RESUMEN
Plants deploy intracellular receptors to counteract pathogen effectors that suppress cell-surface-receptor-mediated immunity. To what extent pathogens manipulate intracellular receptor-mediated immunity, and how plants tackle such manipulation, remains unknown. Arabidopsis thaliana encodes three similar ADR1 class helper nucleotide-binding domain leucine-rich repeat receptors (ADR1, ADR1-L1, and ADR1-L2), which are crucial in plant immunity initiated by intracellular receptors. Here, we report that Pseudomonas syringae effector AvrPtoB suppresses ADR1-L1- and ADR1-L2-mediated cell death. ADR1, however, evades such suppression by diversifying into two ubiquitination sites targeted by AvrPtoB. The intracellular sensor SNC1 interacts with and guards the CCR domains of ADR1-L1/L2. Removal of ADR1-L1/L2 or delivery of AvrPtoB activates SNC1, which then signals through ADR1 to trigger immunity. Our work elucidates the long-sought-after function of SNC1 in defense, and also how plants can use dual strategies, sequence diversification, and a multi-layered guard-guardee system, to counteract pathogen's attack on core immunity functions.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Inmunidad de la Planta , Ubiquitinación , Proteínas Portadoras/metabolismo , Enfermedades de las PlantasRESUMEN
Fungi are an important group of microorganisms that play crucial roles in a variety of ecological and biotechnological processes. Fungi depend on intracellular protein trafficking, which involves moving proteins from their site of synthesis to the final destination within or outside the cell. The soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are vital components of vesicle trafficking and membrane fusion, ultimately leading to the release of cargos to the target destination. The v-SNARE (vesicle-associated SNARE) Snc1 is responsible for anterograde and retrograde vesicle trafficking between the plasma membrane (PM) and Golgi. It allows for the fusion of exocytic vesicles to the PM and the subsequent recycling of Golgi-localized proteins back to the Golgi via three distinct and parallel recycling pathways. This recycling process requires several components, including a phospholipid flippase (Drs2-Cdc50), an F-box protein (Rcy1), a sorting nexin (Snx4-Atg20), a retromer submit, and the COPI coat complex. Snc1 interacts with exocytic SNAREs (Sso1/2, Sec9) and the exocytic complex to complete the process of exocytosis. It also interacts with endocytic SNAREs (Tlg1 and Tlg2) during endocytic trafficking. Snc1 has been extensively investigated in fungi and has been found to play crucial roles in various aspects of intracellular protein trafficking. When Snc1 is overexpressed alone or in combination with some key secretory components, it results in enhanced protein production. This article will cover the role of Snc1 in the anterograde and retrograde trafficking of fungi and its interactions with other proteins for efficient cellular transportation.
Asunto(s)
Proteínas SNARE , Proteínas de Saccharomyces cerevisiae , Proteínas SNARE/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fusión de Membrana , Proteínas R-SNARE/metabolismo , Transporte de Proteínas , Hongos/metabolismoRESUMEN
Immune receptors play important roles in the perception of pathogens and initiation of immune responses in both plants and animals. Intracellular nucleotide-binding domain leucine-rich repeat (NLR)-type receptors constitute a major class of receptors in vascular plants. In the Arabidopsis thaliana mutant suppressor of npr1-1, constitutive 1 (snc1), a gain-of-function mutation in the NLR gene SNC1 leads to SNC1 overaccumulation and constitutive activation of defense responses. From a CRISPR/Cas9-based reverse genetics screen in the snc1 autoimmune background, we identified that mutations in TRAF CANDIDATE 1b (TC1b), a gene encoding a protein with four tumor necrosis factor receptor-associated factor (TRAF) domains, can suppress snc1 phenotypes. TC1b does not appear to be a general immune regulator as it is not required for defense mediated by other tested immune receptors. TC1b also does not physically associate with SNC1, affect SNC1 accumulation, or affect signaling of the downstream helper NLRs represented by ACTIVATED DISEASE RESISTANCE PROTEIN 1-L2 (ADR1-L2), suggesting that TC1b impacts snc1 autoimmunity in a unique way. TC1b can form oligomers and localizes to punctate structures of unknown function. The puncta localization of TC1b strictly requires its coiled-coil (CC) domain, whereas the functionality of TC1b requires the four TRAF domains in addition to the CC. Overall, we uncovered the TRAF domain protein TC1b as a novel positive contributor to plant immunity.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Inmunidad de la Planta , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Mutación , Fenotipo , Enfermedades de las PlantasRESUMEN
