RESUMEN
Histone ubiquitylation/deubiquitylation plays a major role in the epigenetic regulation of gene expression. In plants, OTLD1, a member of the ovarian tumor (OTU) deubiquitinase family, deubiquitylates histone 2B and represses the expression of genes involved in growth, cell expansion, and hormone signaling. OTLD1 lacks the intrinsic ability to bind DNA. How OTLD1, as well as most other known plant histone deubiquitinases, recognizes its target genes remains unknown. Here, we show that Arabidopsis transcription factor LSH10, a member of the ALOG protein family, interacts with OTLD1 in living plant cells. Loss-of-function LSH10 mutations relieve the OTLD1-promoted transcriptional repression of the target genes, resulting in their elevated expression, whereas recovery of the LSH10 function results in down-regulated transcription of the same genes. We show that LSH10 associates with the target gene chromatin as well as with DNA sequences in the promoter regions of the target genes. Furthermore, without LSH10, the degree of H2B monoubiquitylation in the target promoter chromatin increases. Hence, our data suggest that OTLD1-LSH10 acts as a co-repressor complex potentially representing a general mechanism for the specific function of plant histone deubiquitinases at their target chromatin.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteasas de Cisteína , Histonas/genética , Histonas/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Epigénesis Genética , Cromatina/genética , Cromatina/metabolismo , Regulación de la Expresión Génica , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteasas de Cisteína/genética , Proteasas de Cisteína/metabolismoRESUMEN
Functional compensation in response to gene dysfunction is a fascinating phenomenon that allows mutated viruses to regain the capabilities of their wild-type parental strains. In this study, we isolated mutants of tobacco mosaic virus capable of CP-independent systemic movement. These gain-of-function mutants lacked the 16 C-terminal amino acids of the movement protein (MP). Whereas this deletion did not affect the cell-to-cell movement of MP, it dramatically enhanced the viral genomic RNA levels and MP accumulation within the infected cells and altered the subcellular localization of MP from exclusively plasmodesmata (PD) to both PD and plasma membrane. The adapted defective virus suppressed the expression of the ethylene pathway and phloem-associated resistance factors in the inoculated leaves. These findings demonstrate the potential for plant viral MPs to gain a new function that allows viral genomes to move systemically in the absence of the natural viral factor that mediates this spread.
RESUMEN
Epigenetic regulation of gene expression is commonly affected by histone modifying enzymes (HMEs) that generate heterochromatic or euchromatic histone marks for transcriptional repression or activation, respectively. HMEs are recruited to their target chromatin by transcription factors (TFs). Thus, detecting and characterizing direct interactions between HMEs and TFs are critical for understanding their function and specificity better. These studies would be more biologically relevant if performed in vivo within living tissues. Here, a protocol is described for visualizing interactions in plant leaves between a plant histone deubiquitinase and a plant transcription factor using fluorescence resonance energy transfer (FRET), which allows the detection of complexes between protein molecules that are within <10 nm from each other. Two variations of the FRET technique are presented: SE-FRET (sensitized emission) and AB-FRET (acceptor bleaching), in which the energy is transferred non-radiatively from the donor to the acceptor or emitted radiatively by the donor upon photobleaching of the acceptor. Both SE-FRET and AB-FRET approaches can be adapted easily to discover other interactions between other proteins in planta.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Histonas , Transferencia Resonante de Energía de Fluorescencia/métodos , Factores de Transcripción/genética , Epigénesis Genética , FotoblanqueoRESUMEN
The Soybean mosaic virus (SMV) coat protein (CP) is necessary for virion assembly and viral cell-to-cell and long-distance movements in plants. We previously showed that the C-terminal region of the SMV CP is required for CP self-interaction. In the present study, we generated SMV mutants containing CPs with single amino acid substitutions of the charged amino acids in the C-proximal region. Infectivity and cell-to-cell movement of the SMV mutants were examined in soybean plants. Through this genetic approach, we identified three charged amino acid residues (R245, H246, and D250) in the surface-exposed C-terminus of the SMV CP that are critical for virus cell-to-cell and long-distance movement. Our findings suggest that the identified charged amino acids in the surface-exposed C-terminus of SMV CP are critical for CP intersubunit interactions and thereby for cell-to-cell and long-distance movement and virion assembly.
Asunto(s)
Aminoácidos/metabolismo , Proteínas de la Cápside/metabolismo , Potyvirus/fisiología , Internalización del Virus , Liberación del Virus , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Aminoácidos/genética , Proteínas de la Cápside/genética , Análisis Mutacional de ADN , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Potyvirus/genética , Conformación Proteica , Glycine max/virologíaRESUMEN
A rapid, simple, and efficient method for purification of Soybean mosaic virus (SMV) was developed based on immunoprecipitation. Traditional centrifugation-based methods for purification of SMV and other potyviruses require long, complicated procedures and large quantities of infected tissue (100-500 g). The immunoprecipitation procedure described in this study allows the purification of intact SMV virion particles in 4h from 0.5 g of tissue. The reliability of this procedure was demonstrated by RT-PCR and transmission electron microscopy (TEM). This method will be useful for high-throughput examination of the physical and morphological properties of virus particles because intact virion preparations ready for TEM observation can be purified rapidly from very small tissue samples.