RESUMEN
Glycosylinositolphosphorylceramides (GIPCs) are the predominant lipid in the outer leaflet of the plasma membrane. Characterized GIPC glycosylation mutants have severe or lethal plant phenotypes. However, the function of the glycosylation is unclear. Previously, we characterized Arabidopsis thaliana GONST1 and showed that it was a nucleotide sugar transporter which provides GDP-mannose for GIPC glycosylation. gonst1 has a severe growth phenotype, as well as a constitutive defense response. Here, we characterize a mutant in GONST1's closest homolog, GONST2. The gonst2-1 allele has a minor change to GIPC headgroup glycosylation. Like other reported GIPC glycosylation mutants, gonst1-1gonst2-1 has reduced cellulose, a cell wall polymer that is synthesized at the plasma membrane. The gonst2-1 allele has increased resistance to a biotrophic pathogen Golovinomyces orontii but not the necrotrophic pathogen Botrytis cinerea. Expression of GONST2 under the GONST1 promoter can rescue the gonst1 phenotype, indicating that GONST2 has a similar function to GONST1 in providing GDP-D-Man for GIPC mannosylation.
RESUMEN
Two signal molecules, salicylic acid (SA) and N-hydroxypipecolic acid (NHP), play critical roles in plant immunity. The biosynthetic genes of both compounds are positively regulated by master immune-regulating transcription factors SARD1 and CBP60g. However, the relationship between the SA and NHP pathways is unclear. CALMODULIN-BINDING TRANSCRIPTION FACTOR 1 (CAMTA1), CAMTA2, and CAMTA3 are known redundant negative regulators of plant immunity, but the underlying mechanism also remains largely unknown. In this study, through chromatin immunoprecipitation and electrophoretic mobility shift assays, we uncovered that CBP60g is a direct target of CAMTA3, which also negatively regulates the expression of SARD1, presumably via an indirect effect. The autoimmunity of camta3-1 is suppressed by sard1 cbp60g double mutant as well as ald1 and fmo1, two single mutants defective in NHP biosynthesis. Interestingly, a suppressor screen conducted in the camta1/2/3 triple mutant background yielded various mutants blocking biosynthesis or signaling of either SA or NHP, leading to nearly complete suppression of the extreme autoimmunity of camta1/2/3, suggesting that the SA and NHP pathways can mutually amplify each other. Together, these results reveal that CAMTAs repress the biosynthesis of SA and NHP by modulating the expression of SARD1 and CBP60g, and that the SA and NHP pathways are coordinated to optimize plant immune response.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Unión a Calmodulina/metabolismo , Ácidos Pipecólicos/metabolismo , Ácido Salicílico/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/inmunología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Unión a Calmodulina/genética , Inmunoprecipitación de Cromatina , Regulación de la Expresión Génica de las Plantas , Transferasas Intramoleculares/metabolismo , Mutación , Inmunidad de la Planta , Regiones Promotoras Genéticas , Transducción de SeñalRESUMEN
BACKGROUND: Single guide RNA (sgRNA) selection is important for the efficiency of CRISPR/Cas9-mediated genome editing. However, in plants, the rules governing selection are not well established. RESULTS: We developed a facile transient assay to screen sgRNA efficiency. We then used it to test top-performing bioinformatically predicted sgRNAs for two different Arabidopsis genes. In our assay, these sgRNAs had vastly different editing efficiencies, and these efficiencies were replicated in stably transformed Arabidopsis lines. One of the genes, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT), is an essential gene, required for lignin biosynthesis. Previously, HCT function has been studied using gene silencing. Here, to avoid the negative growth effects that are due to the loss of HCT activity in xylem vessels, we used a fiber-specific promoter to drive CAS9 expression. Two independent transgenic lines showed the expected lignin decrease. Successful editing was confirmed via the observation of mutations at the HCT target loci, as well as an approximately 90% decrease in HCT activity. Histochemical analysis and a normal growth phenotype support the fiber-specific knockout of HCT. For the targeting of the second gene, Golgi-localized nucleotide sugar transporter2 (GONST2), a highly efficient sgRNA drastically increased the rate of germline editing in T1 generation. CONCLUSIONS: This screening method is widely applicable, and the selection and use of efficient sgRNAs will accelerate the process of expanding germplasm for both molecular breeding and research. In addition, this, to the best of our knowledge, is the first application of constrained genome editing to obtain chimeric plants of essential genes, thereby providing a dominant method to avoid lethal growth phenotypes.
RESUMEN
Mutations in the microtubule-associated protein tau (MAPT) underlie multiple neurodegenerative disorders, yet the pathophysiological mechanisms are unclear. A novel variant in MAPT resulting in an alanine to threonine substitution at position 152 (A152T tau) has recently been described as a significant risk factor for both frontotemporal lobar degeneration and Alzheimer's disease. Here we use complementary computational, biochemical, molecular, genetic and imaging approaches in Caenorhabditis elegans and mouse models to interrogate the effects of the A152T variant on tau function. In silico analysis suggests that a threonine at position 152 of tau confers a new phosphorylation site. This finding is borne out by mass spectrometric survey of A152T tau phosphorylation in C. elegans and mouse. Optical pulse-chase experiments of Dendra2-tau demonstrate that A152T tau and phosphomimetic A152E tau exhibit increased diffusion kinetics and the ability to traverse across the axon initial segment more efficiently than wild-type (WT) tau. A C. elegans model of tauopathy reveals that A152T and A152E tau confer patterns of developmental toxicity distinct from WT tau, likely due to differential effects on retrograde axonal transport. These data support a role for phosphorylation of the variant threonine in A152T tau toxicity and suggest a mechanism involving impaired retrograde axonal transport contributing to human neurodegenerative disease.