The homeostasis of resistance (R) proteins in plants must be tightly regulated to ensure precise activation of plant immune responses upon pathogen infection, while avoiding autoimmunity and growth defects when plants are uninfected. It is known that CPR1, an F-box protein in the SCF E3 complex, functions as a negative regulator of plant immunity through targeting the resistance (R) proteins SNC1 and RPS2 for degradation. However, whether these R proteins are also targeted by other E3 ligases is unclear. Here, we isolated Arabidopsis MUSE16, which encodes a RING-type E3 ligase, from a forward genetic screen and suggest that it is a negative regulator of plant immunity. Unlike CPR1, knocking out MUSE16 alone in Arabidopsis is not enough to result in defense-related dwarfism, since only RPS2 out of the tested R proteins accumulated in the muse16 mutants. Thus, our study identifies a novel E3 ligase involved in the degradation of nucleotide-binding and leucine-rich repeat (NLR) R proteins, support the idea that ubiquitin-mediated degradation is a fine-tuned mechanism for regulating the turnover of R proteins in plants, and that the same R protein can be targeted by different E3 ligases for regulation of its homeostasis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Arabidopsis/metabolismo , Inmunidad de la Planta/genética , Plantas/metabolismo , HomeostasisRESUMEN
The expression of an intracellular immune receptor gene SNC1 (SUPPRESSOR OF npr1, CONSTITUTIVE 1) is regulated by multiple chromatin-associated proteins for tuning immunity and growth in Arabidopsis. Whether and how these regulators coordinate to regulate SNC1 expression under varying environmental conditions is not clear. Here, we identified two activation and one repression regulatory modules based on genetic and molecular characterizations of five chromatin-associated regulators of SNC1. Modifier of snc1 (MOS1) constitutes the first module and is required for the interdependent functions of ARABIDOPSIS TRITHORAX-RELATED 7 (ATXR7) and HISTONE MONOUBIQUITINATION 1 (HUB1) to deposit H3K4me3 and H2Bub1 at the SNC1 locus. CHROMATIN REMODELING 5 (CHR5) constitutes a second module and works independently of ATXR7 and HUB1 in the MOS1 module. HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 15 (HOS15) constitutes a third module responsible for removing H3K9ac to repress SNC1 expression under nonpathogenic conditions. The upregulation of SNC1 resulting from removing the HOS15 repression module is partially dependent on the function of the CHR5 module and the MOS1 module. Together, this study reveals both the distinct and interdependent regulatory mechanisms at the chromatin level for SNC1 expression regulation and highlights the intricacy of regulatory mechanisms of NLR expression under different environment.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Cromatina , Regulación de la Expresión Génica de las Plantas , Receptores Inmunológicos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Inmunidad de la Planta/genética , Receptores Inmunológicos/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Intracellular immune receptor nucleotide-binding leucine-rich repeats (NLRs) are highly regulated transcriptionally and post-transcriptionally for balanced plant defence and growth. NLR genes often exist in gene clusters and are usually co-expressed under various conditions. Despite of intensive studies of regulation of NLR proteins, cis-acting elements for NLR gene induction, repression or co-expression are largely unknown due to a larger than usual cis-region for their expression regulation. Here we used the CRISPR/Cas9 genome editing technology to generate a series of in situ deletions at the endogenous location of a NLR gene SNC1 residing in the RPP5 gene cluster. These deletions that made in the wild type and the SNC1 constitutive expressing autoimmune mutant bon1 revealed both positive and negative cis-acting elements for SNC1 expression. Two transcription factors that could bind to these elements were found to have an impact on the expression of SNC1. In addition, co-expression of two genes with SNC1 in the same cluster is found to be mostly dependent on the SNC1 function. Therefore, SNC1 expression is under complex local regulation involving multiple cis elements and SNC1 itself is a critical regulator of gene expression of other NLR genes in the same gene cluster.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Regulación de la Expresión Génica de las Plantas , Inmunidad de la Planta/genéticaRESUMEN
