RESUMO
Virus-induced gene silencing (VIGS) has been shown to be of great potential in plant reverse genetics. Advantages of VIGS over other approaches, such as T-DNA or transposon tagging, include the circumvention of plant transformation, methodological simplicity and robustness, and speedy results. These features make VIGS an attractive alternative instrument in functional genomics, even in a high throughput fashion. The system is already well established in Nicotiana benthamiana; however, efforts are being made to improve VIGS in other species, including monocots. Current research is focussed on unravelling the mechanisms of post-transcriptional gene silencing and VIGS, as well as on finding novel viral vectors in order to broaden the host species spectrum. We examined how VIGS has been used to assess gene functions in plants, including molecular mechanisms involved in the process, available methodological elements, such as vectors and inoculation procedures, and we looked for examples in which the system has been applied successfully to characterize gene function in plants.
Assuntos
Inativação Gênica , Genes de Plantas/genética , Nicotiana/genética , Vírus de Plantas/genética , Plantas Geneticamente Modificadas/genética , Transcrição Gênica/genética , DNA Viral , Flores/genética , Vetores Genéticos , Genômica/métodos , Modelos Genéticos , Transformação GenéticaRESUMO
The ABC model for floral development was proposed more than 10 years ago and since then many studies have been performed on model species, such as Arabidopsis thaliana, Antirrhinum majus, and many other species in order to confirm this hypothesis. This led to additional information on flower development and to more complex molecular models. AGAMOUS (AG) is the only C type gene in Arabidopsis and it is responsible for stamen and carpel development as well as floral determinacy. LLAG1, an AG homologue from lily (Lilium longiflorum Thunb.) was isolated by screening a cDNA library derived from developing floral buds. The deduced amino acid sequence revealed the MIKC structure and a high homology in the MADS-box among AG and other orthologues. Phylogenetic analysis indicated a close relationship between LLAG1 and AG orthologues from monocot species. Spatial expression data showed LLAG1 transcripts exclusively in stamens and carpels, constituting the C domain of the ABC model. Functional analysis was carried out in Arabidopsis by overexpression of LLAG1 driven by the CaMV35S promoter. Transformed plants showed homeotic changes in the two outer floral whorls with some plants presenting the second whorl completely converted into stamens. Altogether, these data strongly indicated the functional homology between LLAG1 and AG.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Flores/crescimento & desenvolvimento , Genes Homeobox/genética , Lilium/genética , Proteínas de Domínio MADS/genética , Proteína AGAMOUS de Arabidopsis/genética , Proteína AGAMOUS de Arabidopsis/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , DNA Complementar/química , DNA Complementar/genética , Flores/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes Homeobox/fisiologia , Proteínas de Domínio MADS/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
RNA 2 of Tobacco rattle virus isolate PpK20 encodes the viral coat protein (CP) and two nonstructural proteins of 40 kDa ('40K protein') and 32.8 kDa ('32.8K'). The 40K protein is required for transmission of the virus by the vector nematode Paratrichodorus pachydermus whereas the 32.8K protein may be involved in transmission by other vector nematode species. An antiserum was raised against the 40K protein expressed in E. coli and used to study the expression and subcellular localization of this protein in infected Nicotiana benthamiana plants. The time-course of the expression of the 40K protein in leaves and roots was similar to that of CP and both proteins were similarly distributed over the 1000 g pellet, 30000 g pellet and 30000 g supernatant fractions of leaf and root homogenates. Using the yeast two-hybrid system, a strong interaction between CP subunits was observed and weaker interactions between CP and the 32.8K protein and between CP and the 40K protein were detected. A deletion of the C-terminal 19 amino acids of CP interfered with the CP-40K interaction but not with CP-32.8K or CP-CP interactions, whereas a C-terminal deletion of 79 amino acids interfered with CP-40K and CP-32.8K interactions but not with the CP-CP interaction. As the C terminus of CP is known to be involved in nematode-transmission of tobraviruses, the data support the hypothesis that interactions between CP and RNA 2-encoded nonstructural proteins play a role in the transmission process.
