JA biosynthesis, giving rise to the rapid burst of JA for plant defense. Our findings offer new insights into the highly sophisticated defense systems evolved by plants to defend against herbivory.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas Correpressoras/metabolismo , Ciclopentanos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Oxilipinas/metabolismo , Folhas de Planta/enzimologia , Plantas Geneticamente Modificadas/enzimologia , Spodoptera/fisiologia , Fatores de Transcrição/metabolismo , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sinalização do Cálcio , Calmodulina/genética , Proteínas Correpressoras/genética , Regulação da Expressão Gênica de Plantas , Herbivoria , Peptídeos e Proteínas de Sinalização Intracelular/genética , Complexos Multiproteicos , Fosforilação , Folhas de Planta/genética , Plantas Geneticamente Modificadas/genética , Fatores de Transcrição/genéticaRESUMO
Receptor-like kinases (RLKs) may initiate signaling pathways by perceiving and transmitting environmental signals to cellular machinery and play diverse roles in plant development and stress responses. The rice genome encodes more than one thousand RLKs, but only a small number have been characterized as receptors for phytohormones, polypeptides, elicitors, and effectors. Here, we screened the function of 11 RLKs in rice resistance to the blast fungus Magnaporthe oryzae (M. oryzae) and identified a negative regulator named BDR1 (Blast Disease Resistance 1). The expression of BDR1 was rapidly increased under M. oryzae infection, while silencing or knockout of BDR1 significantly enhanced M. oryzae resistance in two rice varieties. Protein interaction and kinase activity assays indicated that BDR1 directly interacted with and phosphorylated mitogen-activated kinase 3 (MPK3). Knockout of BDR1 compromised M. oryzae-induced MPK3 phosphorylation levels. Moreover, transcriptome analysis revealed that M. oryzae-elicited jasmonate (JA) signaling and terpenoid biosynthesis pathway were negatively regulated by BDR1 and MPK3. Mutation of JA biosynthetic (allene oxide cyclase (AOC)/signaling (MYC2) genes decreased rice resistance to M. oryzae. Besides diterpenoid, the monoterpene linalool and the sesquiterpene caryophyllene were identified as unique defensive compounds against M. oryzae, and their biosynthesis genes (TPS3 and TPS29) were transcriptionally regulated by JA signaling and suppressed by BDR1 and MPK3. These findings demonstrate the existence of a BDR1-MPK3 cascade that negatively mediates rice blast resistance by affecting JA-related defense responses.
Assuntos
Magnaporthe , Oryza , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Transdução de Sinais , Reguladores de Crescimento de Plantas/metabolismo , Oryza/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Resistência à Doença/genética , Magnaporthe/fisiologiaRESUMO
Plant intracellular nucleotide-binding domain, leucine-rich repeat-containing receptors (NLRs) activate a robust immune response upon detection of pathogen effectors. How NLRs induce downstream immune defense genes remains poorly understood. The Mediator complex plays a central role in transducing signals from gene-specific transcription factors to the transcription machinery for gene transcription/activation. In this study, we demonstrate that MED10b and MED7 of the Mediator complex mediate jasmonate-dependent transcription repression, and coiled-coil NLRs (CNLs) in Solanaceae modulate MED10b/MED7 to activate immunity. Using the tomato CNL Sw-5b, which confers resistance to tospovirus, as a model, we found that the CC domain of Sw-5b directly interacts with MED10b. Knockout/down of MED10b and other subunits including MED7 of the middle module of Mediator activates plant defense against tospovirus. MED10b was found to directly interact with MED7, and MED7 directly interacts with JAZ proteins, which function as transcriptional repressors of jasmonic acid (JA) signaling. MED10b-MED7-JAZ together can strongly repress the expression of JA-responsive genes. The activated Sw-5b CC interferes with the interaction between MED10b and MED7, leading to the activation of JA-dependent defense signaling against tospovirus. Furthermore, we found that CC domains of various other CNLs including helper NLR NRCs from Solanaceae modulate MED10b/MED7 to activate defense against different pathogens. Together, our findings reveal that MED10b/MED7 serve as a previously unknown repressor of jasmonate-dependent transcription repression and are modulated by diverse CNLs in Solanaceae to activate the JA-specific defense pathways.