Intracellular immune receptor nucleotide-binding leucine-rich repeats (NLRs) are highly regulated transcriptionally and post-transcriptionally for balanced plant defense and growth. NLR genes often exist in gene clusters and are usually co-expressed under various conditions. Despite intensive studies of the regulation of NLR proteins, cis-acting elements for NLR gene induction, repression or co-expression are largely unknown due to a larger than usual cis-region for their expression regulation. Here we used the CRISPR/Cas9 genome editing technology to generate a series of in situ deletions at the endogenous location of an NLR gene SNC1 residing in the RPP5 gene cluster. These deletions that made in the wild type and the SNC1 constitutive expressing autoimmune mutant bon1 revealed both positive and negative cis-acting elements for SNC1 expression. Two transcription factors that could bind to these elements were found to have an impact on the expression of SNC1. In addition, co-expression of two genes with SNC1 in the same cluster is found to be mostly dependent on the SNC1 function. Therefore, SNC1 expression is under complex local regulation involving multiple cis-elements and SNC1 itself is a critical regulator of gene expression of other NLR genes in the same gene cluster.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas NLR/metabolismo , Inmunidad de la Planta/genética , Factores de Transcripción/metabolismoRESUMEN
Rab GTPases play key roles in defining the identity of the various compartments that comprise the secretory and endocytic pathways. Recruitment of a Rab to a specific compartment requires its localized activation by a guanine nucleotide exchange factor (GEF). This in turn results in the recruitment of a distinct set of Rab effectors that directs the recognition of the appropriate target compartment by a carrier vesicle and their subsequent fusion. A chimeric Rab protein, Ypt1-SW1Sec4, was found to separate GEF specificity from effector specificity (Grosshans BL, et al. Proc Natl Acad Sci U S A 103(32):11821-11827, 2006), but early studies did not observe strong effects of this allele on growth or membrane traffic (Brennwald P, Novick P. Nature 362(6420):560-563, 1993). To resolve this apparent conundrum, yeast strains expressing the chimeric Rab were subjected to a more extensive battery of phenotypic tests. These tests demonstrated that changing the specificity of the GEF interaction does lead to a change in Rab localization and can lead to the ectopic recruitment of an effector, creating trafficking defects that are dependent upon the level of expression (Grosshans BL, et al. Proc Natl Acad Sci U S A 103(32):11821-11827, 2006). Here we describe the methods used in this analysis. Specifically we describe the following: 1. An assay used to quantify the efficiency of export of a cell wall protein Bgl2, 2. The use of thin section electron microscopy to address the morphology of the secretory machinery, 3. The use of a fluorescently tagged vesicle SNARE protein, GFP-Snc1, to follow plasma membrane recycling and. 4. The use of fluorescently tagged Ypt1 effectors, Cog3-GFP, Uso1-GFP, and Sec7-GFP to follow their recruitment by Ypt1-SW1Sec4.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Proteínas R-SNARE , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
The nucleotide-binding and leucine-rich repeat (NLR) proteins comprise a major class of intracellular immune receptors that are capable of detecting pathogen-derived molecules and activating immunity and cell death in plants. The activity of some NLRs, particularly the Toll-like/interleukin-1 receptor (TIR) type, is highly correlated with their nucleocytoplasmic distribution. However, whether and how the nucleocytoplasmic homeostasis of NLRs is coordinated through a bidirectional nuclear shuttling mechanism remains unclear. Here, we identified a nuclear transport receptor, KA120, which is capable of affecting the nucleocytoplasmic distribution of an NLR protein and is essential in preventing its autoactivation. We showed that the ka120 mutant displays an autoimmune phenotype and NLR-induced transcriptome features. Through a targeted genetic screen using an artificial NLR microRNA library, we identified the TIR-NLR gene SNC1 as a genetic interactor of KA120. Loss-of-function snc1 mutations as well as compromising SNC1 protein activities all substantially suppressed ka120-induced autoimmune activation, and the enhanced SNC1 activity upon loss of KA120 functionappeared to occur at the protein level. Overexpression of KA120 efficiently repressed SNC1 activity and led to a nearly complete suppression of the autoimmune phenotype caused by the gain-of-function snc1-1 mutation or SNC1 overexpression in transgenic plants. Further florescence imaging analysis indicated that SNC1 undergoes altered nucleocytoplasmic distribution with significantly reduced nuclear signal when KA120 is constitutively expressed, supporting a role of KA120 in coordinating SNC1 nuclear abundance and activity. Consistently, compromising the SNC1 nuclear level by disrupting the nuclear pore complex could also partially rescue ka120-induced autoimmunity. Collectively, our study demonstrates that KA120 is essential to avoid autoimmune activation in the absence of pathogens and is required to constrain the nuclear activity of SNC1, possibly through coordinating SNC1 nucleocytoplasmic homeostasis as a potential mechanism.
