Caracterização funcional da proteína AtWWP1, componente de uma interconexão de fatores da interação geminivirus-hospedeiro envolvido na formação de corpos subnucleares

• Autor(a) principal: Calil, Iara Pinheiro
• Data de Publicação: 2013
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/4788
• topic: Interação geminivirus-hospedeiro; Corpos nucleares; Geminivirus-host interactome; Nuclear bodies; CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS

Plants are engaged in a continuous co-evolutionary struggle for dominance with their pathogens and the outcomes of these interactions are of particular importance to human activities, as they can have dramatic effects on agricultural systems (Dodds & Rathjen, 2010). Recently, the convergence of molecular studies of plant immunity and pathogen infection strategies is revealing an integrated picture of the plant pathogen interaction (Mukhtar et al., 2011) in which the pathogen effectors interaction converge onto highly connected subgroups of proteins, named hubs. A well-defined hub form plant immune system network corresponds to CSN5A protein, a catalytic subunit of the COP9 signalosome acting as a key regulator in several basic cellular processes. Consistent with the prediction that different effectors from different pathogens target similar connections in plant-pathogen interaction network, it has been shown, independently, that the protein C2 from geminivirus, a DNA virus that infects a wide variety of agronomic crops, interacts to CNS5A (Lozano- Duran et al, 2011). Additionally, it was shown that NIG and the immune receptor NIK, both targets of geminivírus NSP, interact to CSN5A (Machado, 2011; Mukhtar et al., 2011). Based on this information, it is expected that the hub CNS5A is a functional element in the geminivirus-host interaction network. Recently, it was reported that NIG, a cellular partner of CSN5A, also interacts with a unknown function protein, encoded by the locus AT2G41020 in yeast (Machado, 2011). As a possible component from geminivirus-host interaction network converging to CSN5A, AT2G41020 may interact directly or indirectly with virulence factors in defense response or compatibility. The objectives of this research involved biochemical characterization of the protein encoded by the locus AT2G4102, designated AtWWP1, and identification of its possible interactions with viral proteins and host factors. In silico analysis of tWWP1 predicted structure revealed the presence of two WW domains, and a C-terminal domain highly conserved between homologous in plant and animals. Furthermore, it has been shown that AtWWP1 is a nuclear protein capable of forming nuclear bodies via the conserved C-terminal domain. Coimmunoprecipitation and BiFC assays demonstrated that AtWWP1 interacts in vivo with the cytoplasmic protein NIG, redirecting it to nuclear bodies. In order to explore the formative activity of nuclear bodies AtWWP1, the interaction between AtWWP1 and a second protein partner AtMBD2 (methyl CG binding domaincontaining protein) was characterized in vivo. The ability to form nuclear bodies as interaction with AtMBD2 were mapped AtWWP1 occuring via its domain and C-terminal conserved, substantiating the argument that this region of AtWWP1 is responsible for the formation of nuclear bodies. Colocalization assays have shown that nuclear bodies contained in AtWWP1 are distinct from those formed by proteins involved in RNA splicing, but colocalized with nuclear bodies containing CDKC2. Furthermore, it was demonstrated that AtWWP1 does not bind to RNA, but exhibits a binding activity to DNA. These characteristics imply that AtWWP1 should be involved with basic nuclear functions. As a component of a functional hub in geminivirus-host interaction network, it is important to assess whether the viral infection would affect the nuclear bodies formed by AtWWP1.