Asunto(s)
Alelos , Sustitución de Aminoácidos , Variación Genética , Proteínas tau/genética , Proteínas tau/metabolismo , Animales , Animales Modificados Genéticamente , Transporte Axonal , Axones/metabolismo , Caenorhabditis elegans , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Humanos , Ratones , Mutación , Fosforilación , Unión Proteica , Vesículas Sinápticas/metabolismo , Tauopatías/etiología , Tauopatías/metabolismo , Tauopatías/patologíaRESUMEN
Glycosylinositol phosphorylceramides (GIPCs), which have a ceramide core linked to a glycan headgroup of varying structures, are the major sphingolipids in the plant plasma membrane. Recently, we identified the major biosynthetic genes for GIPC glycosylation in Arabidopsis (Arabidopsis thaliana) and demonstrated that the glycan headgroup is essential for plant viability. However, the function of GIPCs and the significance of their structural variation are poorly understood. Here, we characterized the Arabidopsis glycosyltransferase GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) and showed that it is responsible for the glycosylation of a subgroup of GIPCs found in seeds and pollen that contain GlcNAc and GlcN [collectively GlcN(Ac)]. In Arabidopsis gint1 plants, loss of the GlcN(Ac) GIPCs did not affect vegetative growth, although seed germination was less sensitive to abiotic stress than in wild-type plants. However, in rice, where GlcN(Ac) containing GIPCs are the major GIPC subgroup in vegetative tissue, loss of GINT1 was seedling lethal. Furthermore, we could produce, de novo, "rice-like" GlcN(Ac) GIPCs in Arabidopsis leaves, which allowed us to test the function of different sugars in the GIPC headgroup. This study describes a monocot GIPC biosynthetic enzyme and shows that its Arabidopsis homolog has the same biochemical function. We also identify a possible role for GIPCs in maintaining cell-cell adhesion.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glicosiltransferasas/metabolismo , Oryza/crecimiento & desarrollo , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Pared Celular/química , Pared Celular/metabolismo , Ceramidas/metabolismo , Regulación de la Expresión Génica de las Plantas , Glicosiltransferasas/genética , Oryza/genética , Oryza/metabolismo , Filogenia , Plantas Modificadas Genéticamente , Polen/metabolismo , Plantones/genética , Plantones/crecimiento & desarrollo , Semillas/metabolismoRESUMEN
Systemic acquired resistance (SAR) is a defense mechanism induced in the distal parts of plants after primary infection. It confers long-lasting protection against a broad spectrum of microbial pathogens. Lack of high-throughput assays has hampered the forward genetic analysis of SAR. Here, we report the development of an easy and efficient assay for SAR and its application in a forward genetic screen for SAR-deficient mutants in Arabidopsis (Arabidopsis thaliana). Using the new assay for SAR, we identified six flavin-dependent monooxygenase1, four AGD2-like defense response protein1, three salicylic acid induction-deficient2, one phytoalexin deficient4, and one avrPphB-susceptible3 alleles as well as a gain-of-function mutant of CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR3 designated camta3-3D. Like transgenic plants overexpressing CAMTA3, camta3-3D mutant plants exhibit compromised SAR and enhanced susceptibility to virulent pathogens, suggesting that CAMTA3 is a critical regulator of both basal resistance and SAR.
Asunto(s)
Arabidopsis/inmunología , Resistencia a la Enfermedad/genética , Pruebas Genéticas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Enfermedades de las Plantas/inmunología , Alelos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Clonación Molecular , Ciclopentanos/farmacología , Resistencia a la Enfermedad/efectos de los fármacos , Etilenos/farmacología , Mutación/genética , Oxilipinas/farmacología , Peronospora/efectos de los fármacos , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/microbiología , Pseudomonas syringae/efectos de los fármacos , Ácido Salicílico/farmacologíaRESUMEN
Transportin-SR (TRN-SR) is a member of the importin-ß super-family that functions as the nuclear import receptor for serine-arginine rich (SR) proteins, which play diverse roles in RNA metabolism. Here we report the identification and cloning of mos14 (modifier of snc1-1, 14), a mutation that suppresses the immune responses conditioned by the auto-activated Resistance (R) protein snc1 (suppressor of npr1-1, constitutive 1). MOS14 encodes a nuclear protein with high similarity to previously characterized TRN-SR proteins in animals. Yeast two-hybrid assays showed that MOS14 interacts with AtRAN1 via its N-terminus and SR proteins via its C-terminus. In mos14-1, localization of several SR proteins to the nucleus was impaired, confirming that MOS14 functions as a TRN-SR. The mos14-1 mutation results in altered splicing patterns of SNC1 and another R gene RPS4 and compromised resistance mediated by snc1 and RPS4, suggesting that nuclear import of SR proteins by MOS14 is required for proper splicing of these two R genes and is important for their functions in plant immunity.