Asunto(s)
Proteínas de Arabidopsis/inmunología , Arabidopsis/inmunología , Autoinmunidad , Carioferinas/fisiología , Proteínas NLR/metabolismo , Inmunidad de la Planta/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas NLR/antagonistas & inhibidores , Inmunidad de la Planta/inmunologíaRESUMEN
Retrieval of cargo proteins from the endosome towards the trans-Golgi network (TGN) is a crucial intracellular process for cellular homeostasis. Its dysfunction is associated with pathogenesis of Alzheimer and Parkinson's diseases. Myosin family proteins are cellular motors walking along actin filaments by utilizing the chemical energy from ATP hydrolysis, known to involve in pleiotropic cellular trafficking pathways. However, the question of whether myosins play a role in the trafficking of Snc1 and Vps10 has not been addressed yet. The present study assesses the potential roles of all five yeast myosins in the recycling of two membrane cargo, Snc1 and Vps10. It appears that all myosins except Myo2 are not required for the Snc1 traffic, while it was found that Myo1 and 2 play important roles for Vps10 retrieval from the endosome and the vacuole. Multiple myo2 mutants harboring a point mutation in the actin binding or the cargo binding tail domain were characterized to demonstrate abnormal Vps10-GFP and GFP-Snc1 distribution phenotypes, suggesting a severe defect in their sorting and trafficking at the endosome. Furthermore, Vps10-GFP patches in all tested myo2 mutants were found to be near stationary with quantitative live cell imaging. Finally, we found that actin cables in the myo2 mutant cells were considerably disrupted, which may aggravate the trafficking of Vps10 from the endosome. Together, our results provide novel insights into the function of Myo-family proteins in the recycling traffic of Vps10 and Snc1 destined for the TGN.
Asunto(s)
Miosina Tipo V/metabolismo , Proteínas R-SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Red trans-Golgi/metabolismoRESUMEN
IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is one of nine importin-α isoforms in Arabidopsis that recruit nuclear localization signal-containing cargo proteins to the nuclear import machinery. IMP-α3/MOS6 is required genetically for full autoimmunity of the nucleotide-binding leucine-rich repeat immune receptor mutant snc1 (suppressor of npr1-1, constitutive 1) and MOS6 also contributes to basal disease resistance. Here, we investigated the contribution of the other importin-α genes to both types of immune responses, and we analyzed potential interactions of all importin-α isoforms with SNC1. By using reverse-genetic analyses in Arabidopsis and protein-protein interaction assays in Nicotiana benthamiana, we provide evidence that among the nine α-importins in Arabidopsis, IMP-α3/MOS6 is the main nuclear transport receptor of SNC1, and that IMP-α3/MOS6 is required selectively for autoimmunity of snc1 and basal resistance to mildly virulent Pseudomonas syringae in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/inmunología , Resistencia a la Enfermedad/fisiología , Carioferinas/fisiología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Autoinmunidad/fisiología , Carioferinas/metabolismo , Filogenia , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Pseudomonas syringaeRESUMEN
Nuclear pore complexes (NPCs) are main channels controlling nucleocytoplasmic transport and are composed of approximately 30 nucleoporins (NUPs). Emerging evidence suggests that some NUP genes have specialized functions that challenge the traditional view of NPCs as structures of uniform composition. Here, we analysed the role of six outer-ring components of NPC at normal and warm growth temperatures by examining their loss-of-function mutants in Arabidopsis thaliana. All six NUP subunits, NUP85, NUP96, NUP 133, NUP 160, SEH1 and HOS1, have a non-redundant temperature-influenced function in one or more of the processes, including rosette growth, leaf architecture and intracellular immune receptor-mediated disease resistance. At the molecular level, NUP85 and NUP133 are required for mRNA export only at warm temperature and play a larger role in the localization of transcription factor at warm temperature. In addition, NUP96 and HOS1 are essential for the expression of high temperature-responsive genes, which is correlated with their larger activity in facilitating nuclear accumulation of the transcription factor PIF4 at warm temperature. Our results show that subunits of NPC have differential roles at different temperatures, suggesting the existence of temperature-influenced NPC complexes and activities.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/inmunología , Proteínas de Complejo Poro Nuclear/metabolismo , Desarrollo de la Planta , Inmunidad de la Planta , Temperatura , Arabidopsis/genética , Arabidopsis/microbiología , Núcleo Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Mutación con Pérdida de Función , Fenotipo , Transporte de ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fracciones Subcelulares/metabolismo , Transcripción Genética , VirulenciaRESUMEN