Assuntos
Proteínas de Arabidopsis , Imunidade Vegetal , Imunidade Vegetal/genética , Ciclopentanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Complexo Mediador/genética , Complexo Mediador/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMO
GRETCHEN HAGEN 3 (GH3) acyl acid amido synthetases conjugate amino acids to acyl acid hormones to either activate or inactivate the hormone molecule. The largest subgroup of GH3 proteins modify the growth-promoting hormone auxin (indole-3-acetic acid; IAA) with the second largest class activating the defense hormone jasmonic acid (JA). The two-step reaction mechanism of GH3 proteins provides a potential proofreading mechanism to ensure fidelity of hormone modification. Examining pyrophosphate release in the first-half reaction of Arabidopsis GH3 proteins that modify IAA (AtGH3.2/YDK2, AtGH3.5/WES1, AtGH3.17/VAS2), JA (AtGH3.11/JAR1), and other acyl acids (AtGH3.7, AtGH3.12/PBS3) indicates that acyl acid-AMP intermediates are hydrolyzed into acyl acid and AMP in the absence of the amino acid, a typical feature of pre-transfer editing mechanisms. Single-turnover kinetic analysis of AtGH3.2/YDK2 and AtGH3.5/WES1 shows that non-cognate acyl acid-adenylate intermediates are more rapidly hydrolyzed than the cognate IAA-adenylate. In contrast, AtGH3.11/JAR1 only adenylates JA, not IAA. While some of the auxin-conjugating GH3 proteins in Arabidopsis (i.e., AtGH3.5/WES1) accept multiple acyl acid substrates, others, like AtGH3.2/YDK2, are specific for IAA; however, both these proteins share similar active site residues. Biochemical analysis of chimeric variants of AtGH3.2/YDK2 and AtGH3.5/WES1 indicates that the C-terminal domain contributes to selection of cognate acyl acid substrates. These findings suggest that the hydrolysis of non-cognate acyl acid-adenylate intermediates, or proofreading, proceeds via a slowed structural switch that provides a checkpoint for fidelity before the full reaction proceeds.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ácidos Indolacéticos , Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/química , Ácidos Indolacéticos/metabolismo , Ácidos Indolacéticos/química , Oxilipinas/metabolismo , Oxilipinas/química , Reguladores de Crescimento de Plantas/metabolismo , Ciclopentanos/metabolismo , Ligases/metabolismo , Ligases/química , CinéticaRESUMO
Formation of secondary cell wall (SCW) is tightly regulated spatiotemporally by various developmental and environmental signals. Successful fine-tuning of the trade-off between SCW biosynthesis and stress responses requires a better understanding of how plant growth is regulated under environmental stress conditions. However, the current understanding of the interplay between environmental signaling and SCW formation is limited. The lipid-derived plant hormone jasmonate (JA) and its derivatives are important signaling components involved in various physiological processes including plant growth, development, and abiotic/biotic stress responses. Recent studies suggest that JA is involved in SCW formation but the signaling pathway has not been studied for how JA regulates SCW formation. We tested this hypothesis using the transcription factor MYB46, a master switch for SCW biosynthesis, and JA treatments. Both the transcript and protein levels of MYB46, a master switch for SCW formation, were significantly increased by JA treatment, resulting in the upregulation of SCW biosynthesis. We then show that this JA-induced upregulation of MYB46 is mediated by MYC2, a central regulator of JA signaling, which binds to the promoter of MYB46. We conclude that this MYC2-MYB46 module is a key component of the plant response to JA in SCW formation.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição/metabolismo , Ciclopentanos/farmacologia , Ciclopentanos/metabolismo , Oxilipinas/farmacologia , Oxilipinas/metabolismo , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismoRESUMO
Cannabis glandular trichomes (GTs) are economically and biotechnologically important structures that have a remarkable morphology and capacity to produce, store, and secrete diverse classes of secondary metabolites. However, our understanding of the developmental changes and the underlying molecular processes involved in cannabis GT development is limited. In this study, we developed Cannabis Glandular Trichome Detection Model (CGTDM), a deep learning-based model capable of differentiating and quantifying three types of cannabis GTs with a high degree of efficiency and accuracy. By profiling at eight different time points, we captured dynamic changes in gene expression, phenotypes, and metabolic processes associated with GT development. By integrating weighted gene co-expression network analysis with CGTDM measurements, we established correlations between phenotypic variations in GT traits and the global transcriptome profiles across the developmental gradient. Notably, we identified a module containing methyl jasmonate (MeJA)-responsive genes that significantly correlated with stalked GT density and cannabinoid content during development, suggesting the existence of a MeJA-mediated GT formation pathway. Our findings were further supported by the successful promotion of GT development in cannabis through exogenous MeJA treatment. Importantly, we have identified CsMYC4 as a key transcription factor that positively regulates GT formation via MeJA signaling in cannabis. These findings provide novel tools for GT detection and counting, as well as valuable information for understanding the molecular regulatory mechanism of GT formation, which has the potential to facilitate the molecular breeding, targeted engineering, informed harvest timing, and manipulation of cannabinoid production.
Assuntos
Acetatos , Cannabis , Ciclopentanos , Aprendizado Profundo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Oxilipinas , Tricomas , Oxilipinas/farmacologia , Oxilipinas/metabolismo , Ciclopentanos/farmacologia , Ciclopentanos/metabolismo , Cannabis/genética , Cannabis/crescimento & desenvolvimento , Cannabis/metabolismo , Acetatos/farmacologia , Tricomas/genética , Tricomas/metabolismo , Tricomas/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Transcriptoma , Reguladores de Crescimento de Plantas/metabolismoRESUMO
The jasmonic acid (JA) signaling pathway plays an important role in promoting the biosynthesis of tanshinones. While individual transcription factors have been extensively studied in the context of tanshinones biosynthesis regulation, the influence of methyl jasmonate (MeJA)-induced transcriptional complexes remains unexplored. This study elucidates the positive regulatory role of the basic helix-loop-helix protein SmMYC2 in tanshinones biosynthesis in Salvia miltiorrhiza. SmMYC2 not only binds to SmGGPPS1 promoters, activating their transcription, but also interacts with SmMYB36. This interaction enhances the transcriptional activity of SmMYC2 on SmGGPPS1, thereby promoting tanshinones biosynthesis. Furthermore, we identified three JA signaling repressors, SmJAZ3, SmJAZ4, and SmJAZ8, which interact with SmMYC2. These repressors hindered the transcriptional activity of SmMYC2 on SmGGPPS1 and disrupted the interaction between SmMYC2 and SmMYB36. MeJA treatment triggered the degradation of SmJAZ3 and SmJAZ4, allowing the SmMYC2-SmMYB36 complex to subsequently activate the expression of SmGGPPS1, whereas SmJAZ8 inhibited MeJA-mediated degradation due to the absence of the LPIARR motif. These results demonstrate that the SmJAZ-SmMYC2-SmMYB36 module dynamically regulates the JA-mediated accumulation of tanshinones. Our results reveal a new regulatory network for the biosynthesis of tanshinones. This study provides valuable insight for future research on MeJA-mediated modulation of tanshinones biosynthesis.