RESUMEN
Insect herbivory may drive evolution by selecting for trees with heritable resistance against defoliation. The spruce budworm (Choristoneura fumiferana, SBW) is a highly damaging forest insect pest that can affect population structure of white spruce (Picea glauca) in North America. Resistance against SBW was recently described in white spruce and was linked to three constitutive resistance biomarkers: the phenolic compounds piceol and pungenol, and expression of a beta-glucosidase encoding gene (Pgßglu-1). We investigated the phenotypic variability and heritability of these resistance biomarkers and of picein, the precursor of piceol, in the foliage of 874 trees belonging to 33 full-sib families and 71 clonal lines under evaluation in seven field locations in Eastern Canada. We aimed to (i) determine their genetic control, (ii) estimate the genetic and phenotypic correlations among defense biomarkers, and (iii) determine whether their constitutive levels are associated with detrimental trade-offs on growth. Quantitative genetics analyses indicated that all four traits are moderately to highly heritable. The full-sib and clonal analyses showed that additive and non-additive genetic effects play major and minor roles, respectively. Positive genetic and phenotypic correlations between resistance biomarkers and primary growth indicated that there is no trade-off between total height and height increment and resistance traits, contradicting the GDBH (Growth Differentiation Balance Hypothesis). Our findings about the predominant additive genetic basis of the resistance biomarkers show that adaptive evolution of white spruce natural populations to resist to SBW is possible and that potentially important gains could also be expected from artificial selection.
Asunto(s)
Biomarcadores/análisis , Resistencia a la Enfermedad/genética , Evolución Molecular , Interacciones Huésped-Parásitos/genética , Mariposas Nocturnas/patogenicidad , Picea/genética , Picea/parasitología , Animales , Herbivoria , Picea/crecimiento & desarrolloRESUMEN
The endosomal sorting complex required for transport (ESCRT) consists of several multi-protein subcomplexes which assemble sequentially at the endosomal surface and function in multivesicular body (MVB) biogenesis. While ESCRT has been relatively well characterized in yeasts and mammals, comparably little is known about ESCRT in plants. Here we explored the yeast two-hybrid protein interaction network and subcellular localization of the Arabidopsis thaliana ESCRT machinery. We show that the Arabidopsis ESCRT interactome possesses a number of protein-protein interactions that are either conserved in yeasts and mammals or distinct to plants. We show also that most of the Arabidopsis ESCRT proteins examined at least partially localize to MVBs in plant cells when ectopically expressed on their own or co-expressed with other interacting ESCRT proteins, and some also induce abnormal MVB phenotypes, consistent with their proposed functional role(s) as part of the ESCRT machinery in Arabidopsis. Overall, our results help define the plant ESCRT machinery by highlighting both conserved and unique features when compared to ESCRT in other evolutionarily diverse organisms, providing a foundation for further exploration of ESCRT in plants.
RESUMEN
BACKGROUND: Carnation Italian ringspot virus (CIRV) is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication. While the membrane-bound components of the CIRV replication complex, including a 36-kD RNA-binding protein (p36), are known to be essential for these changes in mitochondrial morphology and are relatively well characterized in terms of their roles in nascent viral RNA synthesis, how these proteins are specifically targeted and inserted into mitochondria is poorly defined. RESULTS: Here we report on the molecular signal responsible for sorting p36 to the mitochondrial outer membrane. Using a combination of gain-of-function assays with portions of p36 fused to reporter proteins and domain-swapping assays with p36 and another closely-related viral RNA-binding protein, p33, that sorts specifically to the peroxisomal boundary membrane, we show that the mitochondrial targeting information in p36 resides within its two transmembrane domains (TMDs) and intervening hydrophilic loop sequence. Comprehensive mutational analysis of these regions in p36 revealed that the primary targeting determinants are the moderate hydrophobicity of both TMDs and the positively-charged face of an amphipathic helix within the intervening loop sequence. We show also using bimolecular fluorescence complementation (BiFC) that p36 interacts with certain components of the translocase complex in the mitochondrial outer membrane (TOM), but not with the sorting and assembly machinery (SAM). CONCLUSION: Our results provide insight to how viruses, such as CIRV, exploit specific host-cell protein sorting pathways to facilitate their replication. The characterization of the targeting and insertion of p36 into the mitochondrial outer membrane also sheds light on the mechanisms involved in sorting of host-cell membrane proteins to mitochondria, a process that has been largely unexplored in plants.