Plants have evolved a sophisticated immune system in order to recognize and respond to microbes in their environments. Nucleotide-binding leucine-rich repeat (NLR) proteins detect the presence of specific effector molecules delivered into host cells by pathogens and activate strong defence responses. However, as excessive accumulation of NLRs can result in inappropriate immune responses, their abundance must be tightly regulated. Targeted degradation of NLRs through the ubiquitin proteasome pathway is an important mechanism to limit NLR accumulation. Mutations that perturb NLR degradation can cause autoimmune phenotypes. In this study, we show that the proteasome regulator PTRE1 also contributes to NLR degradation. ptre1 mutant plants exhibit increased defence marker gene expression and enhanced disease resistance against virulent pathogens. The stability of the NLR, SUPPRESSOR OF npr1-1 CONSTITUTIVE 1 (SNC1) is also increased in the ptre1 mutant. Although the mouse homologue of PTRE1 was reported to interact with a Cell Division Control protein 48 (CDC48) homologue in vitro (Clemen et al., 2015), we only observed interaction between PTRE1 and AtCDC48A in a split luciferase assay, but not in co-immunoprecipitation. In addition, a related Arabidopsis protein PTRE1h shares partial redundancy with PTRE1. Together, PTRE1 acts as a negative regulator of plant immunity partly by facilitating the degradation of immune receptors such as SNC1.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Complejo de la Endopetidasa Proteasomal/metabolismo , Mutación/genética , Inmunidad de la Planta , Unión Proteica , Estabilidad ProteicaRESUMEN
Higher plants utilize nucleotide-binding leucine-rich repeat domain proteins (NLRs) as intracellular immune receptors to recognize pathogen-derived effectors and trigger a robust defense. The Activated Disease Resistance 1 (ADR1) family of coiled-coil NLRs (CNLs) have evolved as helper NLRs that function downstream of many TIR-type sensor NLRs (TNLs). Close homologs of ADR1s form the N REQUIREMENT GENE 1 (NRG1) family in Arabidopsis, the function of which is unclear. Through CRISPR/Cas9 gene editing methods, we discovered that the tandemly repeated NRG1A and NRG1B are functionally redundant and operate downstream of TNLs with differential strengths. Interestingly, ADR1s and NRG1s function in two distinct parallel pathways contributing to TNL-specific immunity. Synergistic effects on basal and TNL-mediated defense were detected among ADR1s and NRG1s. An intact P-loop of NRG1s is not required for mediating signals from sensor TNLs, whereas auto-active NRG1A exhibits autoimmunity. Importantly, NRG1s localize to the cytosol and endomembrane network regardless of the presence of effectors, suggesting a cytosolic activation mechanism. Taken together, different sensor TNLs differentially use two groups of helper NLRs, ADR1s and NRG1s, to transduce downstream defense signals.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Proteínas NLR/metabolismo , Inmunidad de la Planta , Transducción de Señal , Autoinmunidad , Citosol/metabolismo , Modelos Biológicos , Mutación/genética , Plantas Modificadas Genéticamente , Multimerización de ProteínaRESUMEN
In mammals, tumor necrosis factor receptor associated factors (TRAFs) are signaling adaptors that regulate diverse physiological processes, including immunity and stress responses. In Arabidopsis, MUSE13 and MUSE14 are redundant TRAF proteins serving as adaptors in the SCFCRP1 complex to facilitate the turnover of nucleotide-binding domain and leucine-rich repeats (NLR) immune receptors. Degradation of MUSE13 is inhibited by proteasome inhibitor, suggesting that the MUSE13 stability is controlled by the 26S proteasome. However, the E3 ligase that regulates MUSE13 level is unknown. Here we report the identification of an F-box protein, SNIPER4 that regulates the turnover of MUSE13 and MUSE14. Protein levels of MUSE13 and MUSE14 are reduced by SNIPER4 overexpression, while higher accumulation of MUSE13 and MUSE14 is observed when dominant-negative SNIPER4 is expressed. Furthermore, SNIPER4 associates with MUSE13 or MUSE14. Taken together, the SCFSNIPER4 complex controls the turnover of TRAF proteins for an optimum immune output.