RESUMO
In Arabidopsis thaliana, seven cyclin-dependent kinase (CDK) inhibitors have been identified, designated interactors of CDKs or Kip-related proteins (KRPs). Here, the function of KRP6 was investigated during cell cycle progression in roots infected by plant-parasitic root-knot nematodes. Contrary to expectations, analysis of Meloidogyne incognita-induced galls of KRP6-overexpressing lines revealed a role for this particular KRP as an activator of the mitotic cell cycle. In accordance, KRP6-overexpressing suspension cultures displayed accelerated entry into mitosis, but delayed mitotic progression. Likewise, phenotypic analysis of cultured cells and nematode-induced giant cells revealed a failure in mitotic exit, with the appearance of multinucleated cells as a consequence. Strong KRP6 expression upon nematode infection and the phenotypic resemblance between KRP6 overexpression cell cultures and root-knot morphology point toward the involvement of KRP6 in the multinucleate and acytokinetic state of giant cells. Along these lines, the parasite might have evolved to manipulate plant KRP6 transcription to the benefit of gall establishment.
RESUMO
The application of recombinant antibodies in plant biology research is limited because plant researchers have minimal access to high-quality phage display libraries. Therefore, we constructed a library of 1.3 x 10(10) clones displaying human single-chain variable fragments (scFvs) that is available to the academic community. The scFvs selected from the library against a diverse set of plant proteins showed moderate to high antigen-binding affinity together with high specificity. Moreover, to optimize an scFv as immunodetection agent, two expression systems that allow efficient production and purification of bivalent scFv-Fc and scFv-CkappaZIP fusion proteins were integrated. We are convinced that this antibody platform will further stimulate applications of recombinant antibodies such as the diagnostic detection or immunomodulation of specific antigens in plants.
Assuntos
Anticorpos Monoclonais/genética , Afinidade de Anticorpos/genética , Biblioteca Gênica , Região Variável de Imunoglobulina/genética , Peptídeos/imunologia , Proteínas de Plantas/imunologia , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Proteínas de Transporte/genética , Expressão Gênica , Humanos , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/isolamento & purificação , Cadeias kappa de Imunoglobulina/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Especificidade por Substrato/genética , Especificidade por Substrato/imunologiaRESUMO
Defining protein complexes is critical to virtually all aspects of cell biology because many cellular processes are regulated by stable protein complexes, and their identification often provides insights into their function. We describe the development and application of a high throughput tandem affinity purification/mass spectrometry platform for cell suspension cultures to analyze cell cycle-related protein complexes in Arabidopsis thaliana. Elucidation of this protein-protein interaction network is essential to fully understand the functional differences between the highly redundant cyclin-dependent kinase/cyclin modules, which are generally accepted to play a central role in cell cycle control, in all eukaryotes. Cell suspension cultures were chosen because they provide an unlimited supply of protein extracts of actively dividing and undifferentiated cells, which is crucial for a systematic study of the cell cycle interactome in the absence of plant development. Here we report the mapping of a protein interaction network around six known core cell cycle proteins by an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, tandem affinity purification adapted for plant cells, matrix-assisted laser desorption ionization tandem mass spectrometry, data analysis, and functional assays. We identified 28 new molecular associations and confirmed 14 previously described interactions. This systemic approach provides new insights into the basic cell cycle control mechanisms and is generally applicable to other pathways in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cromatografia em Gel/métodos , Clonagem Molecular/métodos , Mapeamento de Interação de Proteínas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
The cell cycle is remarkably conserved in yeast, animals, and plants and is controlled by cyclin-dependent kinases (CDKs). CDK activity can be inhibited by binding of CDK inhibitory proteins, designated CKIs. Numerous studies show that CKIs are essential in orchestrating eukaryotic cell proliferation and differentiation. In yeast, animals, and plants, CKIs act as regulators of the G1 checkpoint in response to environmental and developmental cues and assist during mitotic cell cycles by inhibiting CDK activity required to arrest mitosis. Furthermore, CKIs play an important role in regulating cell cycle exit that precedes differentiation and in promoting differentiation in cooperation with transcription factors. Moreover, CKIs are essential to control CDK activity in endocycling cells. So, in yeast, animals, and plants, CKIs share many functional similarities, but their functions are adapted toward the specific needs of the eukaryote.