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Membranas Mitocondriales/metabolismo , Tombusvirus/fisiología , Proteínas Virales/química , Proteínas Virales/metabolismo , Replicación Viral/fisiología , Secuencia de Aminoácidos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/análisis , Microscopía Fluorescente , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Datos de Secuencia Molecular , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , ARN Viral , Proteínas Virales/análisisRESUMEN
Tomato bushy stunt virus (TBSV), a positive-strand RNA virus, causes extensive inward vesiculations of the peroxisomal boundary membrane and formation of peroxisomal multivesicular bodies (pMVBs). Although pMVBs are known to contain protein components of the viral membrane-bound RNA replication complex, the mechanisms of protein targeting to peroxisomal membranes and participation in pMVB biogenesis are not well understood. We show that the TBSV 33-kD replication protein (p33), expressed on its own, targets initially from the cytosol to peroxisomes, causing their progressive aggregation and eventually the formation of peroxisomal ghosts. These altered peroxisomes are distinct from pMVBs; they lack internal vesicles and are surrounded by novel cytosolic vesicles that contain p33 and appear to be derived from evaginations of the peroxisomal boundary membrane. Concomitant with these changes in peroxisomes, p33 and resident peroxisomal membrane proteins are relocalized to the peroxisomal endoplasmic reticulum (pER) subdomain. This sorting of p33 is disrupted by the coexpression of a dominant-negative mutant of ADP-ribosylation factor1, implicating coatomer in vesicle formation at peroxisomes. Mutational analysis of p33 revealed that its intracellular sorting is also mediated by several targeting signals, including three peroxisomal targeting elements that function cooperatively, plus a pER targeting signal resembling an Arg-based motif responsible for vesicle-mediated retrieval of escaped ER membrane proteins from the Golgi. These results provide insight into virus-induced intracellular rearrangements and reveal a peroxisome-to-pER sorting pathway, raising new mechanistic questions regarding the biogenesis of peroxisomes in plants.
Asunto(s)
Retículo Endoplásmico/metabolismo , Peroxisomas/metabolismo , Transporte de Proteínas , Tombusvirus/fisiología , Proteínas Virales/fisiología , Replicación Viral , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Interferencia de ARN , Homología de Secuencia de Aminoácido , Nicotiana/genética , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Fatty acid desaturases (FADs) play a prominent role in plant lipid metabolism and are located in various subcellular compartments, including the endoplasmic reticulum (ER). To investigate the biogenesis of ER-localized membrane-bound FADs, we characterized the mechanisms responsible for insertion of Arabidopsis FAD2 and Brassica FAD3 into ER membranes and determined the molecular signals that maintain their ER residency. Using in vitro transcription/translation reactions with ER-derived microsomes, we show that both FAD2 and FAD3 are efficiently integrated into membranes by a co-translational, translocon-mediated pathway. We also demonstrate that while the C-terminus of FAD3 (-KSKIN) contains a functional prototypic dilysine ER retrieval motif, FAD2 contains a novel C-terminal aromatic amino acid-containing sequence (-YNNKL) that is both necessary and sufficient for maintaining localization in the ER. Co-expression of a membrane-bound reporter protein containing the FAD2 C-terminus with a dominant-negative mutant of ADP-ribosylation factor (Arf)1 abolished transient localization of the reporter protein in the Golgi, indicating that the FAD2 peptide signal acts as an ER retrieval motif. Mutational analysis of the FAD2 ER retrieval signal revealed a sequence-specific motif consisting of Phi-X-X-K/R/D/E-Phi-COOH, where -Phi- are large hydrophobic amino acid residues. Interestingly, this aromatic motif was present in a variety of other known and putative ER membrane proteins, including cytochrome P450 and the peroxisomal biogenesis factor Pex10p. Taken together, these data describe the insertion and retrieval mechanisms of FADs and define a new ER localization signal in plants that is responsible for the retrieval of escaped membrane proteins back to the ER.