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/inmunología , Proteínas F-Box/fisiología , Inmunidad de la Planta , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/fisiología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Resistencia a la Enfermedad , Proteínas F-Box/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Protein recycling is an essential cellular process involving endocytosis, intracellular trafficking, and exocytosis. In mammalian systems membrane lipids, including cholesterol, sphingolipids, and phospholipids, play a pivotal role in protein recycling. To address this role in budding yeast, Saccharomyces cerevisiae, we utilized GFP-Snc1, a v-SNARE protein serving as a fluorescent marker for faithfully reporting the recycling pathway. Here we demonstrate results that display moderate to significant GFP-Snc1 recycling defects upon overexpression or inactivation of phospholipid, ergosterol, and sphingolipid biosynthesis enzymes, indicating that the homeostasis of membrane lipid levels is prerequisite for proper protein recycling. By using a truncated version of GFP-Snc1 that cannot be recycled from the plasma membrane, we determined that abnormalities in Snc1 localization in membrane lipid overexpression or underexpression mutants are not due to defects in the synthetic/secretory pathway, but rather in the intracellular trafficking pathway. We found that membrane lipid imbalance resulted in an accumulation of the late endosome marker Vps10-GFP, indicating trafficking from the endosomes to the Golgi may be being hindered, preventing recycling to the plasma membrane. To elucidate the possible mechanism for this trafficking hindrance, we stained the actin cytoskeleton, then quantified the percentage of cells with visible actin cables. Compared to wild-type cells, membrane lipid mutant cells exhibited lower levels of actin cables, indicating the actin cytoskeleton is disrupted upon membrane lipid imbalance. Taken together, our results show that impairment of proper recycling may be due to disruption of the actin cytoskeleton, which causes trafficking hindrance between the endosomes and Golgi.
Asunto(s)
Membrana Celular/metabolismo , Endocitosis/fisiología , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Lípidos de la Membrana/metabolismo , Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/metabolismo , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Emerging evidence indicates a close connection between cell-cycle progression and the plant immune responses. In Arabidopsis, MODIFIER OF snc1-1 (MOS1) modulates a number of processes including endoreduplication and plant disease resistance, but the molecular mechanism underlying this modulation was not fully understood. Here, we provide biochemical and genetic evidence that TEOSINTE BRANCHED 1, CYCLOIDEA, PCF1 (TCP) transcription factors TCP15 and its homologues are mediators of MOS1 function in the immune response and are likely to be also involved in cell-cycle control. MOS1 and TCP proteins have a direct physical interaction. They both bind to the promoter of the immune receptor gene SUPRESSOR OF npr1-1, CONSTITUTIVE 1 (SNC1) and modulate its expression and consequently immune responses. MOS1 and TCP15 both affect the expression of cell-cycle genes D-type CYCLIN 3;1 (CYCD3;1), which may mediate the MOS1 function in cell-cycle modulation. In addition, CYCD3;1 overexpression upregulates immune responses, and SNC1 expression. This study investigated and revealed a role for MOS1 in transcriptional regulation through TCP15 and its homologues. This finding suggests the coordination of cell-cycle progression and plant immune responses at multiple levels.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta , Factores de Transcripción/metabolismo , Arabidopsis/inmunología , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Ciclo Celular , Ciclinas/genética , Ciclinas/metabolismo , Resistencia a la Enfermedad , Endorreduplicación , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Mapeo de Interacción de Proteínas , Factores de Transcripción/genética , Zea maysRESUMEN
ATP-dependent chromatin-remodeling factors use the energy of ATP hydrolysis to alter the structure of chromatin and are important regulators of eukaryotic gene expression. One such factor encoded by CHR5 (Chromatin-Remodeling Factor 5) in Arabidopsis (Arabidopsis thaliana) was previously found to be involved in regulation of growth and development. Here we show that CHR5 is required for the up-regulation of the intracellular immune receptor gene SNC1 (SUPPRESSOR OF npr1-1, CONSTITUTIVE1) and consequently the autoimmunity induced by SNC1 up-regulation. CHR5 functions antagonistically with another chromatin-remodeling gene DDM1 (DECREASED DNA METHYLATION 1) and independently with a histone mono-ubiquitinase HUB1 (HISTONE MONOUBIQUITINATION 1) in SNC1 regulation. In addition, CHR5 is a positive regulator of SNC1-independent plant immunity against the bacterial pathogen Pseudomonas syringae. Furthermore, the chr5 mutant has increased nucleosome occupancy in the promoter region relative to the gene body region at the whole-genome level, suggesting a global role for CHR5 in remodeling nucleosome occupancy. Our study thus establishes CHR5 as a positive regulator of plant immune responses including the expression of SNC1 and reveals a role for CHR5 in nucleosome occupancy which probably impacts gene expression genome wide.
Asunto(s)
Proteínas de Arabidopsis/inmunología , Arabidopsis/inmunología , Nucleosomas/metabolismo , Inmunidad de la Planta/fisiología , Arabidopsis/crecimiento & desarrollo , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión al Calcio , Proteínas Portadoras/genética , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de la Membrana/genética , Familia de Multigenes , Mutación , Proteínas NLR/genética , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/genética , Plantas Modificadas Genéticamente , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Vast interest exists in developing T. reesei for production of heterologous proteins. Although rich genomic and transcriptomic information has been uncovered for the T. reesei secretion pathway, little is known about whether engineering its key components could enhance expression of a heterologous gene. In this study, snc1, a v-SNARE gene, was first selected for overexpression in T. reesei. In engineered T. reesei with additional copies of snc1, the Aspergillus niger glucose oxidase (AnGOD) was produced to a significantly higher level (2.2-fold of the parental strain). hac1 and bip1, two more component genes in the secretion pathway, were further tested for overexpression and found to be also beneficial for AnGOD secretion. The overexpression of one component gene more or less affected the expression of the other two genes, suggesting a complex regulating mechanism. Our study demonstrates the potential of engineering the secretion pathway for enhancing heterologous gene production in T. reesei.
Asunto(s)
Aspergillus niger/enzimología , Aspergillus niger/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Glucosa Oxidasa/genética , Trichoderma/enzimología , Trichoderma/genética , Biotecnología , Glucosa Oxidasa/metabolismo , Ingeniería Metabólica , Redes y Vías Metabólicas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulación hacia ArribaRESUMEN
Activation of the immune response in plants antagonizes growth and development in the absence of pathogens, and such an autoimmune phenotype is often suppressed by the elevation of ambient temperature. However, molecular regulation of the ambient temperature-sensitive intersection of immune response and growth is largely elusive. A genetic screen identified an Arabidopsis mutant, zed1-D, by its high temperature-dependent growth retardation. A combination of molecular, cytological and genetic approaches was used to investigate the molecular basis behind the temperature-sensitive growth and immune response in zed1-D. A dominant mutation in HOPZ-ETI-DEFICIENT 1 (ZED1) is responsible for a high temperature-dependent autoimmunity and growth retardation in zed1-D. The autoimmune phenotype in zed1-D is dependent on the HOPZ-ACTIVATED RESISTANCE 1 (ZAR1). ZED1 and some ZED1-related kinases (ZRKs) are induced by elevated temperature and function cooperatively to suppress the immune response by modulating the transcription of SUPPRESSOR OF NPR1-1 CONSTITUTIVE 1 (SNC1) in the absence of pathogens. Our data reveal a previously unidentified role of ZRKs in the ambient temperature-sensitive immune response in the absence of pathogens, and thus reveals a possible molecular mechanism underlying the temperature-mediated intersection of immune response and growth in plants.